C. Mac Paper

bioRxiv preprint first posted online Jan. 6, 2016; doi: http://dx.doi.org/10.1101/035998.
The copyright holder for this preprint (which was not

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Deep, Staged Transcriptomic Resources for the Novel Coleopteran Models Atrachya menetriesi
and Callosobruchus maculatus
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Matthew A. Benton1+, Nathan J. Kenny2+, Kai H. Conrads1, Siegfried Roth1* and Jeremy A.
Lynch3*
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50674, Germany
Institute for Developmental Biology, University of Cologne, Zlpicherstrasse 47b, Cologne
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Research of the State Key Laboratory of Agrobiotechnology, The Chinese University of Hong
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Kong, Shatin, Hong Kong
Simon F.S. Li Marine Science Laboratory of School of Life Sciences and Center for Soybean
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States of America
Department of Biological Sciences, University of Illinois at Chicago, Chicago, Illinois, United
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Authors contributed equally
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* Co-corresponding authors
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Matthew A. Benton: matthewabenton@gmail.com
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Nathan J. Kenny: nathanjameskenny@gmail.com
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Kai Conrads: kai.conrads@gmx.de
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Siegfried Roth: siegfried.roth@uni-koeln.de
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Jeremy A. Lynch: jlynch42@uic.edu
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1
bioRxiv preprint first posted online Jan. 6, 2016; doi: http://dx.doi.org/10.1101/035998. The copyright holder for this preprint (which was not
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Abstract:
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Background: Despite recent efforts to sample broadly across metazoan and insect diversity,
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current sequence resources in the Coleoptera do not adequately describe the diversity of the
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clade. Here we present deep transcriptomic data generated with Illumina platform sequencing of
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a number of key stages in the development of two coleopteran species, the false melon beetle
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Atrachya menetriesi (Faldermann 1835) and the cowpea weevil Callosobruchus maculatus
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(Fabricius 1775). These species are both agricultural pests and are of great interest to
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developmental biology, as well as providing additional coleopteran datapoints for comparative
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genomic analysis.
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Results: By sampling at a range of timepoints covering ovary development and several crucial
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phases of embryonic development we assembled five and three timed transcriptomes for A.
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menetriesi and C. maculatus, respectively. We utilized this sequence data to build a
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transcriptomic resource combining all reads into a single, combined assembly for each species,
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and we analysed each of these resources in detail. The combined A. menetriesi assembly
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consists of 228,096 contigs with an N50 of 1,598 bp, while the combined C. maculatus
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assembly consists of 128,837 contigs with an N50 of 2,263 bp. For each of these assemblies,
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we identified 78,879 (34.6%) and 41,744 (32.4%) of contigs by BLASTx against the nr database
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using Blast2GO, and we found that 97% and 98.3% of the BUSCO set of metazoan orthologs
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were present in these assemblies. We also carried out manual analysis of 38 key
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developmental genes, and found that nearly all expected genes were present in our
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transcriptomes. Each of these analyses showed that these transcriptomes are of very high
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quality. Lastly, we performed read mapping using the individual timed RNA samples, allowing us
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to identify up- and down-regulated contigs at key stages in the development of these beetles for
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further analysis.
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Conclusions: The data presented here represent a significant increase in the publicly available
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transcriptomic resources in the Coleoptera. They have also allowed us to note significant
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changes in expression at important embryonic stages, and will provide a firm basis for a variety
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of experimentation, both in developmental biology and in wider exploration of the genomic basis
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of growth and differentiation.
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Key Words: Beetle, Coleopteran, Transcriptome, Ovary, Embryonic, Staged, Atrachya
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menetriesi, Callosobruchus maculatus, Chrysomelidae
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Background:
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The order Coleoptera is the most speciose clade of animals currently known. Despite the best
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efforts of generations of biologists, its species are only sparsely sampled and are yet to be
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comprehensively described, with approximately 90% of coleopteran diversity as yet
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uncategorized (e.g. [1, 2]). A similar discrepancy exists at the molecular level; while several
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genomic resources are available in this clade, their number and phylogenetic distribution is only
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just beginning to accurately sample that of the Coleoptera as a whole. A wide range of
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transcriptomic data is available in whole organisms (for example [35], among others), specific
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body parts (such as [6, 7]), and in several cases for staged embryos following RNA interference-
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mediated gene knock-down [8, 9]. The i5K project [10] will also greatly advance our knowledge
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of the genomic complement of Coleopterans, with 69 species of this Order listed on that
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database as nominated for genomic sequencing as of the 14/09/15 (url:
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http://arthropodgenomes.org/wiki/i5K_nominations) and at least one genome publically available
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[11]. However, the majority of this information is largely still outside of the public domain, the
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Coleoptera are still relatively undersampled compared to the Diptera and Hymenoptera, and, in
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particular, timed embryonic resources (e.g. [12]) are rare in the literature.
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The true phylogeny of the Coleoptera is still under investigation but, in general, four
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suborders, 17 superfamilies, and 168 families are recognised [13]. The structure of the
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coleopteran clade can be seen summarised in Fig 1A. Coleopterans have long been used for
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research into embryology, and in the pre-molecular era, the Chrysomelidae (summarised
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phylogeny shown in Fig 1B) was one of the best studied superfamilies. For example, the first
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functional embryonic experiments in any insect were carried out in the Colorado potato beetle,
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Leptinotarsa decemlineata (sub-family Chrysomelinae, see Fig 1B), leading to the discovery of
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the function of pole cells and the existence of germ plasm in insects [14, 15]. Further, the larval
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cuticle preparation technique, so vital for arthropod developmental biology, was first perfected in
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the bean beetle, Callosobruchus maculatus [16]. Chrysomelid beetles are also interesting from
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an ecological and economic viewpoint, as members of the group are usually pest species,
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perhaps most famously the aforementioned Colorado potato beetle, which is a major pest of
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potato crops in America, Asia and Europe.
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The Chrysomelidae are represented in public databases by a number of ongoing
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transcriptomic and genomic projects. In particular, both L. decemlineata (Bioproject
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PRJNA171749) and C. maculatus [17] are the subject of ongoing genomic sequencing.
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However, while a number of transcriptomes are planned or published in this clade (e.g. [5, 6,
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1822]) none yet sample across embryonic time points in a fashion allowing insight into the
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genetic mechanisms behind key developmental stages and deep sampling of developmentally
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important genes. Here we present transcriptomic sequences for two species of chrysomelid
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beetle, the false melon beetle, Atrachya menetriesi, and the aforementioned C. maculatus (each
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of which are pictured along with their relative phylogenetic positions in Fig 1).
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Compared to C. maculatus, the false melon beetle A. menetriesi (Faldermann), is a
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relatively unknown and understudied species. It is native to Japan, where it is an agricultural
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pest, and feeds on a variety of plants such as clover and lettuce. Although there has only been
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a small amount of research carried out on this beetle, the work that has been done has
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highlighted several interesting developmental traits, including the possibility of generating twin
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embryos after egg bisection [23], or up to four, seemingly complete, embryos following
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treatment with low temperatures [23, 24]. Another interesting trait exhibited by this beetle is that
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almost all eggs enter diapause at a certain stage, and this is only broken in the wild by winter
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conditions. However, a small proportion of eggs skip this diapause and continue to adulthood.
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The ratio of diapause to non-diapause eggs varies in different parts of Japan [25], and is a
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heritable trait [26]. Further, A. menetriesi embryos undergo a very short germ band mode of
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development [27], contrasting strongly with beetles such as C. maculatus (see below). As the
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last common ancestor of these two species is estimated to have existed only 80 million years
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ago [28], how their developmental mechanisms have diverged so greatly is a potentially
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fascinating area for future study. Research on these topics would greatly benefit from modern
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molecular studies.
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C. maculatus (Fabricius) is native to West Africa [29] but is now found worldwide, and is
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a common pest of stored legumes. It is also known as the southern cowpea weevil, however, it
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is not a true weevil. As noted above, this beetle was the focus of active developmental research
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in the pre-molecular era, with special focus on segmentation [30, 31]. Segmentation in C.
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maculatus has also been studied more recently via immunohistochemistry for the even-skipped
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protein [32]. This recent work confirmed previous reports that the embryos undergo the long
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germ mode of development, similar to dipterans like Drosophila melanogaster and
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hymenopterans like Apis mellifera [33]. It is commonly believed that the long germ mode of
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development evolved independently in dipterans and hymenopterans, and given the
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phylogenetic distribution of short and intermediate germ development within the Coleoptera [34,
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35], it seems likely that the long germ mode of development also evolved independently in the
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clade to which C. maculatus belongs. A comparison of the molecular basis of development in C.
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maculatus and other long germ insects, plus with more closely related species that feature short
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germ development, such as well studied flour beetle, Tribolium castaneum (super-family
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Tenebrionoidea, see Fig.1 A) and A. menetriesi, would yield crucial information on how
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developmental pathways have evolved to generate the long germ mode of development. C.
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maculatus has also been studied in other fields and is a useful system for undergraduate lab
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teaching [17].
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In order to facilitate research on A. menetriesi and C. maculatus, as well as wider
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investigations in the Coleoptera and beyond, we present here deep, multi-stage transcriptomic
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resources from a range of key time points in the development of these two beetle species.
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Using RSEM-based methods we have compared transcript abundance across these life stages,
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which will allow the investigation of genes that play key roles in developmental changes in these
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species, particularly at the maternal/zygotic transition. We have carried out extensive searches
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for key genes in developmental patterning and cell signalling pathways, and from our analyses
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we conclude that the transcriptomes for both species are of very high coverage, with almost all
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expected genes being present with full open reading frames. We have already made extensive
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use of these resources for our own studies on the embryonic development of these two beetles,
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and are confident that they will be of broad utility to a range of fields in genomics and
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developmental biology.
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Results and Discussion
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RNA Extraction and Sample Selection
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Ovaries and embryos were collected as described in Materials and Methods, and as seen in Fig
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2. The chosen time-windows cover a variety of important stages in the development of these
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species, and can be expected to contain the majority of embryonically active transcripts in their
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expressed complement. Briefly, RNA was collected from dissected ovaries from each species
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and from four embryonic time windows for A. menetriesi and two embryonic time windows for C.
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maculatus. Samples were sequenced on the Illumina HiSeq platform (one lane per species),
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adaptor trimming was performed, along with preliminary assessment of read quality, and data
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was made available for download from an external server.
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Read Quality and Assembly Metrics:
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Table 1 summarises initial read information, and these reads are available from the NCBI SRA,
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Bioproject Accession numbers: PRJNA293391 and PRJNA293393. To confirm quality of read
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data, FastQC [36] was run on all read data. This showed Phred quality scores were high, with
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median scores always exceeding 28 through to the 101st base, and generally in the mid-30s. A
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slight bias was found in initial nucleotide sequence. To ensure that Illumina adaptors were
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removed in their entirety, Trimmomatic v.0.32 [37] was used to check for residual adaptor
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sequence, but none were observed. We therefore posit that this bias is due to known biases in
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Illumina hexamer binding [38] rather than sequencing-based artifacts.
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Using Trinity [39] as an assembler, Trimmomatic-corrected read assemblies were then
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compared with assemblies based on uncorrected reads. Initial assays of Trimmomatic-treated
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read assemblies empirically found them to be less well assembled than those using un-trimmed
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reads, with a shorter N50 and less overall sequence recovery. Trimmed assemblies were
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therefore discarded in favour of untrimmed assemblies, which were used for all further analyses.
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Assays of initial assemblies noted a small amount of fungal contamination in the data for
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both species. DeconSeq [40] was therefore run on the Trinity output for both species, with high
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stringency as noted in the methods. A total of 336 and 206 contigs with some homology to
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fungal sequence was removed from the A. menetriesi and C. maculatus total assemblies as a
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result, before read mapping was performed. In the A. menetriesi assembly, we observed
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particularly high contamination with the protists, notably Dictyostelium discoideum and
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Naegleria gruberi, and some of this almost certainly remains in the transcriptome. Metrics for
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final assemblies after the removal of contamination can be seen in Table 2, alongside the
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results of assemblies for individual time points.
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The final, combined timepoint, contamination-removed assemblies, comprising 228,096
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and 128,837 contigs for A. menetriesi and C. maculatus respectively, contain a large number of
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well-assembled transcripts, with the number of contigs greater than 1 kb in length (52,217 in A.
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menetriesi, 33,250 in C. maculatus) and a high N50 (1,598 bp A. menetriesi, 2,263 bp C.
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maculatus) indicating a very well assembled dataset. This size is sufficient to span most protein
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coding domains, allowing easy inference of homology, and will be the full length of many
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transcripts. It is important to note that many of the contigs in our assembly will represent splice
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variants of single genes, and some genes will have multiple splice variants, which will affect
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these statistics. However, excellent recovery of splicing variation itself will be useful for a range
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of later analysis. GC content of the final assembled transcriptomes closely mirrors that of reads
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(32.82% in A. menetriesi, 38.7% in C. maculatus; reads 36-38%/40-43% respectively depending
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on library).
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To gain an understanding of whether full-length transcripts were present in our data, we
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ran TransDecoder v 2.01 [41] to identify open reading frames (ORFs) and filtered for the results
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that were at least 100 amino acids long. For the A. menetriesi assembly, this analysis yielded
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71,961 raw and 51,912 filtered contigs, while for the C. maculatus assembly, the analysis
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yielded 65,433 raw and 36,535 filtered contigs. The mean average length of the predicted
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polypeptides, after the filtering step, was 351 (A. menetriesi) and 448 (C. maculatus) amino
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acids. This is long enough for us to be confident that our assembly adequately spans coding
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regions, as the average eukaryotic protein length is 361 amino acids [42]. Together, these
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analyses suggest that we have recovered the vast majority of coding sequence in our combined
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assemblies, with sufficient length to adequately span ORFs, a conclusion further supported by
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gene annotation data as discussed further below. The numbers of ORFs presented here are
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considerably more than the 16,404 gene models observed in the T. castaneum genome
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(Tribolium Genome Sequencing Consortium, 2008), and this is likely due to both spurious ORFs
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in our dataset and to multiple splice variants. All assemblies are available from Figshare online
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(A. menetriesi DOI: 10.6084/m9.figshare.2056464, C. maculatus DOI:
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10.6084/m9.figshare.2056467).
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Timed RNA Expression - Differential Expression Analysis
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Key developmental stages for both A. menetriesi and C. maculatus can be easily observed
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following fixation and nuclei staining (data not shown). Briefly, egg lay to uniform blastoderm
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stage takes approximately 24 hours in A. menetriesi, and 7 hours in C. maculatus. Germband
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formation, gastrulation and elongation occur from 24-100 hours in A. menetriesi, and from 7-24
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hours in C. maculatus. The latter period was subdivided in A. menetriesi according to
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characteristic stages of short germ development pertinent to our research interests. These
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stages, along with assayed sample periods, are shown diagramatically in Fig 2. As well as being
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used for combined assemblies as described earlier, RNA extracted from mature ovaries and
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from embryos collected during the aforementioned time periods was also individually assembled
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using Trinity.
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The timed transcriptome assemblies for each species often possess better assembly
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quality when compared to the combined assemblies by metrics such as N50 and mean contig
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length (Fig 2, Table 2). As a result of being made up of a subset of the total reads, the individual
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assemblies do not possess the breadth of the combined assemblies, with fewer contigs,
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especially at long contig length (e.g. 1kb <). As such, the staged transcriptomes were used
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solely for comparison of expression levels across time, while the combined assemblies were
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used for gene family analyses. We emphasise that no technical replicate was performed for
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these comparisons, and any conclusions drawn from them should hold this consideration in
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regard. With this limitation in mind, we carried out differential expression analyses and observed
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broad trends in expression, as can be seen in Fig 3.
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First, we generated matrices from general comparison between time points in order to
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find the most similar samples (Fig 3 A,B). Generally, these results are congruent with steady
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changes in gene expression across the course of development, with most time points being
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most similar to those immediately preceding and following them. However, a split can be seen in
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A. menetriesi (Fig 3 A,B) between the ovary and 0-24 hour samples and all others, with the
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three later samples resembling each other more closely than the 0-24 hour dataset resembles
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the 24-48 hour sample. This could be due to the maternal:zygotic transition, which will occur at
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some point in this time frame. This cannot be seen for C. maculatus, and could be due to more
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admixture of RNA within the last 7-24 hour sample. Focused analysis on when the
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maternal:zygotic transition occurs in each species is required to resolve this question.
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Next, we clustered the results of our differential expression calculations (Fig 3 C,D). The
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results shown are those with RSEM considering each isoform separately (rather than taking into
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account clustering into genes performed by Trinity). Numerous up- and down-regulated contigs
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can be seen at each time point, with some time points more obviously possessing or lacking a
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subset of genes found in the combined transcriptomes.
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While replicates have not been performed and we have not analysed up and down
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regulated transcripts in detail, these data are available to download from Additional Files 6 and
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7 attached to this document online. These data will act as a good initial guide for those
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interested in tracking differential expression of specific genes across development and will be
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useful for hypothesis building.
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Basic Gene Annotation
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To gain an understanding of the depth of coverage of our datasets we used the BUSCO library
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of well-annotated genes [43], which are known to be highly conserved in single copy across the
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Metazoa, as a basis for comparison with our combined, assembled transcriptomes. Of the
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BUSCO set of 843 metazoan orthologs, the A. menetriesi assembly possesses 801 (95%)
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complete (of which 225, 26%, are duplicated), 16 fragmented (1.8%) and 26 missing genes
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(3.0%), for a total recovery of 97% of the BUSCO dataset. The C. maculatus assembly contains
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815 (96%) complete (with 202, 23%, duplicated), 13 (1.5%) fragmented and 15 (1.7%) missing
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genes (98.3% recovery). This extremely high level of recovery gives us confidence that at least
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all housekeeping genes expected to be present in these species are found in our datasets,
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which strongly suggests that these transcriptomes contain the vast majority of the gene cassette
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of these species.
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The number of duplicate genes in our BUSCO analyses likely reflects the construction of
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our transcriptomes from mixed RNA samples, with the allelic variation that this implies.
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Discerning true duplicates from allelic and splice variant data is largely contingent on the
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availability of well-assembled genomes. However, the recovery of these putative duplicates in
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our assemblies underlines that our RNA sequencing and assembly was of good depth and
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quality (respectively). With this information in-hand, a range of investigations will be made
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possible, particularly into the regulation and expression of developmentally important genes.
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A further understanding of the content of our assemblies was gained from Blast2GO
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analysis [44, 45]. Genes were annotated using b2gpipe, on the basis of BLASTx (E value cutoff,
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10-3) comparisons made against the nr database as downloaded on the 26 January 2015. Of
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228,096 (A. menetriesi) and 128,837 (C. maculatus) contigs in each assembly, 78,879 (34.6%)
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and 41,744 (32.4%) possessed a hit in the nr databases above the threshold. After further
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annotation with ANNEX and Interproscan, a total of 36,315 (15.9%) and 13,096 (10.2%) contigs
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were assigned to one or more GO categories. These numbers, while only a fraction of the total
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number of contigs within our transcriptomes, more closely reflect the expected eukaryotic
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protein complement in number. Blast2GO annotations are given in Additional Files 2 and 3
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Fig 4A shows the distribution of species best hit by BLASTx comparison of contigs from
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A. mentriesi and C. maculatus with the nr database. For both species, T. castaneum is the best
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represented species - a reflection of both the phylogenetic position of this species and its well
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annotated genome. The fact that contigs in our transcriptomes match T. castaneum and other
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coleopteran and insect species more closely than those of other species suggests that gene
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orthology will be easy to assign in many cases, and that a higher than normal rate of molecular
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evolution is not observed in our species.
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The distribution of GO terms within our datasets are shown in Fig 4B, alongside those of
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the well-annotated D. melanogaster and Mus musculus proteomes. In general, our datasets
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resemble one another more than they mirror that of the two sequenced genomes noted. Our
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transcriptomic resources empirically seem under-represented relative to D. melanogaster and
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M. musculus in developmentally interesting categories such as Protein Binding (Molecular
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Function), Multicellular Organism Development and Cell Differentiation (Biological Process).
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Given our results for more targeted investigations, found below, we feel this is likely a result of
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poor annotation of these by Blast2GO, rather than true absence from the transcriptome.
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At a gross level, in the intracellular Cellular Component GO categories, our data
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appears more similar to that seen in the two model species than in Molecular Function or
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Biological Process categories, although this has not been tested statistically. This would
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suggest these well-conserved structural components were more readily assigned GO categories
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than the developmentally interesting categories listed above. While not all GO categories are as
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well-annotated by this process as may be desired, the broad classification of our data into a
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wide range of GO categories of all levels of GO distribution demonstrates that Blast2GO
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annotations of our data are a useful starting point for more focussed investigations and
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identification of specific genes and pathways of interest.
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Gene Family Recovery

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To extend the semi-automated analyses presented above, we performed more targeted
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analysis of individual, developmentally important gene families. Both the Hox family of
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transcription factors and the TGF- cassette were exceptionally well recovered in our dataset.
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The Hox genes, and in particular the ANTP HOXL class, which pattern the anterior-posterior
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body axis, are recovered almost in their entirety in both species when compared to well-
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catalogued databases (e.g. [46]). This can be seen in Fig 5, which shows the phylogenetic
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distribution of recovered ANTP HOXL class sequences from our transcriptomes alongside
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previously annotated members of this class.
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In A. menetriesi, sequences for all the ANTP HOXL class genes are recovered in our
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transcriptome, as can be seen in Fig 5, with sequences and alignments in Additional File 1. We
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note, however, that the A. menetriesi Hox 2 / maxillopedia (mxp)/ proboscipedia (pb) sequence
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bears some BLAST similarity with Hox 4 / Dfd sequences, while the putative Hox 4 / Deformed
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(Dfd) recovered does not include the homeodomain sequence - whether it has lost this crucial
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domain, or if this portion of the sequence is simply not recovered in our assembly is at present
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unknown. The putative A. menetriesi Hox 4 / Dfd sequence is given in Additional File 1,
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although it is not shown in the phylogeny in Fig 5 due to its truncated length.
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While 2 (A. menetriesi) and 4 (C. maculatus) Hox 3 / zerknllt (zen) variants are seen in
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our species, these are identical at the coding level, and therefore seem to be splice or allelic
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variants, rather than the two paralogous genes seen in T. castaneum [47]. Similarly, no
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evidence of the caudal (cad) duplication seen in T. castaneum [48] can be found in our
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transcriptomic assemblies, suggesting this is perhaps specific to the flour beetle lineage. Both
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zen and caudal are important embryonic patterning genes, and comparison of these genes in
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our two species and Tribolium would be an excellent situation in which to study how sub- and
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neo-functionalisation occurs. Likely allelic or splice variants are also observed for other HOXL
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genes in both A. menetriesi and C. maculatus. It should be noted that these could represent
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very recent duplications, or the effect of gene conversion, although this can only be tested fully
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with the advent of a complete genomic resource. No Pdx/Xlox gene was seen, adding further
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circumstantial evidence for the broad scale loss of this gene across the Arthropoda.
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Our C. maculatus transcriptome also contains the full complement of ANTP HOXL
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genes, although, similar to the case of Hox 4 / Dfd in A. menetriesi, the 4 recovered abdominal-
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A (abd-A) homologs lack the whole homeodomain sequence, with several residues missing.
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These truncations excluded them from the phylogeny shown in Fig 5, but the sequences for
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these putative homologs are given in Additional File 1.
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Even more so than in A. menetriesi, a remarkable diversity of potential splice/allelic
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variants are noted in C. maculatus, particularly for the Hox 6/8 superfamily and Hox 9-13 /
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Abdominal-B (Abd-B) gene family. Of the Hox 6/8 gene superfamily, normally represented by
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four genes in the beetle (prothoraxless (ptl), fushi tarazu (ftz), Ultrathorax (Utx) and abd-A), 12
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ptl, 1 ftz, 4 Utx and 4 abd-A representatives were found in our analysis. Furthermore, up to 26
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different potential allelic or splice variants of abdB are recorded. As our transcriptome is made
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of mixed embryonic samples, it is perhaps not surprising that a diversity of splice/allelic variants
347
are observed, but the excellent recovery of this data confirms the deep coverage provided by
348
our sequencing and assembly.
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The TGF- cassettes of the insects have been very well described previously (e.g. [49,
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50]). Our datasets recover almost the full expected complement of the Coleoptera. A slight
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exception to this is Activin (Act), a partial sequence of which is recovered for both species: a
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portion of the propeptide which does not span the mature signal peptide sequence. Whether this
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is a consequence of loss of the mature domain in these species or low levels of expression at
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the sampled timepoints remains to be established. A BMPx ortholog of clear homology to genes
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of that family can be found in C. maculatus, but has been excluded from the tree seen in Fig 6
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as it is incomplete in length. Its sequence can be found in Additional File 1, and we have no
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doubt as to its identity due to high levels of sequence conservation between it and the T.
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castaneum and A. menetriesi orthologs of this gene.

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The glass bottom boat (gbb) duplication observed in T. castaneum cannot be found in
360
our data, but we can recover a range of splice or allelic variants for other genes, especially in A.
361
menetriesi. These do not differ in the protein coding regions, which leads us to suspect that
362
these are not from gene duplications (unless the duplication(s) occurred very recently). The
363
phylogeny shown in Fig 6 confirms the homology of all genes, and splice/allelic variant numbers
364
observed are given there in brackets, with all sequences available in Additional File 1.
365
We also note the discovery of an additional TGF- ligand in C. maculatus. This gene has
366
been previously automatically annotated as derriere in T. castaneum, (XP_008191586.1) and if
367
it is truly of this family, which is also known as GDF1/3/Univin/Vg1, this would be a surprise, as
368
its presence outside the Deuterostomia is controversial [51]. If proof could be found for this
369
being a bona fide GDF1/3/Univin/Vg1, the presence of this gene in more than one coleopteran
370
could suggest that this might in fact be ancestrally present in all bilaterian species, but further
371
investigation is warranted before strong conclusions can be drawn in this regard. We could gain
372
no phylogenetic support for placing either of these beetle sequences in the GDF1/3 clade, and it
373
may well be that these sequences instead represent a coleopteran novelty.
374
Our recovery of not only the full expected complements of these vital developmental
375
genes, but also a remarkable diversity of alternative variants, demonstrates the depth of our
376
assemblies as a resource. Whether used as the basis for simple cloning or more sophisticated
377
analysis of patterns of gene variation and diversification, these transcriptomes will be of wide
378
utility to the field of coleopteran and insect developmental biology.
379
380
Pathway Recovery
381
As well as examining specific gene families, we investigated a number of broader pathways
382
commonly studied in insects [52]. This allows us to both note how well-recovered such
383
pathways are in our species as a measure of transcriptome utility, as well as note interesting
384
differences between these pathways in our species when compared to others. We did this using
15
385
both automated (KEGG KAAS mapping) and manual (BLAST based) methods. Some
386
representative results of KEGG KAAS mapping can be seen in Fig 7, and all KEGG annotations
387
can be downloaded from Additional Files 4 and 5.
388
KEGG KAAS mapping uses BLAST results to annotate known pathways, and gives a
389
rapid overview of the recovery of these. Here we have shown the well-known Wnt, Notch and
390
Hedgehog pathways to indicate the depth of our transcriptomes, and show how they may be
391
useful for future research. However, these maps often use terminology based on vertebrate
392
nomenclature, and contain genes known to be absent from particular clades. We have therefore
393
indicated in Fig 7 (using unshaded boxes as shown in the Key) genes that may be absent
394
ancestrally in the Coleoptera, based on their absence from the T. castaneum pathway. Of genes
395
expected to be present in the Coleoptera we find almost total recovery in our transcriptomes. In
396
the three pathways examined, only three genes noted to be present in T. castaneum were noted
397
as absent from both of our transcriptome datasets, all in the Wnt cascades (Fig 7A). The
398
expected Hedgehog cassette was recovered in toto (Fig 7B) and in the Notch signalling
399
cascade (Fig 7C), only APH-1 was noted to be absent, and only from the C. maculatus
400
transcriptome. We must note that these may not be true absences - KEGG KAAS mapping is
401
based on automatic BLAST assignation, and if these sequences are divergent in our
402
transcriptomes they may have been missed by this analysis.
403
We also examined pathways manually, using reciprocal BLAST hits and closer manual
404
investigation to confirm identity of individual genes, the results of which can be seen in Table 3.
405
The anterior-posterior patterning genes cad (mentioned earlier) and hunchback (hb) are present
406
in both species. Of the germline establishment and localization genes examined, nanos was
407
surprisingly absent in both species, while bruno (bru; also known as arrest), exuperantia (exu),
408
tudor (tud; 2 copies in A. menetriesi), oskar (osk), vasa (vas) and valois (vls) were present.
409
Interestingly, pumilio (pum) is present in C. maculatus in single copy (although it is divided
410
across two contigs), while A. menetriesi possesses a total of seven copies. The different A.
16
411
menetriesi pum copies varied both at the nucleotide level and in their amino acid sequences,
412
strongly suggesting that they are in fact paralogs. In depth analysis of these genes is required to
413
uncover why they have undergone several rounds of duplication. Orthologs of the Drosophila
414
gene swallow (swa) could not be found in either of our transcriptome resources, nor is it present
415
in several other insects (data not shown) and we suggest it may therefore be a schizophoran
416
novelty.
417
Canonical gap genes Krppel (Kr), knirps (kni), giant (gt), huckebein (hkb), tailless (tll; 2
418
paralogs in C. maculatus), buttonhead (btd), empty spiracles (ems) and both orthodenticle
419
orthologs (Otd and Otd2) were recovered in both species examined here. The C. maculatus
420
specific duplication of tll would be another excellent opportunity to study gene duplication and
421
evolution. The pair rule genes even skipped (eve), hairy (h), fushi tarazu (ftz), odd paired (opa),
422
odd skipped (odd), paired (prd), runt (run) and sloppy paired 1 (slp1) were present in single
423
copy. The segment polarity gene cassette was also present in both species, with the notable
424
absence of gooseberry-neuro (gsb-n) from our datasets. The genes armadillo (arm), cubitis
425
interruptus (ci), Engrailed (En), fused (fu), gooseberry (gsb), hedgehog (hh), pangolin (pan),
426
patched (ptc) and wingless (wg) were all present, and their sequences can be found in
427
Additional File 1.
428
All of these pathways are commonly studied in insects, and the annotations provided
429
here, along with preliminary timed expression data, will provide a basis for a wide range of
430
targeted investigations into the embryonic development of these two species, and how these
431
pathways have changed over the course of evolution. Furthermore, the excellent recovery of
432
these pathways by both automated (KEGG-KAAS) and manual annotation gives us high
433
confidence in the completeness of our transcriptomic resources. This confirms the results of our
434
BUSCO analysis, and our datasets are therefore likely to contain the vast majority of transcribed
435
genes in these two species, with only lowly expressed and temporally restricted genes absent
436
from these transcriptome resources.

17
437
438
Conclusions:
439
Our production of deep transcriptomic sequence data for A. menetriesi and C. maculatus will
440
assist in the inference of character gain and loss across the Coleoptera, aid in future
441
phylogenetic efforts, and allow a range of investigations into the embryonic development of
442
these species at the molecular level. The status of these organisms as common agricultural
443
pests also suggests that such resources may allow targeted control mechanisms to be
444
developed for these species. This data will be another key building block in our understanding of
445
the transcriptomic basis to embryological development, and provide a window into the basic
446
biology of the most successful clade of animals.
447
448
Methods:
449
Animal Husbandry
450
A. menetriesi eggs were kindly provided by Dr Yoshikazu Ando, and were reared at 25C on wet
451
sand or soil and fed fresh lettuce. C. maculatus beetles were kindly provided by Dr Joel Savard,
452
and were reared at 30C on dry black-eyed peas.
453
454
RNA Extraction and Sequencing
455
RNA was extracted from ovary and timed embryonic samples using a TRIzol RNA extraction kit
456
according to the manufacturers protocols. RNA quantity and quality was checked using a
457
Thermo Scientific Nanodrop 2000C Spectrophotometer and 1 g was sent for sequencing by
458
the Cologne Centre for Genomics. Adaptor trimming and initial quality control was performed by
459
the commercial provider according to their internal standards. This cleaned data was then made
460
available to us for download from an external server. Paired end read quality after sequencing
461
was assessed using the FastQC program [36].
18
462
463
Transcriptome Assembly and Comparative Expression Analyses
464
Assemblies used in our final analysis were made using Trinity version 2013_08_14 [39], with the
465
default settings. Full assemblies were made using reads from all time points, and individual
466
assemblies were then constructed using each sampled time point individually. All assemblies
467
are available from Figshare online (A. menetriesi DOI: 10.6084/m9.figshare.2056464, C.
468
maculatus DOI: 10.6084/m9.figshare.2056467). DeconSeq standalone version 0.4.3 [40] was
469
run on full assemblies with settings -i 95 -c 95, using the bact, fungi, hsref, and prot databases.
470
Comparative expression analyses were performed using RSEM [53] as packaged in the Trinity
471
module, and results shown here are the as-isoform data, although as-gene data is provided in
472
Additional Files. BUSCO v1.1b1 [43] was used to assess gene complement completeness.
473
474
Functional Annotation
475
Our combined assemblies were automatically assigned homologs and annotated according to
476
gene ontology (GO) terms using Blast2GO [44, 45]. Initially, BLASTx was run using BLAST
477
2.2.29+ against the NCBI nr database as downloaded to a local server on the 17/01/2015, with
478
settings -evalue 0.001 -max_target_seqs 5 -outfmt 5. GO term distribution within the D.
479
melanogaster and H. sapiens genomes were downloaded from B2GO-FAR [54] and calculated
480
using the Combined Graph function of Blast2GO. KEGG KAAS mapping was automatically
481
performed using the KEGG KAAS tool (http:// www.genome.jp/tools/kaas/), single-directional
482
best hit with default BLAST settings, and with the eukaryote dataset as a basis for annotation.
483
484
Gene Identification
485
Gene sequences were manually identified and their homology confirmed by independently using
486
tBLASTn [55] searches using gene sequences of known homology downloaded from the NCBI
487
nr database as queries against standalone databases created on a local server using BLAST
19
488
2.2.29+ or the CLC Main workbench. Genes putatively identified using this method were
489
reciprocally BLASTed against the online NCBI nr database using BLASTx to confirm their
490
identity. Where identity was uncertain, phylogenetic analysis was used to confirm identity.
491
492
Phylogenetic Tree Construction
493
Sequences were aligned using MAFFT 7 [56] unless otherwise stated under the L-INS-i
494
strategy. Alignments were then saved and exported to MEGA 6, where regions of poor
495
alignment were manually excluded and maximum likelihood phylogenetic trees were
496
constructed using the LG model, 1000 bootstrap replicates as indicated, 4 gamma categories
497
and invariant sites, and all other default prior settings [57].
498
499
500
List of Abbreviations Used:
501
BLAST: Basic Local Alignment Search Tool, BUSCO: Benchmarking Universal Single-Copy
502
Orthologs, GDF: Growth Differentiation Factor, KEGG-KAAS: Kyoto Encyclopaedia of Genes
503
and Genomes - Automatic Annotation Server, MAFFT: Multiple Alignment using Fast Fourier
504
Transform, NCBI: National Centre for Biotechnology Information,ORF: Open Reading Frame,
505
RNA: Ribonucleic Acid, RSEM: RNA-Seq by Expectation-Maximization, SRA: Short Read
506
Archive, TGF: Transforming Growth Factor
507
508
Competing Interests:
509
The authors declare that they have no competing interests.
510
511
Authors' contributions:
20
512
MAB, KC and JAL maintained animals, extracted RNA and arranged sequencing. MAB and NJK
513
assembled transcriptomes, analysed raw data and wrote the manuscript. MAB, NJK, KC, JAL
514
and SR designed experiments and contributed to gene family analyses presented in this
515
manuscript. All authors read and approved the final manuscript.
516
517
Acknowledgements:
518
The authors would like to thank the members of their laboratories for their support and
519
discussions. In addition, we thank Dr Y Ando for providing A. menetriesi eggs and advice on
520
establishing cultures, Dr J Savard for providing C. maculatus, and Dr F Marletaz for help in
521
running BUSCO analyses. The efforts of editors and reviewers in considering this manuscript
522
are gratefully acknowledged. MAB was supported by a Humboldt Fellowship for Postdoctoral
523
Researchers. KHC, SR and JAL were supported by the DFG Collaborative Research Center
524
grant 680.
525
526
Availability of data and materials
527
The datasets supporting the conclusions of this article are available in the NCBI SRA repository
528
[Bioproject Accession numbers: PRJNA293391 and PRJNA293393] and in the Figshare
529
repository [DOIs: 10.6084/m9.figshare.2056464 and 10.6084/m9.figshare.2056467]. NOTE:
530
DATA EMBARGOED UNTIL PUBLICATION
531
532
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Figure legends
684
Figure 1: A) Cladogram of Coleoptera simplified from that determined by Hunt et al. (2007) [13].
685
Red asterisk indicates the superfamily in which the Chrysomelidae are located. B) Cladogram of
686
Chrysomelidae simplified from that determined by Gomez-Zurita et al. (2007) [28]. Black and
687
grey asterisks indicate the sub-families in which A. menetriesi and C. maculatus are located
688
(respectively). C) A. menetriesi and D) C. maculatus adults.
689
690
Figure 2: Summary of RNA sources, life stages sampled and sequencing results, with
691
A.menetriesi data presented at left and C. maculatus data at right.
27
692
693
Figure 3: Overview of results of differential expression analysis performed by RSEM within the
694
Trinity framework. A) and B) show the sample correlation matrix for A. menetriesi and C.
695
maculatus respectively. C) and D) show relative expression of each differentially expressed
696
contig, considered as isoforms, across time.
697
698
Figure 4: Blast2GO results A) Distribution of BLASTx best hits by species, showing metazoans
699
only, for A. menetriesi in orange, C. maculatus in blue. B) Distribution of GO terms expressed as
700
a percentage of annotated contigs which were assigned a term within each of the three
701
(Molecular Function, Cellular Component, Biological Process) categories of GO ID.
702
703
Figure 5: Phylogenetic inter-relationships of ANTP HOXL class genes, as reconstructed by
704
MEGA 6 using the LG+Freqs model with 4 gamma categories and invariant sites, based on a 59
705
amino acid alignment spanning the homeodomain. Numbers at base of nodes represent
706
bootstrap percentages of 1000 replicates. Scale bar at base of phylogeny gives substitutions
707
per site at given unit distance. Red underline indicates A. menetriesi and C. maculatus
708
sequences, coloured boxes are used to delineate known gene families (and in the case of Hox
709
6/7/8, a superfamily).
710
711
Figure 6: Maximum Likelihood Phylogeny of TGF- ligands, as determined using MEGA under
712
the LG+Freqs model with 4 gamma categories and invariant sites, on the basis of a 72 amino
713
acid alignment of mature peptide sequences. The given scale depicts the number of
714
substitutions per site per unit length. Bootstrap percentages (of 1000 replicates) are given at
715
base of nodes. A. menetriesi and C. maculatus sequences are underlined in red. Coloured
716
boxes represent known gene families with representatives in our transcriptomic resources, while
717
all gene families, including those not found in our datasets, are indicated at right.
28
718
719
Figure 7: KEGG style pathway maps showing recovery in our transcriptome resources of A) the
720
Wnt signalling pathway in canonical and non-canonical contexts, B) the Hedgehog signalling
721
pathway and C) the Notch signalling pathway. Coloration of genes indicates presence, absence
722
or ancestral absence from the Coleoptera as detailed in the key, which also gives other
723
information as noted.
724
725
Tables
726
Table 1: Basic read data metrics

A. menetriesi
Library: (hpf=
C. maculatus
Ovary
0-24 hpf
24-48 hpf
48-72 hpf
72-100 hpf
Ovary
0-7 hpf
7-24 hpf
44,695,199
56,139,626
39,911,618
46,217,614
45,213,862
67,026,913
61,060,357
55,273,927
101
101
101
101
101
101
101
101
7.3
9.5
6.8
7.8
7.7
10.8
9.8
8.8
38
36
37
37
37
43
41
40
hours post
fertilization)
Number of
Paired-end
Reads
Read Length
(bp)
Amount of
Data
(Gigabytes)
GC content
(%)
727
728
729
730
731
29
732
Table 2: Assembly metrics for individual time point and combined transcriptomic resources
A. menetriesi
Ovary
C. maculatus
0-24 hpf
24-48 hpf
48-72 hpf
72-100
Combined
Ovary
0-7 hpf
7-24 hpf
Combined
hpf
Number
100,084
106,548
120,497
140,868
126,400
228,096
98,082
57,879
80,665
128,837
20,759
17,765
18,398
17,591
17,180
20,757
30,742
22,941
22,956
29,933
983.95
1027.36
924.27
879.03
914.2
844.66
1074.86
1216.55
1106.56
991.04
471
490
453
426
448
401
416
526
474
391
1,894
1,993
1,721
1,643
1,686
1,598
2,450
2,570
2,308
2,263
14,196
15,200
17,511
19,814
18,105
31,196
11,695
7,874
10,978
15,155
28,255
31,944
32,307
34,520
33,002
52,217
27,763
19,919
25,529
33,250
# bases,
98,477,17
109,463,3
111,371,6
123,827,2
115,555,1
192,664,2
105,424,2
70,412,72
89,260,49
127,682,5
total
62
55
26
46
13
20
07
# bases in
68,737,07
78,526,34
75,208,10
80,796,00
77,199,41
122,986,5
78,519,05
55,032,54
67,296,56
91,466,09
contigs >
47
of contigs
Max
contig
length
(bp)
Mean
contig
length
(bp)
Median
contig
length
(bp)
N50
contig
length
(bp)
# contigs
in N50
# contigs
> 1kb
1kb
30
GC
33.54
33.14
33.14
33
33.17
32.82
39.26
39.21
38.59
38.7
Content
% (2dp)
733
734
735
736
737
738
Table 3: Genes identified by manual annotation

Pathway/Gene
A. menetriesi
C.maculatus
Maternal Effect:
Pathway/Gene
A. menetriesi
C.maculatus
Pair rule:
caudal
present
present
even skipped
present
present
hunchback
present
present
fushi tarazu
present (see Hox)
present (see Hox)
hairy
present
present
odd paired
present
present
Germline:
bruno/arrest
present
present
odd skipped
present
present
exuperantia
present
present
paired
present
present
nanos
absent
absent
runt
present
present
oskar
present
present
sloppy paired 1
present
present
pumilio
present - 7 copies
present
swallow
absent
absent
Segment Polarity:
tudor
present - 2 copies
present
armadillo
present
present
valois
present
present
cubitis interruptus
present
present
vasa
present
present
Engrailed
present
present
31
Gap genes:
fused
present
present
gooseberry
present
present
buttonhead
present
present
gooseberry-neuro
absent
absent
empty spiracles
present
present
hedgehog
present
present (but on 3
contigs)
giant
present
present
pangolin
present
present
huckebein
present
present
patched
present
present
knirps
present
present
wingless
present
present
Krppel
present
present
orthodenticle 1
present
present
orthodenticle 2
present
present
tailless
present
present - 2 copies
739
740
741
Additional Files:
742
Additional File 1: Sequences of all genes referred to in text, and alignments used in
743
phylogenetic analyses (.xls)
744
Additional File 2: Annotations of transcriptome (Blast2GO .annot file) A. menetriesi
745
Additional File 3: Annotations of transcriptome (Blast2GO .annot file) C. maculatus
746
Additional File 4: KEGG data, A. menetriesi
747
Additional File 5: KEGG data, C. maculatus
748
Additional File 6: Comparative expression data, A. menetriesi
749
Additional File 7: Comparative expression data, C. maculatus
32
A)
Coleoptera
B)
Chrysomelidae
C)
Atrachya
menetriesi
2 cm
D)
Callosobruchus
maculatus
1 cm
Atrachya menetriesi
Sample 1: Mixed Ovarian
Developing Gamete
Ovarian
Development
44,695,199 paired end reads

100,084 contigs
Sample 2: 0-24 hpf
Callosobruchus maculatus
Sample 1: Mixed Ovarian

98,082 contigs
Early Egg to
Blastoderm Stage

106,548 contigs
Sample 2: 0-7 hpf

57,879 contigs
Sample 3: 24-48 hpf

120,497 contigs
Sample 4: 48-72 hpf

140,868 contigs
Embryo Formation,
Gastrulation,
Germband Elongation
Sample 3: 7-24 hpf

80,665 contigs
Sample 5: 72-100 hpf

126,400 contigs
Samples 1-5 Combined:

228,096 contigs
Final, Combined
Assemblies
Samples 1-3 Combined:

128,837 contigs
02
06
CoVa
orue
Key
go
Co or Key
yeK royeoK
C ro oC
mp
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mp
mono pm o
pm
m no
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Samp e Co e a on Ma x
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001_27
27_84
6 02 0
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0 66 0 8 1
1
20
Vaa ue
Re
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S m ar y
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24_48
ova
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72_100
72_100
24_48
72_100
24_48
0_24
0-24 hour
D)
-5 0
0 55 10
10
Vaatue
Re
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express on
7_24
7-24 hour
Co or Key
samp es vs ea u es
Ca
osobruchus
macu atus
so o msc us e ed ma x og2 cen e ed
Cont g Re at ve Express on
0_24
-10
10
0_7
0-7 hour
ova y
ovary
72_100
24_48
ova y
24_48
0_24
ovary
48_72
24_48
ova y
0_24
48_72
ova y
24_48
0_24
24_48
samp es vs ea u es
Atrachya
menetr es
so o msc us e ed ma x og2 cen e ed
Cont g Re at ve Express on
0-7 hour
0_7
ova y
ova y
72_100
0_24hour
72-100
0_24
48_72hour
48_72
0-24
24_48
ovary
84_42
og
84_42
dm
on m
27_84
24_48
72_100
ova
y
48_72
ovary
27_84
u
o
ova y
72_100
0_24
24-48 hour
48_72
0_24
24_48
001_27
001_27
y avo
y avo
Co or
Co
orKey
Key
-5
5
0 55
0
Re
Vaatueve
express on
7-24 hour
7_24
0-7 hour
0_7
0_24
72-100 hour
ovary
48-72 hour
48_72
0-24 hour
24_48
ovary
comp86476_c0_s
comp111618_c1_seq10
comp111618_c1_seq12
comp111618_c1_seq13
comp111618_c1_seq9
comp111618_c1_seq1
comp111618_c1_seq3
comp111618_c1_seq14
comp111618_c1_seq6
comp86579_c2_seq1
comp92303_c0_seq1
comp109712_c0_seq1
comp73271_c0_seq1
comp111925_c0_seq22
comp111618_c1_seq2
comp63201_c0_seq1
comp100088_c0_seq1
comp99616_c0_seq3
comp54614_c0_seq1
comp97104_c0_seq1
comp103563_c2_seq2
comp109922_c1_seq6
comp36407_c0_seq1
comp101297_c0_seq2
comp97615_c0_seq6
comp19619_c1_seq1
comp91877_c0_seq2
comp91877_c0_seq1
comp94921_c0_seq1
comp78259_c0_seq1
comp94921_c0_seq2
comp111698_c2_seq2
comp97214_c0_seq3
comp80407_c0_seq1
comp108382_c0_seq3
comp109142_c1_seq1
comp109142_c0_seq1
comp97293_c0_seq4
comp94097_c0_seq1
comp92193_c0_seq1
comp103118_c0_seq12
comp90155_c0_seq1
comp79141_c0_seq1
comp108292_c3_seq1
comp83140_c1_seq1
comp109210_c0_seq1
comp99761_c0_seq2
comp104281_c0_seq2
comp110523_c0_seq5
comp112032_c1_seq2
comp112318_c0_seq1
comp112318_c0_seq2
comp112032_c1_seq3
comp105249_c2_seq1
comp102392_c0_seq1
comp111014_c0_seq1
comp95123_c0_seq2
comp107616_c3_seq1
comp112669_c0_seq1
comp112901_c0_seq1
comp112901_c0_seq2
comp110388_c0_seq5
comp112535_c0_seq5
comp89538_c0_seq1
comp105267_c0_seq4
comp108665_c0_seq3
comp106426_c0_seq1
comp104777_c0_seq1
comp99083_c1_seq1
comp110641_c0_seq2
comp90591_c0_seq3
comp103124_c0_seq1
comp96887_c1_seq8
comp98158_c1_seq4
comp112516_c0_seq4
comp90591_c0_seq1
comp101683_c0_seq1
comp105106_c1_seq3
comp96125_c0_seq6
comp112483_c0_seq6
comp101909_c0_seq3
comp106285_c0_seq4
comp105040_c2_seq2
comp96838_c0_seq2
comp92433_c0_seq1
comp101922_c0_seq4
comp101922_c0_seq2
comp111965_c0_seq1
comp105781_c0_seq4
comp112483_c0_seq11
comp112483_c0_seq7
comp97193_c0_seq1
comp112483_c0_seq4
comp109204_c1_seq1
comp108304_c0_seq2
comp79350_c0_seq1
comp107274_c0_seq2
comp105627_c2_seq4
comp105565_c0_seq2
comp101714_c1_seq1
comp89770_c0_seq1
comp112375_c0_seq6
comp97909_c0_seq1
comp104183_c0_seq1
comp110155_c0_seq5
comp93561_c0_seq1
comp103737_c0_seq1
comp75591_c0_seq1
comp106635_c0_seq1
comp112375_c0_seq4
comp105781_c0_seq5
comp112483_c0_seq9
comp112516_c0_seq2
comp112483_c0_seq2
comp96749_c0_seq1
comp88969_c0_seq1
comp103439_c0_seq3
comp106297_c0_seq1
comp97734_c1_seq1
comp96749_c0_seq2
comp108913_c0_seq3
comp108913_c0_seq1
comp96033_c0_seq1
comp102823_c5_seq2
comp55179_c0_seq1
comp98640_c0_seq1
comp84657_c0_seq4
comp84657_c0_seq5
comp90725_c0_seq1
comp90741_c0_seq1
comp112655_c0_seq1
comp107793_c1_seq1
comp100296_c0_seq3
comp94579_c0_seq6
comp95553_c0_seq1
comp91580_c1_seq2
comp111960_c0_seq4
comp112483_c0_seq3
comp101103_c0_seq1
comp102740_c0_seq1
comp99435_c1_seq1
comp107972_c0_seq2
comp97867_c0_seq1
comp97902_c0_seq3
comp105794_c0_seq8
comp106310_c0_seq7
comp106310_c0_seq10
comp79975_c0_seq2
comp105023_c0_seq4
comp106310_c0_seq8
comp86745_c0_seq1
comp107891_c0_seq2
comp111473_c0_seq1
comp104370_c0_seq5
comp83099_c0_seq1
comp111151_c0_seq1
comp98829_c2_seq4
comp89402_c0_seq1
comp96678_c0_seq1
comp106310_c0_seq3
comp102598_c0_seq2
comp97423_c0_seq1
comp98515_c0_seq1
comp108868_c2_seq3
comp97077_c0_seq1
comp97077_c0_seq4
comp101067_c0_seq1
comp85025_c0_seq1
comp97077_c0_seq3
comp107267_c1_seq2
comp108782_c0_seq3
comp106227_c1_seq1
comp83044_c0_seq1
comp92257_c0_seq2
comp105310_c2_seq5
comp97055_c0_seq1
comp97055_c0_seq2
comp100405_c2_seq2
comp86527_c0_seq1
comp109035_c0_seq1
comp102403_c0_seq10
comp92372_c0_seq1
comp88651_c0_seq1
comp111543_c0_seq7
comp111422_c3_seq1
comp109237_c1_seq1
comp75880_c0_seq1
comp103824_c0_seq11
comp99703_c1_seq1
comp73244_c0_seq1
comp105794_c0_seq6
comp106192_c1_seq1
comp104264_c0_seq2
comp104894_c3_seq1
comp106245_c2_seq3
comp104825_c0_seq4
comp104894_c3_seq2
comp100032_c0_seq1
comp96783_c1_seq1
comp98693_c0_seq2
comp96783_c1_seq3
comp111174_c1_seq5
comp109161_c1_seq15
comp84657_c0_seq6
comp103743_c1_seq2
comp98729_c0_seq1
comp86914_c0_seq1
comp103743_c1_seq1
comp94958_c0_seq1
comp92377_c0_seq1
comp109784_c1_seq5
comp109784_c1_seq1
comp93383_c0_seq2
comp97895_c0_seq8
comp95597_c0_seq1
comp106510_c0_seq1
comp95597_c0_seq2
comp101554_c0_seq1
comp78259_c0_seq2
comp104284_c0_seq4
comp102354_c0_seq1
comp106999_c0_seq1
comp108106_c1_seq5
comp104233_c4_seq4
comp104233_c4_seq9
comp92765_c0_seq1
comp71953_c0_seq1
comp92961_c0_seq3
comp104701_c0_seq1
comp97599_c0_seq2
comp98637_c0_seq1
comp111111_c2_seq1
comp103332_c0_seq3
comp104737_c0_seq1
comp110155_c0_seq1
comp104284_c0_seq1
comp111543_c0_seq3
comp105501_c0_seq13
comp111111_c1_seq2
comp91664_c0_seq1
comp104284_c0_seq2
comp101985_c0_seq4
comp104425_c0_seq1
comp85712_c0_seq1
comp97803_c0_seq1
comp64793_c0_seq1
comp71566_c0_seq1
comp93814_c0_seq3
comp99517_c0_seq2
comp93814_c0_seq1
comp105247_c0_seq1
comp111230_c0_seq6
comp101985_c0_seq2
comp98829_c2_seq3
comp103167_c0_seq1
comp97520_c0_seq1
comp94248_c0_seq1
comp97599_c0_seq1
comp104284_c0_seq3
comp104602_c0_seq1
comp104602_c0_seq4
comp104602_c0_seq2
comp98048_c1_seq2
comp103489_c0_seq1
comp102278_c0_seq2
comp110439_c0_seq1
comp105676_c0_seq1
comp107770_c0_seq2
comp103350_c0_seq1
comp104602_c0_seq3
comp103588_c1_seq1
comp103588_c0_seq1
comp111646_c0_seq2
comp112516_c0_seq1
comp95190_c0_seq1
comp106424_c0_seq3
comp111646_c0_seq5
comp104231_c0_seq1
comp111646_c0_seq3
comp101696_c0_seq5
comp104231_c0_seq7
comp86017_c0_seq1
comp97909_c0_seq2
comp105886_c1_seq1
comp108888_c0_seq5
comp84704_c0_seq1
comp99952_c3_seq1
comp97520_c0_seq2
comp106424_c0_seq2
comp105683_c0_seq3
comp104231_c0_seq3
comp106424_c0_seq1
comp105274_c1_seq4
comp106099_c0_seq1
comp97741_c0_seq1
comp108843_c0_seq1
comp104386_c0_seq7
comp110213_c0_seq3
comp97077_c0_seq2
comp106691_c0_seq4
comp96615_c0_seq1
comp105310_c2_seq1
comp107334_c2_seq6
comp111664_c0_seq8
comp94483_c0_seq1
comp108998_c0_seq3
comp108843_c0_seq2
comp108782_c0_seq2
comp105886_c1_seq3
comp104370_c0_seq1
comp97215_c0_seq1
comp105310_c2_seq13
comp92598_c0_seq1
comp97754_c0_seq2
comp99413_c0_seq1
comp105794_c0_seq5
comp94483_c1_seq1
comp108846_c0_seq1
comp112880_c0_seq1
comp111664_c0_seq3
comp109398_c1_seq1
comp104059_c0_seq2
comp104140_c0_seq1
comp109970_c1_seq2
comp109970_c1_seq1
comp101369_c0_seq1
comp111664_c0_seq2
comp111664_c0_seq5
comp111664_c0_seq4
comp111432_c2_seq3
comp111664_c0_seq7
comp111664_c0_seq1
comp111664_c0_seq6
comp111664_c0_seq9
comp108626_c0_seq5
comp103758_c0_seq2
comp103758_c0_seq1
comp109564_c0_seq3
comp93478_c0_seq1
comp101442_c0_seq1
comp107955_c0_seq15
comp103830_c0_seq1
comp111807_c0_seq7
comp102452_c0_seq3
comp111865_c1_seq1
comp111865_c1_seq2
comp112752_c0_seq14
comp82755_c0_seq1
comp92232_c3_seq1
comp100021_c0_seq1
comp89440_c1_seq1
comp108292_c0_seq3
comp112982_c0_seq2
comp110739_c0_seq1
comp101804_c0_seq8
comp107439_c0_seq1
comp105310_c2_seq6
comp97767_c0_seq1
comp109908_c2_seq2
comp32549_c0_seq1
comp100439_c2_seq9
comp112413_c0_seq11
comp97989_c0_seq1
comp112413_c0_seq4
comp112413_c0_seq16
comp111692_c0_seq19
comp90532_c0_seq2
comp104425_c0_seq2
comp111692_c0_seq28
comp101571_c0_seq1
comp110445_c0_seq1
comp106459_c0_seq4
comp110213_c0_seq1
comp105794_c0_seq1
comp101804_c0_seq17
comp108721_c0_seq10
comp102094_c2_seq6
comp34039_c0_seq1
comp102419_c3_seq3
comp89279_c0_seq1
comp109035_c0_seq3
comp106133_c0_seq2
comp106691_c0_seq7
comp100519_c0_seq3
comp101442_c0_seq2
comp109035_c0_seq2
comp81109_c1_seq2
comp106003_c0_seq12
comp104430_c0_seq3
comp111432_c2_seq2
comp89474_c0_seq1
comp93580_c0_seq1
comp108626_c0_seq11
comp108626_c0_seq8
comp108626_c0_seq7
comp108016_c0_seq4
comp98124_c0_seq1
comp86021_c0_seq1
comp87413_c0_seq1
comp88224_c0_seq4
comp108779_c1_seq2
comp92066_c0_seq1
comp108754_c2_seq6
comp112595_c0_seq2
comp110445_c0_seq2
comp88832_c0_seq2
comp105794_c0_seq7
comp105090_c0_seq6
comp101510_c0_seq1
comp89236_c0_seq1
comp98413_c0_seq1
comp109775_c1_seq2
comp103425_c0_seq3
comp100817_c1_seq1
comp92257_c0_seq1
comp100807_c0_seq2
comp103425_c0_seq2
comp94456_c1_seq1
comp109822_c0_seq1
comp106225_c0_seq5
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comp72249_c1_seq1
comp84271_c0_seq1
comp101344_c0_seq1
comp101210_c1_seq4
comp101210_c1_seq10
comp104690_c0_seq10
comp104690_c0_seq8
comp101210_c1_seq8
comp104690_c0_seq5
comp101210_c1_seq2
comp101210_c1_seq6
comp101210_c1_seq5
comp88076_c0_seq2
comp88076_c0_seq1
comp104690_c0_seq19
comp104690_c0_seq6
comp94905_c0_seq1
comp101210_c1_seq13
comp101210_c1_seq3
comp101210_c1_seq1
comp101210_c0_seq1
comp72743_c0_seq1
comp104690_c0_seq4
comp104690_c0_seq16
comp104690_c0_seq20
comp104690_c0_seq15
comp104690_c0_seq11
comp104690_c0_seq3
comp104690_c0_seq12
comp104690_c0_seq1
comp104690_c0_seq18
comp13992_c0_seq1
comp86976_c0_seq1
comp86158_c0_seq1
comp37458_c1_seq1
comp104690_c0_seq2
comp106270_c0_seq6
comp102947_c0_seq4
comp72335_c0_seq1
comp104690_c0_seq13
comp99132_c0_seq1
comp101210_c1_seq12
comp87445_c0_seq1
comp97638_c0_seq1
comp87445_c0_seq2
comp99133_c0_seq1
comp108348_c1_seq1
comp103318_c0_seq1
comp109178_c0_seq4
comp108913_c0_seq2
comp109178_c0_seq2
comp103261_c0_seq1
comp105267_c0_seq1
comp111816_c0_seq1
comp105941_c0_seq1
comp95123_c0_seq1
comp105356_c0_seq1
comp109757_c0_seq2
comp109757_c0_seq1
comp106388_c1_seq2
comp98821_c0_seq2
comp104548_c1_seq2
comp106489_c0_seq1
comp104824_c0_seq1
comp98821_c0_seq1
comp111020_c0_seq1
comp92411_c0_seq1
comp103957_c0_seq1
comp108280_c1_seq10
comp91932_c0_seq1
comp101947_c2_seq1
comp99090_c0_seq1
comp110974_c1_seq1
comp78963_c0_seq1
comp78963_c1_seq1
comp71539_c0_seq1
comp93329_c0_seq1
comp95214_c0_seq1
comp112862_c0_seq1
comp100622_c0_seq1
comp97615_c0_seq3
comp94419_c0_seq1
comp93973_c0_seq2
comp93973_c0_seq1
comp103000_c0_seq1
comp111020_c1_seq1
comp111020_c0_seq2
comp110804_c0_seq3
comp108248_c1_seq4
comp71539_c1_seq1
comp72335_c0_seq2
comp108248_c0_seq1
comp99514_c0_seq5
comp106485_c1_seq1
comp97486_c0_seq1
comp99214_c0_seq2
comp99214_c0_seq1
comp57165_c0_seq1
comp88048_c0_seq1
comp110961_c1_seq1
comp99127_c0_seq4
comp99127_c0_seq2
comp94430_c0_seq1
comp111798_c1_seq2
comp96790_c0_seq1
comp105037_c0_seq1
comp95639_c0_seq1
comp88732_c0_seq1
comp61236_c0_seq1
comp102403_c0_seq8
comp100829_c0_seq2
comp103064_c1_seq1
comp108163_c0_seq1
comp98980_c3_seq4
comp106054_c0_seq1
comp106995_c0_seq2
comp102403_c0_seq4
comp97697_c0_seq1
comp71686_c0_seq1
comp92937_c0_seq1
comp97326_c0_seq1
comp99147_c0_seq1
comp100104_c3_seq2
comp110413_c1_seq1
comp94302_c1_seq1
comp98606_c1_seq8
comp99495_c1_seq6
comp102947_c0_seq3
comp61731_c0_seq1
comp88684_c0_seq1
comp102403_c0_seq13
comp37500_c1_seq1
comp15383_c0_seq1
comp78115_c0_seq1
comp87713_c0_seq2
comp105528_c3_seq6
comp87713_c0_seq3
comp87713_c0_seq4
comp87713_c0_seq1
comp113065_c0_seq1
comp93430_c0_seq1
comp88108_c0_seq1
comp37471_c0_seq1
comp20156_c0_seq1
comp15575_c0_seq1
comp102061_c0_seq2
comp61006_c0_seq1
comp78116_c0_seq1
comp88752_c0_seq1
comp91491_c0_seq1
comp37352_c0_seq1
comp99776_c0_seq1
comp99776_c0_seq2
comp84382_c0_seq1
comp108601_c0_seq1
comp92526_c0_seq1
comp84300_c0_seq1
comp61062_c0_seq1
comp88506_c0_seq1
comp88506_c0_seq2
comp15519_c0_seq1
comp84327_c1_seq1
comp15523_c0_seq1
comp106657_c1_seq2
comp106657_c1_seq1
comp72247_c0_seq1
comp92582_c1_seq1
comp84327_c1_seq2
comp15564_c0_seq1
comp37411_c0_seq1
comp107387_c0_seq6
comp107387_c0_seq5
comp107387_c0_seq4
comp107387_c0_seq7
comp107387_c0_seq1
comp99404_c0_seq1
comp84316_c0_seq1
comp72623_c0_seq2
comp72623_c0_seq1
comp11873_c0_seq1
comp108589_c0_seq1
comp102313_c1_seq2
comp106950_c0_seq3
comp77718_c0_seq1
comp38193_c0_seq1
comp107522_c2_seq3
comp91910_c0_seq1
comp113232_c0_seq1
comp113259_c0_seq1
comp107041_c0_seq1
comp107387_c0_seq2
comp109364_c1_seq1
comp93704_c0_seq1
comp97600_c2_seq2
comp17431_c0_seq1
comp110659_c0_seq1
comp78058_c0_seq1
comp88656_c0_seq1
comp82564_c0_seq1
comp72176_c0_seq2
comp107522_c0_seq1
comp63774_c0_seq1
comp107522_c3_seq1
comp60251_c1_seq1
comp106426_c0_seq8
comp106426_c0_seq13
comp113198_c0_seq1
comp112397_c1_seq4
comp61761_c0_seq1
comp101176_c0_seq1
comp17861_c0_seq1
comp101901_c0_seq1
comp72351_c0_seq1
comp37502_c0_seq1
comp83885_c0_seq2
comp96868_c1_seq1
comp83885_c0_seq1
comp63774_c1_seq1
comp113133_c0_seq1
comp72176_c0_seq1
comp72351_c0_seq3
comp95818_c0_seq1
comp37290_c0_seq1
comp72351_c0_seq4
comp89253_c0_seq1
comp102977_c0_seq1
comp72579_c0_seq2
comp11899_c0_seq1
comp113104_c0_seq1
comp99032_c1_seq3
comp110659_c1_seq4
comp14961_c0_seq2
comp102313_c0_seq1
comp107972_c0_seq1
comp14961_c0_seq1
comp15488_c0_seq1
comp61354_c0_seq1
comp15323_c0_seq1
comp113070_c0_seq1
comp72313_c0_seq1
comp106471_c5_seq1
comp71948_c0_seq1
comp108820_c3_seq8
comp108820_c3_seq1
comp72302_c1_seq1
comp108820_c3_seq4
comp60221_c0_seq2
comp37377_c0_seq1
comp108601_c1_seq2
comp102947_c0_seq2
comp102313_c1_seq1
comp103221_c0_seq1
comp20532_c0_seq1
comp105985_c0_seq1
comp88864_c0_seq1
comp37502_c0_seq2
comp89595_c0_seq1
comp105985_c3_seq1
comp72176_c0_seq3
comp72351_c0_seq2
comp100632_c0_seq1
comp72351_c0_seq5
comp60959_c0_seq1
comp90730_c0_seq1
comp100621_c1_seq7
comp78845_c0_seq1
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Species'Hit'
Atrachya(menetriesi(
Callosobruchus(maculatus(
Drosophila(melanogaster(
Mus(musculus(
60"
50"
40"
30"
20"
10"
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Atrachya menetriesi comp103230 c0 seq2 Utx (10 variants)

93 Callosobruchus maculatus comp41872 c0 seq15 Utx (4 variants)
Tribolium castaneum Utx

Drosophila melanogaster Ubx
Drosophila melanogaster abd-A
58
Tribolium castaneum abd-A
89
Atrachya menetriesi comp139998 c0 seq1 abd-A
16
Branchiostoma floridae Hox8
37
Drosophila melanogaster Antp
Tribolium castaneum ptl
49 Atrachya menetriesi comp109961 c0 seq1 ptl (2 variants)
Callosobruchus maculatus comp41872 c0 seq2 ptl (12 variants)
Drosophila melanogaster ftz
Tribolium castaneum ftz
5850
Atrachya menetriesi comp92490 c0 seq1 ftz
66
Callosobruchus maculatus comp42069 c2 seq12 ftz
Tribolium castaneum Cx
76 52 Drosophila melanogaster Scr
83 Atrachya menetriesi comp75090 c0 seq2 Cx
Callosobruchus maculatus comp41872 c0 seq9 Cx (5 variants)
15
Callosobruchus maculatus comp37257 c0 seq2 comp37257 c1 seq1 Dfd
Drosophila melanogaster Dfd
98
22
Tribolium castaneum Dfd
Branchiostoma floridae Xlox
Tribolium castaneum zen1
67
Atrachya menetriesi comp102511 c0 seq2 zen (2 variants)
65 Callosobruchus maculatus comp34354 c0 seq6 zen (4 variants)
Tribolium castaneum zen2
54 Drosophila melanogaster Abd-B
99 Tribolium castaneum Abd-B
Callosobruchus maculatus comp44633 c0 seq26 Abd-B (26 variants)
84
6
Atrachya menetriesi comp146889 c0 seq1 Abd-B
77
Branchiostoma floridae Cdx
41
Drosophila melanogaster cad
43
Tribolium castaneum Cad2
99
Tribolium castaneum Cad1
Atrachya menetriesi comp39955 c0 seq1 Cad
9
76
Callosobruchus maculatus comp45520 c0 seq1 Cad
Atrachya menetriesi comp42215 c0 seq1 mxp (NB Dfd like)
33 Callosobruchus maculatus comp37995 c0 seq3 mxp (3 variants)
22 Tribolium castaneum mxp
Drosophila melanogaster pb
14
Drosophila melanogaster lab
97
Tribolium castaneum lab
75 Atrachya menetriesi comp74830 c0 seq2 lab (2 variants)
Callosobruchus maculatus comp42069 c2 seq15 seq1 lab
Drosophila melanogaster zen
69
Drosophila
melanogaster zen2
9
Drosophila melanogaster ind
47
Branchiostoma floridae Gsx
Callosobruchus maculatus comp26552 c0 seq2 Ind
95
Tribolium castaneum Ind
79 Atrachya menetriesi comp108136 c0 seq1 Ind (2 variants)
Callosobruchus maculatus comp38822 c1 seq3 Btn
Drosophila melanogaster btn
92
Atrachya menetriesi comp68233 c0 seq1 Btn
67
Tribolium castaneum Btn
50
Branchiostoma floridae M ox
50
Branchiostoma floridae Evxb
Branchiostoma floridae Evxa
56
99
Tribolium castaneum Eve
89
Drosophila melanogaster eve
22
Atrachya menetriesi comp98599 c0 seq1 Eve (2 variants)
57
Callosobruchus maculatus comp39876 c0 seq1 Eve
Tribolium castaneum Unpg
81
Atrachya menetriesi comp127691 c0 seq1 Unpg
70
Callosobruchus maculatus comp2890 c0 seq2 Unpg
98
Drosophila melanogaster unpg
Branchiostoma floridae Gbx
82 Branchiostoma floridae M nxa
Branchiostoma floridae M nxb
31 Drosophila melanogaster exex
Callosobruchus maculatus comp12077 c1 seq1 Exex
93
Tribolium castaneum Exex
Atrachya menetriesi comp84723 c0 seq1 Exex
Drosophila melanogaster bcd
Branchiostoma floridae Pitx
Drosophila
melanogaster
Ptx1
99
76 Tribolium castaneum Ptx1
81
0 .2
Hox6/7/8
Utx/Ubx
Abd-A
Ptl/Antp
Ftz
Hox5 / Cx/Scr
Hox4 / Dfd
Hox3 / Zen
Hox9-13 / Abd-B
Cdx / Cad
Hox2 / Mxp/Pb
Hox1 / Lab
Gsx / Ind
Mox / Btn
Evx / Eve
Gbx / Unpg
Mnx / Exex
Branchiostoma floridae BMP 2/4 AAC97488.1
37
Mus musculus BMP4 AAC37698.1
66
Saccoglossus kowalevskii BMP24

Mus musculus BMP2 NP031579.2
50
38
Apis mellifera Dpp BMP2/4 XP001122815.2

Drosophila melanogaster Dpp AAN10431.1
Lottia gigantea Dpp/BMP2/4 205842
70
Nematostella vectensis BMP2/4 AAR13362.1
Dpp / BMP2/4
Tribolium castaneum NP 001034540.1 Dpp
26
Callosobruchus maculatus comp33819 c0 seq2 Dpp
42
Atrachya menetriesi comp100246 c0 seq7 Dpp (6 variants)
Saccoglossus kowalevskii univin-like

Branchiostoma floridae GDF 5/6/7 XP002602867.1
Nematostella vectensis GDF5 AAR27581.1
Mus musculus GDF5 NP032135.2
23
13
99
GDF 5/6/7

Nematostella vectensis BMP5/8 XP 001638358.1
Callosobruchus maculatus comp40430 c0 seq1 Gbb
78
92
Atrachya menetriesi comp89425 c0 seq1 Gbb
47
Tribolium castaneum NP 001107813.1 Gbb1
28
Apis mellifera Gbb XP394252.1
21
30
Tribolium castaneum EFA04646.1 Gbb2
Drosophila melanogaster Gbb AAF47075.1

Saccoglossus kowalevskii BMP5/8
54
25
32
Gbb /BMP 5/6/7/8
Lottia gigantea BMP5/8 195882

Branchiostoma floridae BMP5/7/8 C3ZTY2
51
86

Drosophila melanogaster Scw AAN11056.2
Lottia gigantea BMP10 111943
15
6
44
66
Mus musculus BMP9 AAD56961.1

Apis mellifera BMPx (BMP9/10) XP001120039.1
49
BMPx / BMP 9/10
Atrachya menetriesi comp170252 c0 seq1 BMPx (BMP9/10)
65
Tribolium castaneum XP 973577.1 BMPx (BMP9/10)
99
Nematostella vectensis ADMP related AFP87424.1

74
98
GDF 1/3
Saccoglossus kowalevskii BMP3

Branchiostoma floridae ADMP XP002604737.1
83
37
Lottia gigantea ADMP 110168

99
Saccoglossus kowalevskii ADMP
ADMP / BMP3
Danio rerio ADMP NP571951.2

Saccoglossus kowalevskii Nodal A
6
Lottia gigantea Nodal ACB42423.1
47
Branchiostoma floridae Nodal AAL99367.1
56
Mus musculus Nodal AAI28019.1
87
Atrachya menetriesi comp106563 c0 seq1 Maverick (3 variants)
97
Tribolium castaneum XP 001811434.1 Maverick
99
Apis mellifera BMP2B/Maverick XP001122118.2

20
Nodal
Callosobruchus maculatus comp48645 c0 seq1 Maverick
95
Maverick
Drosophila melanogaster Maverick AAF59328.1
97
Saccoglossus kowalevskii TGFB2 ADB22639.1

Mus musculus TGFB 2 NP033393.2
100
83
63
TGF
100
Mus musculus Myostatin AAO46885.1

69
Branchiostoma floridae Myostatin XP002599461.1

Tribolium castaneum XP 966819.1 Myostatin
11
50
Callosobruchus maculatus comp39049 c0 seq1 Myo
88
Atrachya menetriesi comp104142 c0 seq2 Myo (2 variants)
39
26
Saccoglossus kowalevskii GDF8/11 XP 002734819.1
51
57
Myostatin / GDF 8/11
Nematostella vectensis Myostatin XP 001641598.1
39
Lottia gigantea Myostatin 82990

Drosophila melanogaster Myostatin AAF59319.1
Mus musculus Inhibin e NP032408.2
11
Apis mellifera Activin XP001123044.2

100
Tribolium castaneum XP 008194984.1 Act

Drosophila melanogaster Activin NP651942.2
81
Inhibin / Activin
Mus musculus Inhibin a NP032406.1

Mus musculus Inhibin b NP032407.1
88
Saccoglossus kowalevskii Lefty NP001164679.1
10
87
Branchiostoma floridae Inhibin beta XP002606835.1

Saccoglossus kowalevskii Activin/Inhibin NP001161496.1
38
16
Nematostella vectensis Activin ABF61781.1

Lottia gigantea Activin 151945
37
Apis mellifera ALP XP001122210.1

Drosophila melanogaster Dawdle (ALP) NP722840.1
94
Tribolium castaneum XP 970355.1 Dawdle (ALP)

Callosobruchus maculatus comp42632 c3 seq1 Dawdle (ALP)
99
66
Atrachya menetriesi comp92923 c0 seq1 Dawdle (ALP) (2 variants)
Callosobruchus maculatus comp30559 c0 seq2 putative Derriere (2 variants)

Tribolium castaneum XP 008191586.1 putative Derriere
43

94

Mus musculus Neurturin NP 032764.1
Danio rerio GDNF NP 571807.1
100
99
65
0 .5
Lefty
Inhibin / Activin
Mus musculus GDNF NP 034405.1

Xenopus laevis GDNF NP 001090196.1
Dawdle / ALP
Unknown Derriere-Like
BMP15 / GDF9
A) Wnt Signalling:
MAPK Signalling
Pathway
Hip
Canonical Pathway:
PS-1
+P
TAK1
NLK
Gas1
PKA
IDAX
c-myc
+P
Duplin
FRP
Cer1
Hedgehog
Ptc
Wnt
c-jun
CKI
Frizzled
Scaffold
GBP
Porc
Dvl
Pontin52
+P
GSK3
-catenin
TCF/LEF
Wnt
CK2
LRP5/6
Axin
DNA
SMAD4
CKI
APC
XSox17
CtBP
-TrCP
p53
Fu
Cholesterol
Su(fu)
Zic2
Rab23
PKA
Slmb
Uterine
GSK3
CK1
Phosphorylation
Independence
SIP
Cul1
Skp1
Rbx1
C) Notch Signalling:
Ubiquitin Mediated
Proteolysis
+u
Co-activators
Proteolysis
TBL1
Dvl
Fringe
MAML
Numb
Planar Cell Polarity

Pathway:
Focal Adhesion
Frizzled
RhoA
Cytoskeletal
Changes
ROCK2
PSE2
Serrate
Dvl
Rac
NCSTN
Gene
Transcription
JNK
PSEN
CtBP
Groucho
Co-repressors
Via MAPK/RAS
HDAC
MAPK Signalling
Wnt/Ca2+ Pathway:
CaMKII
Frizzled
PLC
Ca2+
CaN
G Protein?
PKC
-P
NFAT
DNA
Onward Effects
Key:
Present
Both
Present
A.menetriesi
Cell
Membrane
Present
C. maculatus
Internal
Membrane
Absent
Both
Coleopteran
Absence (?)
Up-regulation
or Movement
Other
Pathway
Down-regulation
State
Change
HES1/5
PreT
SMRT
Others
APH-1
MAPK Signalling
Pathway
Wnt5
DNA
CSL
Hairless
Secretase
Complex
TASE
Knypek
SKIP
Notch
Intracellular
Domain
Notch
Delta
Prickle
Daam1
Wnt11
HATS
Delta
Sfom
Wnt Signalling
TGF Signalling
Ptc
Megalin
Skp1
Nkd
Phosphorylation
Independence
Dpp
TGF Signalling
Siah-1
APC
Ci
Cell Cycle
+P
p53 Signalling
Pathway
PAR-1
Cos2
Smo
PPAR8
Groucho
BAMBI
Dkk
fra-1
cyc-D
SMAD3
ICAT
Axam
SOST
Microtubule
CBP
Dally
Wif-1
B) Hedgehog Signalling:
Adherens
Junctions
Alzheimers
Disease
Disassociation
CIR

C. Mac Paper

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C. Mac Paper

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bioRxiv preprint first posted online Jan. 6, 2016; doi: http://dx.doi.org/10.1101/035998.

The copyright holder for this preprint (which was not

and Callosobruchus maculatus

Institute for Developmental Biology, University of Cologne, Zlpicherstrasse 47b, Cologne

Kong, Shatin, Hong Kong

Department of Biological Sciences, University of Illinois at Chicago, Chicago, Illinois, United

Authors contributed equally

Matthew A. Benton: matthewabenton@gmail.com

Nathan J. Kenny: nathanjameskenny@gmail.com

Kai Conrads: kai.conrads@gmx.de

Siegfried Roth: siegfried.roth@uni-koeln.de

Jeremy A. Lynch: jlynch42@uic.edu

developmental biology, as well as providing additional coleopteran datapoints for comparative

menetriesi and C. maculatus, respectively. We utilized this sequence data to build a

of growth and differentiation.

Key Words: Beetle, Coleopteran, Transcriptome, Ovary, Embryonic, Staged, Atrachya

menetriesi, Callosobruchus maculatus, Chrysomelidae

comprehensively described, with approximately 90% of coleopteran diversity as yet

database as nominated for genomic sequencing as of the 14/09/15 (url:

http://arthropodgenomes.org/wiki/i5K_nominations) and at least one genome publically available

potato crops in America, Asia and Europe.

The Chrysomelidae are represented in public databases by a number of ongoing

transcriptomic and genomic projects. In particular, both L. decemlineata (Bioproject

Compared to C. maculatus, the false melon beetle A. menetriesi (Faldermann), is a

relatively unknown and understudied species. It is native to Japan, where it is an agricultural

germ mode of development, similar to dipterans like Drosophila melanogaster and

development evolved independently in dipterans and hymenopterans, and given the

clade to which C. maculatus belongs. A comparison of the molecular basis of development in C.

In order to facilitate research on A. menetriesi and C. maculatus, as well as wider

Results and Discussion

RNA Extraction and Sample Selection

was made available for download from an external server.

Read Quality and Assembly Metrics:

Bioproject Accession numbers: PRJNA293391 and PRJNA293393. To confirm quality of read

Illumina hexamer binding [38] rather than sequencing-based artifacts.

Using Trinity [39] as an assembler, Trimmomatic-corrected read assemblies were then

compared with assemblies based on uncorrected reads. Initial assays of Trimmomatic-treated

results of assemblies for individual time points.

The final, combined timepoint, contamination-removed assemblies, comprising 228,096

menetriesi, 33,250 in C. maculatus) and a high N50 (1,598 bp A. menetriesi, 2,263 bp C.

(32.82% in A. menetriesi, 38.7% in C. maculatus; reads 36-38%/40-43% respectively depending

To gain an understanding of whether full-length transcripts were present in our data, we

(A. menetriesi DOI: 10.6084/m9.figshare.2056464, C. maculatus DOI:

Timed RNA Expression - Differential Expression Analysis

stage takes approximately 24 hours in A. menetriesi, and 7 hours in C. maculatus. Germband

hours in C. maculatus. The latter period was subdivided in A. menetriesi according to

broad trends in expression, as can be seen in Fig 3.

maternal:zygotic transition occurs in each species is required to resolve this question.

subset of genes found in the combined transcriptomes.

useful for hypothesis building.

Basic Gene Annotation

availability of well-assembled genomes. However, the recovery of these putative duplicates in

evolution is not observed in our species.

transcriptomic resources empirically seem under-represented relative to D. melanogaster and

M. musculus in developmentally interesting categories such as Protein Binding (Molecular

Function), Multicellular Organism Development and Cell Differentiation (Biological Process).

At a gross level, in the intracellular Cellular Component GO categories, our data

wide range of GO categories of all levels of GO distribution demonstrates that Blast2GO

identification of specific genes and pathways of interest.

Gene Family Recovery

To extend the semi-automated analyses presented above, we performed more targeted

previously annotated members of this class.

these putative homologs are given in Additional File 1.