www.sciencemag.org/content/356/6338/642/suppl/DC1

Supplementary Materials for Male sex in houseflies is determined by Mdmd, a paralog of the generic

splice factor gene CWC22 Akash Sharma,* Svenia D. Heinze,* Yanli Wu, Tea Kohlbrenner, Ian Morilla, Claudia

Brunner, Ernst A. Wimmer, Louis van de Zande, Mark D. Robinson, Leo W. Beukeboom, Daniel Bopp†

*These authors contributed equally to this work.

†Corresponding author. Email: daniel.bopp@imls.uzh.ch

Published 12 May 2017, Science 356, 642 (2017) DOI: 10.1126/science.aam5498

This PDF file includes:

Materials and Methods Figs. S1 to S8 Tables S1 and S2 Protein Sequences References

2  

Materials and Methods

Musca domestica strains and culturing

The following strains were used: (I) aabys: multi-marked XY strain: homozygous for a recessive

visible mutation on each of the 5 autosomes (ali curve (ac)I, aristapedia (ar)II, brown body

(bwb)III, yellow eyes (ye)IV, snip wings (snp)V). Males are XY and carry the M-factor on the Y,

females are XX; (II) XY: wildtype strain from Siat, Switzerland. Males are XY and carry the M-

factor on the Y, females are XX; (III) Md-traD: females carry the dominant gain-of-function Md-

traD allele on chromosome IV. Females and males are homozygous for MIII; (IV) Arrhenogenic:

females carry the Ag mutation on chromosome I and produce only sons due to lack of maternal

Md-tra; (V) MI: males carry the M-factor on the first autosome linked to the wt allele ali curve ac+

(normal wings); MI/+; ac+/ac. Females are homozygous for ac (alicurve); +/+; ac/ac; (VI) MII:

males carry the M-factor on chromosome II linked to the wildtype allele of aristopedia (ar+); MII,

ar+/ar. Females are homozygous ar/ar; (VII) MIII: males carry the M-factor on chromosome III

linked to the wildtype alleles of brown body (bwb+) and pointed wings (pw+) (pw+, MIII, bwb+/pw,

bwb). Females are homozygous pw, bwb/pw, bwb; (VIII) MV: males carry the M-factor on

chromosome V linked to the wildtype allele of ocra (ocra+); MV, ocra+/ocra. Females are

homozygous ocra/ocra. All strains (9,15,22) were cultured at 25°C in beakers containing 140g

food-mixture (150g flour, 50g yeast, 120g milk powder, 1000g bran) dissolved in 185ml water

and 4ml Nipagin (66g Nipagin, 123g Nipasol in 2l of 96% Ethanol) until hatching and then

transferred to cages at room temperature or kept in beakers at 18°C. As low density of larvae on

standard medium can cause substantial decrease in survival rates, we reared injected embryos and

the surviving larvae on porcine manure. To avoid contamination with mites, parasites and wild

housefly populations, manure was stored at -70° C for at least two weeks prior to use.

3  

RNA sequencing and Identification of male-specific transcripts

Total RNA was prepared from 0-6 hrs and 1-8 hrs unisexual embryos (Fig. S1) with TRIzol

reagent (Invitrogen) according to the manufacturer’s protocol. Libraries for sequencing were

generated with the TruSeq RNA Sample Preparation kit (Illumina). Clones were Illumina

sequenced with 50bp paired ends (GATC Biotech, Konstanz, Germany) yielding 140 million male

and 120 million female reads for 0-6 h embryos and 80 million male and 120 million female reads

for 1-8 hrs old embryos. To create the transcriptome catalog, all reads from the 4 samples were

concatenated into a single file and Trinity (23) as run with default parameters. Transcript-level

quantifications for each sample were done against this catalog using RSEM (24) (table S1).

Differential gene expression was performed using edgeR (25) by fitting a negative binomial

generalized linear model (GLM) that accounts for time (0-6h/1-8h) as well as gender

(male/female). To find male-specific transcripts, the main effect for gender was tested using the

likelihood ratio test (26) from the NB GLM within edgeR. Transcripts from the catalog were then

mapped against the Musca domestica aabys genome with GMAP (27) to provide an additional

filter.

Genomic DNA and cDNA amplifications

Genomic DNA was extracted from one male and one female of each strain in 1ml extraction

buffer (0.1M Tris-HCL, pH9.0; 0.1M EDTA; 1% SDS, and 0.5%-1% DMDC added freshly).

After incubation at 70°C for 30 minutes, samples were lysed by adding 140 µl Potassium acetate,

re-incubated for 30 min on ice, centrifuged, precipitated in ½ volume isopropanol, washed with

70% ethanol and eluted in 100 µL 10mM Tris + 1 µl RNAse A (10mg/ml stock). Genomic PCRs

were performed with primers Mdmd_F1/ Mdmd_R4 for Mdmd and Md-ncm_6/ Md-ncm_7 for

Md-ncm. For cDNA amplifications primer pairs Mdmd_7s/Mdmd_8as were used for Mdmd, and

Md-ncm_1s/ Md-ncm_2as and Md-ncm_9s/Md-ncm_10as for Md-ncm (table S2). PCR was

4  

performed in 50 µl volume containing 10 µl 5x GoTaq Reaction Buffer, 1.5 µl 25 mM MgCl2, 1.5

µl 10 mM of each dNTP, 0.3 µl GoTaq DNA Polymerase (Promega), 1.5 µl 10µM of each primer

and 2 µl genomic DNA. PCR thermal cycling consisted of 2 min initial denaturation at 94°C,

followed by 35 cycles of 30 s at 92°C, 30 s at 59°C and 30s at 72°C, and a final elongation of 3

min at 72°C. For expression analysis of the downstream genes Md-dsx and Md-tra total RNA was

extracted and cDNA prepared from single flies as described above. Amplifications were

performed on the cDNA with primers Md-tra9 / Md-tra20 for Md-traF, Md-dsx6 / Md-dsx12 for

Md-dsxF and Md-DSX-62 / Md-DSX-59B for detecting Md-dsxM transcripts.

Phylogenetic analysis

To determine the phylogenetic origin and position of the Mdmd genes (Fig. 1E and Fig. S3), the

protein sequence of MdmdV was used to perform tblastn on the NCBI web page (28) against the

full genome of a number of yeast, vertebrate, and insect species: budding yeast Saccharomyces

cerevisiae (Sc), fission yeast Schizosaccharomyces pombe (Sp), zebrafish Danio rerio (Dr),

African clawed frog Xenopus laevis (Xl), domestic chicken Gallus gallus (Gg), house mouse Mus

musculus (Mm), human Homo sapiens (Hs), jewel wasp Nasonia vitripennis (Nv), silk moth

Bombyx mori (Bm), red flour beetle Tribolium castaneum (Tc), yellow fever mosquito Aedes

aegypti (Aa), vinegar fly Drosophila melanogaster (Dm), Mediterranean fruit fly Ceratitis

capitata (Cc), Oriental fruit fly Bactrocera dorsalis (Bd), stable fly Stomoxys calcitrans (Stc), and

the female genome of M. domestica (Md). In each genome, the best hit corresponded to the

ortholog of CWC22, which is termed nucampholin (ncm) in ecdysozoa (29,30). No specific

ortholog to Mdmd was found in the entire NCBI database. The identified protein sequences are

listed in Supplementary Information: Protein Sequences.

To align the protein variants of the four different M. domestica strains, MdmdY, MdmdIII,

MdmdII, and MdmdV, with the identified sixteen CWC22/NCM homologs, we used the MUSCLE

5  

alignment tool (31,32) within Geneious version 10.0 (Biomatters, Auckland, New Zealand) with a

maximum of eight iterations. From this alignment (Fig. S3A), we trimmed off the variable N (up

to alignment position 435) and C (from alignment position 1005) termini, to use only the

conserved central parts of the proteins for phylogenetic analysis. The twenty trimmed sequences

were then analyzed by Geneious Tree Builder (Fig. 1E) using a Jukes-Cantor Neighbor-Joining

method and resampling the tree with a Bootstrap method of one thousand replications within

Geneious version 10.0 (Biomatters, Auckland, New Zealand). A virtually identical tree (Fig. S3B)

was calculated by applying MrBayes (33) using default settings as implemented in Geneious

version 10.0 (Biomatters, Auckland, New Zealand).

Embryonic RNAi

Two dsRNAs templates, 33840_c0 and 22793_c0 (table S2), were designed based on the Mdmd

ORF sequences in the amino-terminal region and carboxy-terminal region respectively, and

synthesized by in vitro transcription of DNA templates comprising the T7 promoter sequences on

both ends. Total RNA was extracted from a single MIII, MII, MV and MY male with the NucleoSpin

RNAII Kit (Machery-Nagel) and cDNA was isolated with the Transcription High Fidelity cDNA

Synthesis Kit (Roche). PCR was performed under the following conditions: an initial denaturation

step at 94°C for 2 min was followed by 35 amplification cycles (denaturation at 92°C for 30 sec,

annealing at 59°C for 30 sec and elongation at 72°C for 30 sec) and a final elongation step at 72°C

for 3 min. The 25 µl reaction mixtures contained 10µl 5× GoTaq reaction Buffer, GoTaq reaction

Buffer (Promega), 10 pmol of each primer, 10mM dNTP mix and 25mM MgCl2. PCR products

were purified (JETquick, PCR Product Purification Spin Kit/ 250) and 1 µg of each was used as a

template for in vitro transcription of dsRNAs (MEGAscript RNAiKit, High Yield Transcription

Kit do dsRNA, Ambion).

6  

Embryos of the MIII, MII, MV and MY strains were collected one hour after fertilization

and injected with dsRNA 33840_c0 and 22793_c0 separately and in combination at 1 µg/µl and

500 ng/µl concentration. dsRNA M122 was used as a control. After microinjection embryos

were transferred to a sieve, dechorionated with 3% NaOCl for 3 minutes under a compound

microscope after rinsing with tap water, and placed in Ringer’s solution (7.2 g sodium chloride,

0.37g potassium chloride and 0.17g calcium chloride). Embryos were aligned on a coverslip in

vertical rows with the posterior end of the embryo at 0.5 mm from the coverslip border under a

compound microscope. Coverslips were placed in silica gel for 5 minutes for dehydration and

covered with 10S Voltalef Prolabo oil. dsRNAs solution was injected with a glass capillary and

a micromanipulator (Femtojet-Eppendorf) under a dissecting microscope. After injection,

coverslips with embryos were incubated on an apple-juice agar plate at 25°C for 24 hrs.

Surviving embryos were collected and placed into a beaker filled with pig dung at 25°C until

hatching.

For dsRNAs template Mdncm5_7, synthesis of dsRNA solution and procedure for

microinjections were same as described above (primers listed in table S2).

dsRNA injected flies were dissected under a microscope 3 to 5 days after hatching. The gonads

were placed into the well of a microtiter plate filled with phosphate buffered-solution (PBS),

fixed by 3.6% paraformaldehyde for 30 minutes at RT, rinsed with PBST (PBS+ 0.1% Triton

X-100) and permeabilized in PBS containing 1% Triton X-1000 and 10mg/ml BSA for 1 hour

at RT. The samples were then incubated in a 1:500 dilution of DAPI (DNA dye) overnight in

the dark (O/N) and transferred onto a slide containing 50% glycerol. The tissues were observed

under fluorescence microscopy and stored at 4°C.

Quantitative Mdmd expression studies

7  

Quantitative real-time PCR was used to measure relative expression levels of Mdmd in 10 hrs and

20 hrs old male embryos collected from homozygous MIII males crossed to pw, bwb females. RNA

isolation was done by the NucleoSpin RNAII Kit (Machery-Nagel) and was subsequently reverse

transcribed with the Transcription High Fidelity cDNA Synthesis Kit (Roche). Subsequent qPCR

was done with a 1:3 cDNA dilution and PerfeCTaTM SYBR®Green (300nM) mix (Quanta

Biosciences, Gaithersburg, USA) on an Applied Biosystems 7300 Real Time PCR System with

250 nM Mdmd qPCR primers 22793_c0_qF1 and 22793_c0_qR1 (table S2). β-Actin was used as

internal control for relative gene quantification (34) with 250 nM of primers β-Actin _F1 and β-

Actin_R1. All primers sets were developed with Primer3Plus. The qPCR profile was as follows:

95°C for 3 min, 40 cycles of 95°C for 15s, 56°C for 30s and 72°C for 30s followed by a standard

ABI7300 dissociation curve to control for nonspecific amplification.

LinRegPCR 11.0 (35) was used for base-line correction and calculation of N0 value with

PCR efficiencies per amplicon from raw fluorescence data generated by 7300 System SDS

Software (Applied Biosystems, CA, USA). Relative expression levels of Mdmd in M112-dsRNA

injected and Mdmd-dsRNA injected male embryos were determined by dividing Mdmd N0 values

by β-Actin N0. The expression levels of Mdmd decreased to 70% after injecting Mdmd-dsRNA

and compared with that of control M112-dsRNA injected embryos in 10h samples. Three

biological samples were used and three technical replicates from each of the 10h control, 10h

Mdmd-dsRNA and 20h Mdmd-dsRNA samples. Two biological samples and three technical

replicates from each were used for the 20h control samples injected with M112-dsRNA. The

average of the relative expression (RE) values and their standard errors were calculated to

construct the graph by using Sigmaplot (Fig. 2D). Mdmd expression differences between the

Mdmd-dsRNA injected and control M112-dsRNA samples were compared with the Mann-

Whitney U test.

8  

sgRNA synthesis and RNP complex assembly

sgRNAs were designed with MultiTargeter Website (http://www.multicrispr.net). Possible

OFF-target sites were excluded with the program Cas-OFFinder (http://www.rgenome.net/cas-

offinder/) and by directly BLASTing selected sequences against the published housefly genome

sequence (15). sgRNA was synthesized according to instructions of the Megatranscript T7 kit

(Ambion) with 400 ng of target template and a 5' flanking T7 promoter as starting material.

After RNA synthesis, template was removed by incubating with TurboDNase (Mmessage

Mmachine T7 Ultra Kit, Ambion) for 15 min at 37 °C. Cas9 was expressed as an His-tagged

protein and purified from bacteria (36). The injection cocktail was prepared by mixing 1.5 µl

purified Cas9 protein (9 mg/ml) with 2µl of sgRNA in 1.36 µl of 2M KCl in a volume of 10µl.

Prior to injection, the mix was incubated for 10 min at 37° C (37). A 1:1 mix of sgY3 preloaded

Cas9 complex and sgY2 preloaded Cas9 complex was injected.

Microinjection of sgRNA-Cas9 complexes

Embryos of the MIII host strain were collected 1 hrs after egg laying and the chorion membrane

removed by incubating in 3% sodium hypochlorite solution (NaOCl) for 1.5 min.

Dechorionated embryos were rinsed thoroughly with water and Ringer's solution. Embryos

were aligned on a cover slip with posterior ends pointing to the injection site, dehydrated for 4

min in a silicagel chamber and covered with 3S/10S (1:4) Voltalef oil (Prolabo). A glass needle

was filled with the preloaded sgRNA-Cas9 mix and injected into the posterior end of 0-1 hrs

old embryos. After injection excess Voltalef oil was carefully removed and cover slips were

placed on an agar plate overnight at 25°C. Surviving larvae were transferred to a small beaker

with porcine manure. G0 males were collected shortly after eclosion and crossed with untreated

virgin females of the MIII strain.

9  

Genomic analysis of CRISPR-Cas9 mediated lesions in Mdmd

To examine for the presence of lesions in Mdmd genomic DNA was extracted following the

protocol described above. PCR products were purified with the Wizard® Genomic DNA

Purification Kit (Promega), subcloned in the pGEM®-T Easy Vector (Promega) and

sequenced. To test whether pw+, bwb+ females carry Mdmd sequence primers 1s and 1as were

used which are shared by multiple Mdmd copies. To test for lesions in the CRISPR-Cas9 target

sites flanking primer pairs Md-MIII-FsgF3/Md-MIII-FsgR3 and Md-MIII-FsgFA/Md-MIII-

RsgFA (table S2) were used. These sequences are also present in other copies of Mdmd. To test

specifically for lesions in MdmdIII primer pairs F1 and R4 (table S2) were used for

amplification.

Author Contributions

DB, EAW, LWB, LvdZ and MR conceived, designed and supervised the project. AS, YW, SDH,

TK and CB performed molecular biological experiments. MR and IM performed the transcriptome

assembly, differential expression analysis and some additional sequence analyses. All of the

authors discussed the data and helped manuscript preparation. DB, LWB, and AS wrote the

manuscript with intellectual input from all authors.

10  

     

                           1    2    3    4    5    6  

Fig. S1.  

Generation of unisexual progeny for differential expression analysis. (A) Only-female

progeny was obtained by crossing wildtype females to no-M males that were collected from the

Arrhenogenic (Ag) strain. This strain produces no-M males because the Ag mutation (chromosome

I) in females prevents maternal expression of Md-tra which is required to activate the female

promoting function of Md-tra in the zygote (11). (B) Only-male progeny was obtained by

crossing wildtype females to homozygous MIII males collected from the Md-traD strain. Males in

this strain can be kept in homozygous state for MIII because of the presence of a dominant gain-of

A   B  

C

11  

function Md-traD allele in females which overrides repression by M (11). (C) RT-PCR

amplifications of collected RNA samples (only female 1: 1-8 hrs AEL, 2: 1-5 hrs AEL, 3: 0-6 hrs

AEL, only male 4: 1-8 hrs AEL, 5: 1-5 hrs AEL, 6: 0-6 hrs AEL). Male-specific transcripts of Md-

tra, Md-traM5 and Md-traM1, only detected in male samples (primers Mdtra12Bs and Mdtra20as).

Female-specific transcripts of Md-tra, Md-traF1 (primers Mdtra9s and Mdtra24as) are detected in

all samples because of maternal expression (11)  

12  

Mdmd III TCACTCGTTTCAGAACTTTGGGTATTAAAAGACGGATTACTTACCATTGTTCTCTTAATG Md-ncm ------------------------------------------------------------

Mdmd III GCAATATATTTCTTAGATTCGAAACCTTTTGTCGATATATTAAGAAAATAACCTGGCGGT Md-ncm ------------------------------------------------------------

Mdmd III TAATCTGCACGTAACACCCGCAGTTTATCATATTCTCATAGGATTTCAATAA-------- Md-ncm ------------------------------------------CTGCCAATGAGGAGAAGA

* **** *

Mdmd III --CGACTCTCGAATAAACAATAAACAGATCCTGACAACAACAAAAATATGAATGCCA--- Md-ncm ATGGACGCCAGAAAAAGGAAAAATC----------------AAAAACA--CCTTCAAAAG

*** * *** ** ** ** * ***** * * * *

Mdmd III CCGACGCCGAATCTCGAAAACCGGAAAATAAACCTAGTTCTGAGTCTTCGT--------- Md-ncm AGGACAAGAAATCACGGAAAAAGAAAAGTGAGTCCAGTTCTGAGTCTTCATCGTCTTCTG

*** **** ** *** * *** * * * ************** *

Mdmd III ---------CGTCGGGGTCCACAAGTGGATCAAGTGATGGAGAAGTGTCTTCCAAAACAT Md-ncm ATTCCGACTCGTCGGAGTCCTCAAGTAGTTCAAGTGATGGCGAAGTATCTACCAGCTCCG

****** **** ***** * *********** ***** *** *** *

Mdmd III ATT---------------------TCAA---------AAATAACAAATCAAAAGTTCTAT Md-ncm GTTCATCGTCAGACAGTGAGAAGGTTAAAAGTAAAGTCAAAAACAAATCCAAAAGTCCAT

** * ** ** ******** *** ** **

Mdmd III CAGGGCAAAGGGAAGTCGTA---TTGGAAGTAGTACGTGATCTATCTTATACTATATGCA Md-ncm CAGCACAAAGGGAAGTAGTCCAAAAGGAAACAGTTACAGATACGCCTGATAATATATCCA

*** *********** ** **** *** *** ** *** ***** **

Mdmd III AGGAAGCAGAAGAAAAATTAGTTGAAAGATTTCCTAGAAAGGATGGATCGAATGAAATGC Md-ncm AGGAAACACAAGAGAAGTTGGTTGAAGATATTCCTAAGGAGAATGAATTGAATGTAAATT

***** ** **** ** ** ****** ****** ** *** ** ***** **

Mdmd III TTCCCAAGGAAGATAGCATAAATACTAACCATAATTACACAACGGATTCAAATGAACACC Md-ncm TGGCAAAGGAAGATAGCGTTAATGCCAAGCAAAATGACACAGAGGCTTCAAATGAGAACG

* * ************ * *** * ** ** *** ***** ** ********* **

Mdmd III CAGTAGAACTAACGACA---------------------AAGACAGAAGAATGTAAAAATA Md-ncm TAGCGGAAGAAATAACAAAACCGCCTCAGAAGCAACCTGACACTGAAGAAGGTGAAATTA

** *** ** *** * ** ****** ** *** **

Mdmd III CAGAGAAGACTAAAAAAAAATCTTTTGTGCGAGCTCTCTCCAAAGATAAACAACTATCCG Md-ncm CAGAAAATAATGAAAAAAATTCTTCARCGAGATCTCCTTCCAAAGAAAAACAACTGTCCG

**** ** * * ******* **** * ** *** ******** ******** ****

Mdmd III CCTATAGAAGCCGTTCTAGAAGCACACGTTTAAGTTATTCTGGACACATTAGCCGAACTC Md-ncm CCAATAGAAGCCGTTCTAGAGAAAGA---AGAAGTCATTCTGGAGACAGTAGAGGARCTC

** ***************** * * **** ******** *** *** ** ***

Mdmd III ATTCAGTAGAAAA------------------------AAGTCTATCAAGAT--------- Md-ncm ATTCAGGAGACAAAGGAAGTCCTTCAAGACTTAAAAGAAGTCCTTCAAGATCTAAAAAAA

****** *** ** ***** *******

Mdmd III ------------ATAAAAAAAGTGTATTAAGAAATCGAAGAACTTCCTTTGGACACGGCC Md-ncm GTCCATCACGTTCTAAAAGGAGTGTATCAAGAAAC---AGAAGTTCCTCCAGACACGGCC

***** ******* ****** **** ***** *********

Mdmd III GAGATTCTTCTACGACCAAAAGGTCTGTCTCCAGGGACAAGGACAATCGACTAAGACGAA Md-ncm GAGATTCATCTAGGAACAGACGAAGTGTTTCCGGCGATAAGGAGAACCAACTAAGACGGA

******* **** ** ** * * *** *** * ** ***** ** * ********* *

Mdmd III GA------ATAGGATCTAGCAGAAGCCATACTAGATCTCACAG------TCGATTTCGAA Md-ncm AATCAAGGTCAAGATCTAGCAGAAGTCGTAGTAGATCTCGCAGTCGCAGTCGTTCTCGAA

* * ************* * ** ******** *** *** * *****

13  

Mdmd III GGTCGGAAAAGAAGCTTCCATCAAGATCTCCACGACGAATTCGTTCACAAGAAAGACGAC Md-ncm GACCAGAACGTAAACACCGATCAAGAACACCACGACGATCACGTTCACGAGAAAGGCGAC

* * *** ** * * ******* * ********* ******* ****** ****

Mdmd III ATGAACGACGTCGTTCGATGTCGTCAGATTATGAAAGGA---TAGCGTTACGCCGATCTG Md-ncm ATGAACGACGTCGTTCGGTGTCATCAGATTATGATGCGAGAAGACGATCTCGTCGCTCTG

***************** **** *********** ** * * ** ** ****

Mdmd III AGCCAATCAAAAGAAGA------------------------------------------- Md-ncm AGTCAATCGAAAGAAGACGTGAGGAACGTAAGAGACGTCATGCAGAACGTGATGAAAGGG

** ***** ********

Mdmd III -----------------------GATAAAGATGAATTCTTCAAAAACAATAAGAAAGTAT Md-ncm AAAAATCAAAACGATCCAGACGAGATGAAGATGATTCGTTCAAGACA---AATAAAGTAT

*** ****** * ***** * ** *******

Mdmd III CTGGCGATATAAAAAAGGGAAAGGGGAACGATAATGGTACTGTAGCAGAACTCGAGGCCA Md-ncm CTGCTGAAATGGAAAAAGATAAAGAGAATGATAATGCCACGGTAACTGATCCCAAGGCCA

*** ** ** **** * ** * *** ******* ** *** * ** * * ******

Mdmd III AGATAACAGAACGTCAAAGGAAAAGTCTAGATATACTAACATCACGTACAGGCGGTGCTT Md-ncm AGATTACAGAACGTCAAAGGAAAACTGTAGACATACTTACATCACGTACCGGTGGAGCCT

**** ******************* * **** ***** *********** ** ** ** *

Mdmd III GTTTAACACCCGATAAATTGCGTATGATACAAGCAGAAATTACAGACAAATCATCGGCTG Md-ncm ATATACCACCAGCTAAGTTGCGTATGATGCAAGCAGAAATTACGGACAAAGCATCGGCTG

* ** **** * *** *********** ************** ****** *********

Mdmd III CATATCAAAGTATAGCTCGGGAAGCTCTTAAGAAATACATACATGGTTACATCAATAAAG Md-ncm CATATCAACGTATTGCTTGGGAAGCTCTTAAGAAATCCATACATGGTTACATTAATAAAG

******** **** *** ****************** *************** *******

Mdmd III TCAATGTGGATAGTGTTGCAGTTATCACCCGAAAATTGCTTAAGGATAATATAGTACGTG Md-ncm TCAATGTGGATAATATTGCAATTATTACCCGAGAACTGCTAAAGGAAAATATAGTACGTG

************ * ***** **** ****** ** **** ***** *************

Mdmd III GTAGAGGCGTGCTTTGCCATTCCATAATACAAGCTCAAGCTACATCGCCAACATTTACCC Md-ncm GTAGAGGCTTGCTTTGCCGTTCCATAATACAGGCTCAAGCTGCATCGCCGACATTTACCC

******** ********* ************ ********* ******* **********

Mdmd III ATGTTTATGCCGCCATGGTGGCCATTATTAATTCAAAATTTCCCAATATTGGGGAATTGC Md-ncm ATGTTTATGCCGCCTTGGTGGCTATTATTAATTCGAAATTTCCCAATATTGGAGAATTGC

************** ******* *********** ***************** *******

Mdmd III TGTTGAAACGTTTGGTCATACAGTTCAAACGAGCATTTGGGTGTAACGATAAGACCGTTT Md-ncm TGTTGAAACGTTTGGTAATACAATTTAAACGAGCCTTTCGACGTAACGATAAGACTGTGT

**************** ***** ** ******** *** * ************* ** *

Mdmd III GTTTGACTTCTTCACATTTTATTGCCCACTTGGTTAATCAAAGGGTGGCCCATGAAATTT Md-ncm GTTTGTCATCTTCACGTTTCATAGCCCACTTGGTCAATCAAAGGGTGGCCCATGAAATCT

***** * ******* *** ** *********** *********************** *

Mdmd III TGGCCCTAGAAATTCTAACACTCTTAATTGAATCGCCGACTGATGATAATGTTGAAGTGG Md-ncm TGGCCTTGGAAATTCTAACGCTTTTGGTTGAATCGCCGACAGACGATAGTGTTGAAGTGG

***** * *********** ** ** ************* ** **** ***********

Mdmd III CCATAACATTTCTTAAGGAATGTGGTATGAAATTGACAGAGGTATCATCGGATAGGGTTG Md-ncm CCATAGCATTTCTTAAGGAATGTGGTATGAAGTTGACAGAGGTCTCATCGAAGGGCATTG

***** ************************* *********** ****** * * ***

Mdmd III GAGGCATTTTTGAATTGCTTAAAAATATCTTGCATCAAGGCAAGTTGGATAAACGTGTAC Md-ncm GAGCCATTTTTGAAATGCTCAAAAATATCTTGCATGAGGGCAAGTTAGATAAACGTGTGC

*** ********** **** *************** * ******** *********** *

Mdmd III AATATATGATTAAAGTTTTATTTCAAGTACGCAGAGATGGCTTCAAGGACCATCAATCGA Md-ncm AGTATATGATTGAAGTTGTTTTCCAAGTACGCAAAGATGGTTTTAAGGATCATCAATCGG

* ********* ***** * ** ********** ****** ** ***** *********

14  

Mdmd III TTATTGAGTCATTAGAATTGGTAGAAGAATATGCTCAATTCACTCATTTGCTATTGTTGG Md-ncm TCATWGAGTCCTTGGAATTGGTAGAAGAGGATGATCAATTTACACATTTACTAATGTTGG

* ** ***** ** ************** *** ****** ** ***** *** ******

Mdmd III AAGATGTCACATACCCGAAAGATATATTGAACGAATTTAAATTCGACGATCAGTACGAGAMd-ncm ATGATGCCACATCCCCAGAAGATGTACTGAATGCTTTTAAATTTGATGAACAATACGAGG

* **** ***** *** ***** ** **** * ******** ** ** ** ******

Mdmd III CCAATGAAGAGAAATATAAAGCACTTAGTAAGAATATTTTGGGTAGCCATGCTTCAGATT Md-ncm CCAATGAAGAAAAATACAAAGGGCTTAGTAAAGAGATTTTGGGTAGTGATGCCTCAGATT

********** ***** **** ******** * *********** **** *******

Mdmd III CGGATGGCTCCTTCGGTTCGGGTAGTAATTCCGAAACTGCATTATCTGACTGTGATAAGG Md-ncm CGGATGGCTCCTCCGGTTCAGGTAGTGATTCCGACTCTGAATCATCTGACTCGGATGAGG

************ ****** ****** ******* *** ** ******** *** ***

Mdmd III GCAAAAATGAGGTTAATGATAAATACACGAGTGGTGACATTATTGATGAAACGAAACCAA Md-ncm ACAAAAATGAGGGGGATGAKAAACCCACAGCTGGCGATATTATTGATAATACGGAAACCA

*********** **** *** *** *** ** ********* * *** ** * *

Mdmd III ATCTGATTGCGTTAAGAAGAACAATATATCTTACCTTAAATTCCTGTTTGGATTATGAGG Md-ncm ATCTGATTGCGTTAAGGCGAACYATATATCTGACCATTAACTCCAGTTTGGATTATGAGG

**************** **** ******** *** * ** *** ***************

Mdmd III AATGTGCCCAAAAATTAATGAAAATGCAATTGAAAACTTGTCAACAAAATGAGTTTTGCC Md-ncm AGTGCGCCCATAAGCTTATGAAAATGCAATTGAAACCCGGCCAAGAAATAGAGCTTTGCC

* ** ***** ** * ****************** * * *** *** *** ******

Mdmd III AAATATTGTTAGATTGCTGCGCTGAACAAAGAACCTATGAGAAGTTCTATGGCCTTTTAA Md-ncm ATATGTTTTTAGATTGCTGCGCTGAACAACGRACTTATGAGAAGTTCTATGGTCTGTTGG

* ** ** ********************* * ** ***************** ** **

Mdmd III CTCACCGAATTTGTAAAATGAACAAGTCTTTTATAGAACCATTCAAAGAAATCTTCAAGG Md-ncm CTCAGAGATTTTGTAACATCAACAAATCGTATATAGAACCGTTTGAGGAAATCTTCAAGG

**** ** ******* ** ***** ** * ********* ** * *************

Mdmd III ATATCTGTCAGACTACGCATTGTTTAGATACAAATCGTTTGCGGAATATAAGCAAATTCT Md-ncm ATACCTATCAGACCACACATCGTTTGGATACAAATCGTTTGCGRAATGTAAGCAAATTCT

*** ** ****** ** *** **** ***************** *** ************

Mdmd III TTGCTCATTTGTTGTTTACCGATGCAATTAGTTGGGATGTTTTAGACTGCATAAAGCTAA Md-ncm TTGCTCATTTGTTGTTTACCGATGCCATAAGTTGGGATGTTTTGGACTGCATAAAGCTGA

************************* ** ************** ************** *

Mdmd III CTGAAGATGAGGCAATAACATCACGTTGTATTTTTATAAAAAGTTTCTTTCAAGAATTGG Md-ncm ATGAAGACGACACAACATCATCAAGTCGTATTTTCATAAAAATTCTTTTTCAAGAGTTGG

****** ** *** * ***** ** ******* ******* * * ******** ****

Mdmd III TCGAATACATGGGTCTGTATCACTTTAATAAAAAACTTAAGACTGAAGTTTTAGCTGGAA Md-ncm CCGAGTACATGGGTCTGGGCCAATTGAATAAAAAACTTAAGGATGAAGTTTTAGCAGAGA

*** ************ ** ** *************** ************ * *

Mdmd III CTTTGGCGGGACTATTCCCCAAAGATAATCCCAGAAATATACGCTTCTCCATTAACTTTT Md-ncm GTTTGGCCGGGCTATTTCCCAAAGATAACCCCAGAAATACCCGATTTTCCATTAACTTTT

****** ** ***** *********** ********** ** ** *************

Mdmd III TCACATCTATTGGTTTGGGCGGAATAACTAATGAATTGTGTCAACTCCTAAAGATTGCTC Md-ncm TCACATCCATTGGTTTGGGTGGCCTAACTGATGAATTGCGTCAATTCCTAAAGAATGCCC

******* *********** ** ***** ******** ***** ********* *** *

Mdmd III CGAAATCTGCACCCTCGTCCTCATCATCAAGTTCTTTATCATCGGAATTGTCTGCACCAT Md-ncm CGAAATCTGTACCCGCCATA-AATGCTGAAATTCT------------------GGCCAA-

********* **** * ** * ** **** * * * Mdmd III CCGACGACGATTCTTCAAGTGATTCGGAAAATAAGAAAAAACATAAAGGCAAAAATAAGA Md-ncm -------------------------------TA------AACCT--------------GT

** *** * *

^

15  

Mdmd III AAATGACCAAGAAGAAAAATCCTTCGAAAAAAAAGGAAAAAACTAAAAAATTTGTAGGTA Md-ncm AGATG--------------------------------------TCTCAAATTCGTCGTC-

* *** * ***** ** *

Mdmd III AAAATAAAATAGCCGCTAAGAATAAAACTATAAAGCGAAGGACTGACAAAGACAACTCTT Md-ncm -----------------------------------------------------TCCTCCT

*** *

Mdmd III CTTCAAAAGATAATTTTTTGAAAAGCGAAAGTAGCAGCAACGAATCAATATCCTTAGATA Md-ncm CRTCAT--CATCATCT--TCAACCTCG--------------TCTTCATCATCATCAAGTT

* *** ** ** * * ** ** *** *** * * *

Mdmd III GTTTATCTTCGGAATTGTTTGCACCATCGTCTT------ATTCGTCGTCGGAATCGTCAA Md-ncm CTTCATCTTCGGAATCGTCTGCATCATCATCTTCTAGTGATTCGTCGTCGGATTCTTCAA

** *********** ** **** **** **** ************* ** ****

Mdmd III ACGATTCAGAAA---GTAAGGAAAAACATAAAGGCAAAAATAAGAAGATGACCAAAAAGA Md-ncm GTGATTCAGAAGTGGATAAGAAAAAACGTAAAGGCAAGGTTAAGAAGA---CYAARAAGA

********* **** ****** ********* ******** * ** ****

Mdmd III AAAATCCTTCGAATAAAAAGGAAAAAACAAAAAAAAAATTAAGTAAAAATAAAAAAGCCC Md-ncm AGAAAWCTTCAAATAAAAAGGAGAAAACTAAAAAATCGAAAARTAAAAATAAAAAAGACR

* ** **** *********** ***** ****** ** ************** *

Mdmd III CAAACAAAAATACTAAGAAGCGAATGACTGAAAAGGACATTTCTTCTTCTG--------- Md-ncm CCAAGAAAAAGRCCAAGAAGCGAAAGTCCAAAAAAGSCASCACTTCTTCWGAARATGATT

* ** ***** * ********** * * **** * ** ******* *

Mdmd III ------------AAAGTAGCATCAGTGAATCAAAATCTTTAAATTGTTCGGCCTCGAACC Md-ncm CTTCGAAAAGCGAAAGTAGTAGYAGTGAATCAAAATCTTCAGACAGTTCGGACTCGGAGC

******* * **************** * * ****** **** * *

Mdmd III AAAATGAGAATGAAAAACGGAAGAAAAGGGTTACATCGAAATCAAGAACAAAACGGGTAA Md-ncm AAAGTGGAAATGATAAATCGAAGAAGAAGACTAAATCGAAATCAAAAACAAAACCGAATA

*** ** ***** *** ****** * * ** *********** ******** * *

Mdmd III AGATGTTTAAACAATGTCAATGGGTTGACGCGGACAATCAACGAGATATTAAACGCAAGA Md-ncm AGAAGTCCAAACGATCTGACTCAGAAGACGTTGAAAATGAACGAGATATTAAACGTAAGA

*** ** **** ** * * * * **** ** *** **************** ****

Mdmd III AAAGGGCAGAATATAGATATGAACCTCTTGTATATAGAAAAAGAAATGAGGAATGTTTAA Md-ncm AAAGGGAAGATTTCGGAAATTACATTCATGAAGATAGAAGAAGAGACGAGGAATATTCAA

****** *** * ** ** * ** ** * ****** **** * ******* ** **

Mdmd III AGAAGGGCGCACCAAATTGCAGGAAAGATAACTATGGTAA---TAGGCAGAATCATGAAA Md-ncm AGAATGGTGGAAGAGATCGCAAAAAGGATAATCACGGTAATAATAAGGAGAAACGGGAAA

**** ** * * * ** *** ** ***** * ***** ** * **** * ****

Mdmd III TATCACAACGTCATGATAGTGAAATTAAAAGACGCCGGGAAGAGCGAAAAAAACGCCACC Md-ncm TACCACAACGTCGCGATAGCGAAATTGAAAGGCGTCGAGAGGAGCGCGAAAAACGCCATC

** ********* ***** ****** **** ** ** ** ***** ********** *

Mdmd III ATGAAAA------AAATCACTCACGTGAATATAAACGTTCTAAATTAGG-GCTCTGCCAG Md-ncm GTGAAAGAGAAAGAAATTTCTCACGT------------TCCAGATCAAGATCTCN-----

***** **** ******* ** * ** * * ***

Mdmd III AGGGAATATTTTTTATATATGTGCTGTCAGTTTTATTATCCATGTACATTTCAATGTTTA Md-ncm ------------------------------------------------------------

Mdmd III TGTCAAAATTGTCATTTTACATTCTACTCCTAATTCTTGCTATCAAACACWWWCACTATG Md-ncm ------------------------------------------------------------

Mdmd III TGA Md-ncm ---

16  

Fig. S2.

Nucleotide alignment of the open reading frames of MdmdIII and Md-ncm.

RNA was isolated from embryos of the MIII/+ strain. cDNAs of Mdmd and Md-ncm were

amplified with end-specific primers F1 and R4 for Mdmd and Md_ncm5end and Md_ncm3end for

Md-ncm for sequence analysis. ^ indicates the position of a conserved small spliced out intron.

A

1004

436

B

18  

Fig. S3

Mdmd alignment with CWC22/ncm homologs and phylogenetic relationship. (A) MUSCLE

Protein alignment of Mdmd variants with an intact open reading frame of the four different M.

domestica strains, MdmdY, MdmdIII, MdmdII, and MdmdV, with the sixteen CWC22/ncm homologs

identified by tblastn searches (see Methods and Supplementary Information: Protein sequences).

From this alignment, the variable N (up to alignment position 435) and C (from alignment position

1005) termini (indicated by red lines) were trimmed, to use only the conserved central parts of the

proteins for phylogenetic analysis. (B) The twenty trimmed sequences were then analyzed by

Geneious Tree Builder using a Jukes-Cantor Neighbor-Joining method (Fig. 1E) and

independently with MrBayes (B; branch label: posterior probability).

19  

MIII      MY      MV      MII      MI

3  kb

2  kb

1  kb

6  kb

B  

A

C

1.89  kb 1.64  kb

1.24  kb

20  

Fig. S4

Multiple copies of Mdmd arranged in tandem repeats. (A) The divergent primers pairs, 6as and

1as, were used to amplify sequences between Mdmd copies. (B) Several bands were detected in

males of strains MIII, MY, and MII indicating that their M regions are composed of multiple

tandemly repeated copies of Mdmd, with most copies not carrying an intact open reading frame.

Neither these primers nor any of the Mdmd-specific primers used in this study produced amplicons

in males of the MI strain (M located on chromosome I) suggesting that Mdmd is either absent or

highly diverged in sequence. (C) The intergenic fragments which were isolated from different M

strains display size differences (numbers on the right) that are due to insertions and deletions in an

otherwise highly conserved sequence (blue lines indicate identity). The origin of sequenced

fragments is indicated on the left.

21  

Fig. S5

Model for stepwise evolution of the M region in M. domestica. We propose that in a first step a

duplicate of the essential gene Md-ncm, Md-ncm2, translocated to a new chromosomal location.

Following this event the Md-ncm2 paralog acquired a male-determining activity and became

Mdmd. Its presence defined this chromosome as the new Y chromosome. We propose that the

gradual loss of recombination of this proto Y chromosome posed a high risk to coding genes.

Local amplification may have been a response to preserve the function of Mdmd that is

particularly vulnerable due to its above average long ORF. The presence of multiple nonfunctional

copies of Mdmd could be a manifestation of the gradual erosion of this region due to accumulation

of deleterious mutations and heterochromatization. Empirical evidence for deleterious effects of

gene conversion on tandem arrays in non-recombining regions was previously found in studies of

the mammalian Y chromosome (38,39). We propose that for these reasons translocation of Mdmd

22  

to new autosomal sites e.g. chromosome III, may have aided in evading the weakening effects of

gene conversion and heterochromatization at its original location on the Y.  

23  

 

Fig. S6

Silencing of Mdmd by embryonic RNAi. (A) sequences of dsRNA templates 33840_c0 and

22793_c0 are specific to Mdmd and not present in Md-ncm. (B) dsRNA of 33840_c0 and

22793_c0 were injected separately or together into syncytial embryos of the MIII, MII, MV, MI and

strain   dsRNA   conc.  M/+  with  

testes  

M/+  with  

ovaries  

+  /+  with  

ovaries  

MIII   33840_c0   1  μg/μl   68  (0.32)   147  (0.68)   180  

MIII   33840_c0   2  μg/μl   12  (0.44)   15  (0.56)   36  

MIII   22793_c0   1  μg/μl   54  (0.33)   106  (0.67)   170  

MIII   33840_c0+22793_c0   1  μg/μl   7  (0.17)   34  (0.83)   33  

MII   22793_c0   0.5  μg/μl   3  (0.15)   17  (0.85)   14  

MV   33840_c0+22793_c0   1  μg/μl   4  (0.26)   11  (0.73)   5  

MY   33840_c0+22793_c0   1.2  μg/μl   2  (0.22)   7  (0.88)   6  

MI   33840_c0+22793_c0   1  μg/μl   28   0   18  

MIII   M122   1  μg/μl   48   0   45  

B  

A  

24  

MY strains. The majority of surviving M/+ individuals are phenotypically normal males with

differentiated ovaries with the notable exception of MI males which all developed into normal

fertile males. Combined injections of both dsRNA resulted in a higher penetrance of male ovary

phenotype. As expected +/+ females are not affected by Mdmd silencing, they have normally

differentiated ovaries and are fertile. As a control we injected dsRNA of a male-specific sequence

unrelated to Mdmd, M122. Number of examined adults are indicated, the fraction of males with

testes or with ovaries are shown in brackets.

25  

F1   pw+,  bwb+  males  

pw+,  bwb+  females  

pw,  bwb  females  

M6   34   4   34  

M21   22   1   8  

M25   34   1   32  

M27   30   3   20  

M29   42   6   45  

M30   35   1   30  

M31   29   6   23  

M32   35   2   33  

A

B

C

26  

Fig. S7

CRISPR induced lesions in MIII individuals. (A) Experimental strategy to identify sex

transformed individuals based on linkage of MIII with dominant phenotypic markers pw+ and

bwb+. sgRNA/Cas9 complexes were injected into pw+ bwb+ males of the MIII strain and these

males were outcrossed with untreated females of the same strain. The F1 progenies were screened

for the occurrence of rare pw+ and bwb+ females. (B) The table depicts different lines of G0 males

which sired pw+ and bwb+ female progeny. (C) Genomic amplification of pw+ and bwb+ females

with primers sgF3 and sgFA. Mdmd sequences are present in many lines but the banding pattern

suggests that CRISPR induced large structural changes in the Mdmd cluster. Structural

organization of M32 and M29#6 seem to be unaffected.

27  

strain   dsRNA   Concen-­‐tration.  

number  of  injected  embryos  

number  of  hatching  larvae  

number  of  adult  

females  

number  of  adult  males  

%  of  larvae  surviving  to  adulthood  

MIII   Mdncm5_7   1  μg/μl   341   0   0   0   0  

MIII   Mdncm5_7   0.20  μg/μl   880   154   0   3   0.01  

MIII   Mdncm5_7   0.10  μg/μl   590   89   5   5   0.11  

MIII   Mdncm5_7   0.05  μg/μl   506   54   4   6   0.18  

MIII   Mdncm5_7   0.025μg/μl   857   72   21   16   0.51  

MII   Mdncm5_7   1  μg/μl   56   0   0   0   0  

MIII   M122   1  μg/μl   507   149   45   48   0.62  

Fig. S8

Silencing of Md-ncm by embryonic RNAi. (A) dsRNA template Mdncm5_7 was prepared from

a region at the 5' end of Md-ncm which has low similarity to Mdmd. (B) dsRNA of template

Mdncm5_7 was injected into syncytial embryos of the MIII and MII strain. Increasing the

concentration of dsRNA leads to higher levels of lethality between the larval and adult stage in

both males and females. The highest concentration of 1µg/µl caused 100% lethality already during

embryonic development. Control injections with dsRNA of M122 result in normal survival rates

similar to those observed in Mdmd silencing experiments.

A  

B  

28  

Table S1: List of contigs of male-biased orphan reads.

All reads were assembled in 14,392 contigs that also passed a low expression threshold after

mapping back to the contigs and quantifying with RSEM. Of these, we found 39 contigs (shown in

this list) that did not map to sequences of the previously published female genome draft (15), have

a positive fold change (higher in males) and FDR < 5%. In the top 14, we found 5 orphan contigs

(grey shading) of the same transcriptional unit (see Fig. 1A).

id female 1-8h

male 1-8h

female 0-6h

male 0-6h scaffold logFC logCPM LR PValue FDR

comp64077_

c2_seq2 0 3.89 0 4.83 not_found 12.70694547 1.127387117 48.5834239 3.17E-12 4.56E-08

comp64077_

c5_seq1 0 2.05 0 3.27 not_found 12.03311927 0.419659566 43.8389407 3.57E-11 2.57E-07

comp64077_

c6_seq2 0 1.57 0 2.37 not_found 11.58931956 -0.014398535 41.4789742 1.19E-10 4.61E-07

comp64077_

c3_seq1 0 0.9 0 1.6 not_found 10.95821666 -0.660700448 37.78868168 7.88E-10 2.27E-06

comp66948_

c0_seq5 23.62 388.89 0.01 0.84 not_found 4.729860471 6.690970297 33.20015019 8.31E-09 1.71E-05

comp65705_

c8_seq4 0.03 2.91 0.01 3.31 not_found 7.63560747 0.647002095 31.51819349 1.98E-08 2.84E-05

comp66112_

c0_seq1 0 0.28 0 0.52 not_found 9.329689612 -2.259155615 29.59941192 5.31E-08 5.46E-05

comp66112_

c1_seq1 0 0.16 0 0.85 not_found 9.739113106 -1.922779568 29.36842218 5.98E-08 5.74E-05

comp66112_

c6_seq1 0 0.09 0 1.05 not_found 9.953201423 -1.732634656 27.98924966 1.22E-07 0.000103276

comp58560_

c0_seq1 0.07 5.79 0 0.87 not_found 7.366389398 0.738006618 27.62889895 1.47E-07 0.000117508

comp56711_

c1_seq1 0.03 3.04 0.05 3.82 not_found 6.453091959 0.799011746 27.21782292 1.82E-07 0.000127121

comp66112_

c7_seq1 0 0.09 0 0.68 not_found 9.367798786 -2.283778992 26.25648374 2.99E-07 0.000175063

comp66112_

c4_seq1 0 0.08 0 0.7 not_found 9.406084378 -2.249612933 26.10237573 3.24E-07 0.000179228

comp64077_

c6_seq1 0 1.06 0.03 1.97 not_found 6.974017472 -0.372546802 25.33982814 4.81E-07 0.000230598

comp66112_

c3_seq1 0 0.07 0 0.55 not_found 9.081017758 -2.552625368 24.60558664 7.03E-07 0.000326594

comp517191

_c0_seq1 0 4 0 0 not_found 10.82152232 -0.026669173 22.94230608 1.67E-06 0.000686445

comp108752

9_c0_seq1 0 0.07 0 0.31 not_found 8.335043956 -3.229131818 22.02499356 2.69E-06 0.001075894

29  

comp44611_

c0_seq1 0.05 23.47 0 0 not_found 8.495645732 2.550672689 21.37399994 3.78E-06 0.001414534

comp49039_

c0_seq1 0 0.04 0 0.36 not_found 8.468150916 -3.120524882 21.34520583 3.84E-06 0.001414534

comp66112_

c2_seq1 0 0.06 0 0.36 not_found 8.500193568 -3.08119601 20.90047449 4.84E-06 0.001619183

comp174825

_c0_seq1 0.04 13.83 0 0 not_found 8.134000154 1.785296687 20.20511504 6.96E-06 0.00213022

comp64776_

c0_seq1 0.03 7.61 0 0.01 not_found 7.829555161 0.918843148 20.07342357 7.45E-06 0.002234512

comp42865_

c0_seq1 0 1.76 0 0 not_found 9.645406858 -1.238620477 19.96253838 7.90E-06 0.002241335

comp51856_

c1_seq1 0 1.26 0 0 not_found 9.183623987 -1.735757111 19.95166507 7.94E-06 0.002241335

comp64150_

c2_seq1 0.01 0.29 0.04 4.13 not_found 5.640569171 0.185097932 18.93394906 1.35E-05 0.003337824

comp63084_

c1_seq2 0 1.12 0 0 not_found 8.984979441 -1.900883429 17.06280389 3.62E-05 0.006848266

comp23538_

c0_seq1 0 0.01 0 0.18 not_found 7.415057104 -4.042122375 15.56594341 7.97E-05 0.011821751

comp61718_

c0_seq1 5.16 139.29 0.01 0.03 not_found 3.945898406 5.173921113 15.22636345 9.54E-05 0.013724614

comp52867_

c0_seq1 0.01 1.14 0 0.01 not_found 6.08453022 -1.849045938 15.11957543 0.000100911 0.014207114

comp3145_c

0_seq1 0 0.01 0.01 0.54 not_found 5.952886899 -2.671278371 14.27317797 0.000158102 0.019447871

comp57279_

c0_seq1 0.01 0.49 0 0.03 not_found 5.187918342 -2.970062849 14.19960475 0.000164405 0.019883339

comp59360_

c0_seq1 2.83 52.39 0 0.02 not_found 4.298997469 3.785436854 13.57315248 0.000229444 0.024828224

comp39474_

c0_seq1 0.03 1.33 0 0.01 not_found 5.425047803 -1.608560396 13.2758852 0.000268842 0.027636949

comp52318_

c1_seq3 18.68 451.95 2.03 6.65 not_found 3.156510898 6.904581915 12.89108903 0.00033015 0.032996685

comp57141_

c4_seq1 0.03 0.61 0.01 0.09 not_found 4.043548082 -2.491355462 12.76661691 0.00035286 0.034560967

comp53439_

c0_seq1 0.03 0.53 0.04 0.36 not_found 3.729868824 -2.045511985 12.5128477 0.000404163 0.036193155

comp43327_

c0_seq1 0.03 1.03 0 0.01 not_found 5.094334631 -1.966933993 12.49794762 0.000407399 0.036193155

comp63560_

c12_seq1 0 0.09 0.03 0.4 not_found 4.264731554 -2.809482084 11.93266763 0.000551581 0.046423154

comp58952_

c1_seq1 0.02 1.37 0 0 not_found 5.747997698 -1.58071779 11.917512 0.000556087 0.046530257

30  

Table S2. Oligonucleotides used in this study.

Primer name Primer sequence (5'-3') comments

Md-ncm_6 AGAAGAATGGACGCCAGAAA Md-ncm specific

Md-ncm_7 GTCAGGTTGCTTCTGAGGCG Md-ncm specific

Md_ncm5end TTTATGTCGCGTAGCAGTCG Md-ncm specific

Md_ncm3end TTGACTTTTTGGGGGTTTCA Md-ncm specific

Mdmd_F1 CACTCGTTTCAGAACTTTGGGT Mdmd specific

Mdmd_R4 GTGTTTGATAGCAAGAATTAGGAGT Mdmd specific

Md-ncm_1s CGCAGAGATGGCTTTAAGGA cDNA

Md-ncm_2as TTTTTGGGCACATTCCTCAT cDNA

Md-ncm_9s CTCATCGAAGGGCATTGGAGC cDNA

Md-ncm_10as AATAATATCGCCAGCTGTGGGTTT cDNA

Mdmd_7s ATCATCGGATAGGGTTGGAGG cDNA

Mdmd_8as ATAATGTCACCACTCGTGTATTTA cDNA

Mdmd_6s GCTCTTCCCGGCGTCTTTTA eRNAi

Mdmd_6as GGTTGACGCGGACAATCAAC eRNAi

Mdmd_1s ATCAGGGCAAAGGGAAGTCG eRNAi

Mdmd_1as GATTGGCTCAGATCGGCGTA eRNAi

Md-ncm5 TGAAAGCGAAACAAATGCTG eRNAi

Md-ncm7 GTCAGGTTGCTTCTGAGGCG eRNAi

22793_c0_qF1 TGGTGCGCCCTTCTTTAAAC qPCR

22793_c0_qR1 GTTGACGCGGACAATCAACG qPCR

β-Actin_F1 ATGGGTTGGTATGGGACA qPCR

β-Actin_R1 CACGATTAGCCTTGGGAT qPCR

Mdtra-12Bs GTGATCCTAACAATCAGCTAG RT-PCR

Mdtra-20as TTGCTGCTGGGGGAATGTG RT-PCR

Mdtra-24as GATGCATTTTGTGCATCGCAA RT-PCR

Md-tra2_F1 TTGCTTGAGTTGCCTGCTGC ATA Md-tra2

Md-tra2_R1 CGTCCCCTGTAAACACCTGGG Md-tra2

Mdtra9 CTGCTACAGAAAAGAAAGGCC Md-traF

Md-tra20 TTGCTGCTGGGGGAATGTG Md-traF

Md-dsx6s CTAAAAGATGCCGGTGTTGAC Md-dsxF

Md-dsx12 CGATAACTTTAAGCACCTTG Md-dsxF

Md-DSX-62 CAGAACATTTAAATCAACTGACA Md-dsxM

Md-DSX-59B GCTGCTGGGTCGACTATTC Md-dsxM

CYP6D3-1 GTTCGGTAATATTTGGCTTGG control

CYP6D3-2 CCCGTATTCCGTAGTTGAATT control

31  

CRISPR primers:

Mdmd crispr-sgFA (targets the coding strand)

5’GAAATTAATACGACTCACTATAGGAATTACTACCCGAACCGAGTTTTAGAGCTAGAAATAGC–3’

Mdmd crispr-sgF3 (targets the non-coding strand)

5’GAAATTAATACGACTCACTATAGGCGATATAAAAAAGGGAAAGTTTTAGAGCTAGAAATAGC–3’

Reverse primer

5’AAAAGCACCGACTGCGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCT

AGCTCTAAAAC–3’

Supplementary Information Protein Sequences:

>Aa_NCM [Aedes aegypti] MSDGEQSSRSRQEKRDRSASRDSDGKQQEERRKKLSRRGRDSRSRSRSRSRSRSRSVERRRRRDDRRRKTRSRSTSRSRGRRDDDSRRRRRERRDSSRESGERSRSKSADRKPAQEEKKVPEKQEQEGDEALLKNEKPAIRRTVDLLTSKTGGAYIPPAKLRMMQAEITDKTSAAYQRISWEALKKSIHGYINKVNVDNIGIITRELLHENIVRGRGLLCRSIIQAQAASPTFTHVYAALVAIINSKFPNIGELLLKRLVIQFKRGFRRNDKSICLSSSRFVAHLVNQRVAHEILALEILTLLVENPTDDSVEVAIAFLKEVGQKLTEVSGKGINAIFEMLKNILHEGKLDKRVQYMIEVVFQIRKDGFKDHTAVVEALELVEEDDQFTHLIMLDEATDAQDILNVFKVDNEYEENESKYKAISKEILGSDASDDEDGGGSSSGSGSGSDSDDEDASGSGEEGDETKQQLIVDNTETNLIALRRTIYLTIHSSLDYEECAHKLMKMELKPGQESELCHMFLDCCAEQRTYEKFYGLLAQRFCMINKIYIEPFEQIFQDTYTTTHRLDTNRLRNVSKFFAHLLFTDSIGWDVMRCIRLNEEDTTSSSRIFIKILFQELAEYMGLYKLNARFKDPTLAEPFSGLFPRDNPKNTRFAINFFTSIGLGGLTDELRDFLKNAPKQIVQPVVAPVPEEEADSSSSSSSSSSSSSSSSSSDSSSETSSSSDSDSEDDRRKKKGKKDKRPTKSKSSTSKDKHSSKYKDERRAENGNGSRRDYEASRRDQDRREKDDRESNRREDRPRERNYDDRGRKEYRDREADRGRRDRDDRRRDEAHSSSSRRRRSEERW

>Bd_NCM [Bactrocera dorsalis] MADSDTDSSSSSSESESVTSSSSSSSSSADSAPSPKESSAKKDVTTTENGKDYKSLSVPSLEEKKSLTQSEDYLPEKVNVKSCENREGNDEVRLTNTVSADRNKELSDSTGTAECDSNENKASGELEEGEITNNEAHTQVVDDKERESEGFNGPNENIEPTPTSDKSVENTFDLSENKDQPSEKKSESTSAKKSDENNKIQETKETRIANKSRRRSRSRSREPIKSSRRNRSRNRSISRQRSRSRSRKRSRRSVSLERRQEERKRRHAERERGENEDRNKKRRERGKSLSAESSPKRNKLSPTRKEKDDNKNDDNDNATVTDPNAKITDRQRRTVDLLTSRTGGAYIPPAKLRMMQAEITDKASAAYQRIAWEALKKSIHGYINKVNVDNIAIITRELLKENIVRGRGLLCRSIIQAQAASPTFTHVYAALVSIINSKFPNIGELLLKRLVIQFKRAFRRNDKAVCLSSSRFVAHLVNQRVAHEILALEILTLLVETPTDDSVEVAIAFLKECGMKLTEVSSKGIGAIFEMLKNILHEGKLDKRVQYMIEVVFQVRKDGFKDHQSIIESLELVEEDDQFTHLLMLDEATNPEDVLNAFKFDDQYENNEEKYKGLCKEILGSDASGSGSGSSSSSDSESGSGDSDSENKGEDGGQKTAGDIIDNTETNLIALRRTIYLTINSSLDYEECAHKLMKMQLKPGQEIELCHMFLDCCAEQRTYEKFYGLLAQRFCAINKIYIEPFEEIFKDTYQTTHRLDTNRLRNVSKFFAHLLFTDAIGWDVLECIKLNEDDTTSSSRIFIKILFQELAEYMGLGKLNTKLKDEVLTESLAGLFPKDNPRNTRFAINFFTSIGLGGLTDELRQFLKNAPKSVPAINVEILAVKPEESSSSSSSSSSSSSSSSSDSSSTTSDTSDSSDSEDEKGKKKKRGKAKTDKKKGSNKVSKREKVKRKAHSRKNKPKKISEHNSSSDSDNSSDSNSSTSDDESSSGSSSSSSDDNRRNRDRKKSKDSSTKSKKKSSRANDDKLDKIQRNRHINVDEDNRKASKVKRDKRDKSERNERNDEEKSRNDNRYSDRHMEHRRQRDKSPDRKREEIRERKERAKEYVRHSDEERRKDRENRRQRSRSRSRSRDRNRKDRNYYDKRSVRSTRNGSKERG

>Bm_NCM [Bombyx mori] MEKTNTKQKKDKYEEDDERRRSKRSRRARSRSKSLEEQQNREYRSKRRRSKSRSPHYKAREDKRVKDTQKEPETKKDASTKPERRAKDTDMLNTRTGGAYIPPARLRMMQAQITDKSSIAYQRLAWEALKKSIHGHINKINVGNIGIIIKELLKENIVRGRGLLCRSVIQAQAASPTFTNVYAALVSAVNSRFPNIGELLLKRLVIQFKRGFKRNDKSICISSASFIAHLVNQRVAHEILALELLTLLVESPTDDSVEVAIAFLKECGQKLTEVSSKGVNAIFEMLRNILHEGQLDKRVQYMIEVMFQVWKDGFKDHPALIEELELVPEEEQFTHLLMLDDATDPQDILNVFKFDEKYEENEQKYKNLCKEILGSDAESGDEDGSGESGSEESDEEDEEQPKKEMTIIDNTETNLVALRRTIYLTINSSLDFEECAHKLMKMQLKPGQEVELCHMFLDCCAEQRTYEKFYGLLAQRFCNINRIYIGPFEEIFKDSYSTAHRLDTNRLRNVSKFFAHLLFTDSISWESLECVKLNEEDTTSSSRIYIKILFQELAEYMGLKKLNDRLQDPTLQEAFAGLFPRDNPRNTRFSINFFTSIGLGGLTDELREHLKQMPKNTPAPPTEIKIDSDSSSDSSSDSSSSSDSSSSESEEETQKKKKEKNKNKEKSPEPEKPQDNKVERWGHEGFYDTYGRDARRHDPRDRGDGGRGGAHRDTDQQGRRARDENGRGLRRRDEDHEQDPRDRRAPEKDDRRNRKEGEKSRDGNSRKDIDRDSEKEKDKRSRKDDDRVTDREKDKRQTRNSEEDSRRRDSEYNRKGEKEKEKKRKNARDDEHKKETYHNDEYSGCETRPSPGELRTREATSSIIITNLQEYNFLKLVRHLSLVDGTKTESFPGMAKSPHKVLLQLDDELMNQCMMQRESRYVESEARRMTNVKCYAAMLDSKSNSGSVSSAALRDKAASDPSCAPQNVTEAQIKPQISAPSYFYSLKENEALPFKRPEKEPRPKLLLNSVVNENDVVAMIAAHDANRTEGLKKHIGKLLVEKQIVVNYNLSERKFVTNLLCETIDYAAQRDFSAFKLACLLTTYVTVHSYFKWYYWQAPVSVWMYFKELMIRLTIEDSPDGEAIFEPDECYDILSHFHTMYIKNLPLIHVLTFGVRRLKFLWPFKRK

>Cc_NCM [Ceratitis capitata] MSESDSDSSTSSSGSGSSSSSTSSTSSDESANSPKVSTVEKKLSDSATEKDSSLSANTEERENYPAKNDSEVKSGQEEVIESKVEIENGVSKDQSTNEKKEADQKNTSEEIEEGELDNEGSNDKEKLDDTTSGENEVQLKNDKEKINVVVVEEPAAGDKLEDETPVVDQDPQTVGENISEKEGEPKGDNNKIQEIKHNGTERRRRQRSRSRSHNREQTKSRRRSRSRNQSYSRQRSRTRSLSRKRSRRSASLERRREERKRRHAERELRDNEDRNKRRRERGKKSRSAEKSPKSKAASTVRKEADAPNIKDTENDNATVTDPKAKITERQRRTVDLLTSRTGGAYIPPAKLRMMQAEITDKASAAYQRIAWEALKKSIHGYINKVNVDNIAIITRELLKENIVRGRGLLCRSIIQAQAASPTFTHVYAALVSIINSKFPNIGELLLKRLVIQFKRAFRRNDKAVCLSSSRFVAHLVNQRVAHEILALEILTLLVETPTDDSVEVAIAFLKECGMKLTEVSSKGIGAIFEMLKNILHEGKLDKRVQYMIEVVFQVRKDGFKDHQSVIESLELVEEDDQFTHLLMLDDATNPEDALNAFKFDDQYENNEDKYKGLSKEILGSDASGSENGSGSGSDSDSESGDSSDSDNEGKEEGQPTA

GDIIDNTETNLIALRRTIYLTINSSLDYEECAHKLMKMQLKPGQEIELCHMFLDCCAEQRTYEKFYGLLAQRFCSINKIYIEPFEEIFKDTYQTTHRLDTNRLRNVSKFFAHLLFTDAIGWDVLECIKLNEDDTTSSSRIFIKILFQELAEYMGLGKLNTKLKDEVLSESLAGLFPKDNPKNTRFAINFFTSIGLGGLTDELRQFLKNAPKSVPAINVEILAQKPVNSASSTSSSSSDTSSSSSSSSESSTESSSSSSDTTSSSDSENEKRKKKNLKGKAGDKSNNRKKAATKETMSKKSKNNKNRRKKRSKTESTSSSGDSSDAKSSESDVSSSESSSSSDDNKRKKKQKIQSKKHKKTLTKENNESSKPKVTKSTKAERNGRNVAVNSRSDDKHSDTYTDRRRRKDKSPERKAREDREPNRDHRERKRDRDSRTDYQKDRDSRRHRSRSRSRSRGRKEPTNYDKRSIRSTRNASKERG

>Dm_NCM [Drosophila melanogaster] MGESDAESDSSSNSSSSDTSSGSDSDARSESSSSESSGREEEEAKQEESAKDAKKTDDTDRGEKRAKERDAGQDEQPTEQKKTPAAEPRSERQHISHSAGVEKQQEEAVAAAEAESEKLNEAKKVETPVQRKEEAEASSVTKELNSPKAQEENAARELEERRKDEEQPVTTNGSSKESPVEAAAETVKPPTADHIEEGEITDKDEDDLPTKEEKKAVASKSPPKETQRKQSRSPDGKRRRPRSSSRSPSPSSRRRRRSRSKGSRTRSRSKSPIRRRSNSLERRRVERQRRHEERDKRDEERAKEREKRHQKGEPTSSRRRDDSREKKRSPERKRDRSSSTPKSKSSKTPRHTETTETNADNETVTEPAAKITERQRKTVDVLTSRTGGAYIPPAKLRMMQSQITDKSSAAYQRIAWEALKKSIHGYINKVNVTNIAIITRELLRENIVRGRGLLSRSIIQAQAASPTFTHVYAALVSIINSKFPNIGELLLKRLVIQFRRAFRRNDKMVCMSATRFIGHLVNQRVAHEILALEILTLLVETPTDDSVEVAIAFLKECGMKLTEVSSKGIGAIFEMLRNILHEGKLDKRVQYMIEVLFQIRKDGFKDHQAVVPELELVEEDDQFTHLMMLDEATETEDILNVFKFDDNYAENEDKYKGLSREILGSDDGSSSGSGSGSDSDSDSDGESGSDAEKKAEAGDIIDSTETNLIALRRTIYLTINSSLDYEECAHKLMKMQLKPGQEIELCHMFLDCCAEQRTYEKFYGLLAQRFCNINKIYIPPFEEIFKDTYQTTHRLDTNRLRNVSKFFAHLLFTDAISWDVLECIQLNEDDTTSSSRIFIKILFQELAEYMGLGKLNAKLKDDVLVESIAGLFPKDNPRNTRFSINFFTSIGLGGLTDDLRRFLKNAPKSVPAINAEILANAGGNPFRDGSAPAGNTKVAPSSSSSSSSSSDTDSEDSSEEDSSSDSSSESSSSDSSSEPKKKRKRKDKDKKKSKKATKEKSKKTKNKKKKKKAEKEQEKEKEKQRKSKKEKEKDKKRKKEEKKAAKKKSKHRRKSQESSDSSGSEDSDKSTSESSDSSNSSSDESDAEPQAKIKRQEHVEKNKFRGRTQDSDEFNLEGPGSKNRFQPNGNGQRRRDNSTGRERNRENSSYDRERNRGNSSYDRERKRGNSSYDRERNRESSYDKERKNRNAVAHDRQRKRDRSRSYERPTIRENSAPREKRMESSRSEKDSRRGDRSSRNERSDRGERSDRGERSDRGERSDRGERSDRGERSDRGERSDREKERSRAKERERDRDRDLKGQRERKRERDDGSRDRSRRERSSRRSKGRS

>Dr_CWC22 [Danio rerio] MAAESNESQTAMNMGPTSRDEPPSTEEMEQRQRKASTSSSEDGEHEDNDRRRVVGSPGSRDGSPRVGSPVARASPRAKRDQKSSDSDSDSSDSDDGVMRKIRSSVMHIRRTSDEETKNRERSSSPDRHEKKSKSRSRSRSRSRSRSRSRSPRERYRRARRSRERERDRYGDRERYERRSSRERDWEHRRRGRSASPDKNDKPPTEEPPVKKRKETLDPILTRTGGAYIPPAKLRMMQAQITDKSSLEYQRMSWEALKKSINGLINKVNVSNIANIIQELLQENIVRGRGLLARSILQAQAASPIFTHVYSAVVAIINSKFPQIGELILKRLILNFRKGYRRNDKQQCLTASKFVGHLINQNVAHEVLCLEMLTLLLERPTDDSVEVAISFLKECGLKLTEVSPRGINAIFERLRNILHESEIDKRVQYMIEVMFAIRKDGFKDHPIIPEGLDLVEEEDQFTHMLPLEDEYNTEDILNVFKLDPNFLENEEKYKTIKREILDEGSSDSGDDAGGSGDDEDDDEEDEEAAAGEEQEKVTIFDQTEVNLVAFRRTIYLAIQSSLDFEECAHKLIKMDFPESQTKELCNMILDCCAQQRTYEKFFGLLAGRFCLLKKEYMESFEAIFQEQYETIHRLETNKLRNVARIFAHLLYTDSVPWSVLECVRMSEDTTTSSSRIFVKILFQELCAYMGLPKLNERLKDTTLQPFFEGLFPRDNPRNTRFAINFFTSIGLGGLTDELREHLKNAPKMIMTQNQEVESSDSSSSSSSSSDSSSSSGSSSESDSSESDSDSSSDSDSSSSSGSSSDSDSRRKKASGKKKDKSKKSSKAANQRSPLEERPTKRHENRRQDASKEDRRGSDKHNRDPQRRGQQDESPPARPRGEPERIRGQKEPHRHAQDHQDRPTDSGRHKDDGKNSRANKDKDRRRSREKEPLRRSRDRSKSRERSRKEMDSRDSYGNGLERADKENRHSDRYKESRRKDDRRHR

>Gg_CWC22 [Gallus gallus] MKSRVTQINHGSSHERKERYSPRHRSLTPEDRDVERDRSRSPRRRRYSDDSRYDQEYSRREYYDDRSSEGRRMERGRDRYYEKWEERDYDRRRKRRLSSPDHRSPERSTVQGSNTHEEATSKKKKEEVDPILTRTGGAYIPPAKLRMMQEQITDKNSLAYQRMSWEALKKSINGLVNKVNVSNIENIIYELLQENIVRGRGLLSRSILQAQGASPIFTHVYAALVAIINSKFPNIGELILKRLILNFRKGYRRNDKQLCLTSSKFVAHLMNQNVAHEVLCLEMLTLLLERPTDDSIEVAIGFLKESGLKLTEVSPRGINAIFDRLRHILHESKIDMRVQYMIEVMFAVRKDGFKDHPIIPEGLDLVEEEDQFTHMLPLEDEYNPEDVLNVFKMDPNFMENEEKYKALKKEILDEGDSESEPDQEAGSSDEEEEEDEEEDEDGQKVTVHDKTEINLVSFRRTIYLAIQSSLDFEECAHKLLKMDFPESQTKELCNMILDCCAQQRTYEKFFGLLAGRFCMLKKEYMESFEAIFKEQYDTIHRLETNKLRNVAKMFAHLLYTDSIPWSVLECIILSEETTTSSSRIFVKIFFQELSEYMGLPNLNARLKDITLQPFFEGLLPRDNPRNTRFAINFFTSIGLGGLTDELREHLKNAPKMIMTQKQDVESSDSSSSSETDSSSDSDSDSSSSSSESSSSSDSSSSSSDSSDSDASKAKKRRTQKKNRESDKASRKKQEKKRKSLEKKTGRRRQEDRSDSESKSERNHRNLREAHRRDDTSKYHHRDESNGRDDYEAYRRDDVSKYHQRDESNGRDNYHSGRDRDHERSKDLENKHSNSKLKKAERRASFSDDESYRHGSRDNGHRSRKRERSKSGERSYNKSSPREEEDDHRYRNGSERLREKYNHYSDQYRESRKHEDRRREYSPHRRK

>Hs_CWC22 [Homo sapiens] MKSSVAQIKPSSGHDRRENLNSYQRNSSPEDRYEEQERSPRDRDYFDYSRSDYEHSRRGRSYDSSMESRNRDREKRRERERDTDRKRSRKSPSPGRRNPETSVTQSSSAQDEPATKKKKDELDPLLTRTGGAYIPPAKLRMMQEQITDKNSLAYQRMSWEALKKSINGLINKVNISNISIIIQELLQENIVRGRGLLSRSVLQAQSASPIFTHVYAALVAIINSKFPQIGELILKRLILNFRKGYRRNDKQLCLTASKFVAHLINQNVAHEVLCLEMLTLLLERPTDDSVEVAIGFLKECGLKLTQVSPRGINAIFERLRNILHESEIDKRVQYMIEVMFAVRKDGFKDHPIILEGLDLVEEDDQFTHMLPLEDDYNPEDVLNVFKMDPNFMENEEKYKAIKKEILDEGDTDSNTDQDAGSSEEDEEEEEEEGEEDEEGQKVTIHDKTEINLVSFRRTIYLAIQSSLDFEECAHKLLKMEFPESQTKELCNMILDCCAQQRTYEKFFGLLAGRFCMLKKEYMESFEGIFKEQYDTIHRLETNKLRNVAKMFAHLLYTDSLPWSVLECIKLSEETTTSSSRIFVKIFFQELCEYMGLPKLNARLKDETLQPFFEGLLPRDNPRNTRFAINFFTSIGLGGLTDELREHLKNTPKVIVAQKPDVEQNKSSPSSSSSASSSSESDSSDSDSDSSDSSSESSSEESDSSSISSHSSASANDVRKKGHGKTRSKEVDKLIRNQQTNDRKQKERRQEHGHQETRTERERRSEKHRDQNSSGSNWRDPITKYTSDKDVPSERNNYSRVANDRDQEMHIDLENKHGDPKKKRGERRNSFSENEKHTHRIKDSENFRRKDRSKSKEMNRKHSGSRSDEDRYQNGAERRWEKSSRYSEQSRESKKNQDRRREKSPAKQK

>Md_NCM [Musca domestica] MTGSQSENNTSTSSNSSEDTNNDSRNESETNADKNVVETKTTSDNNNKMMNAAETAANEEKNGRQKKEKSKTPSKEDKKSRKKKSESSSESSSSSDSDSSESSSSSSDGEVSTSSGSSSDSEKVKSKVKNKSKSPSAQREVVQKETVTDTPDNISKETQEKLVEDIPKENELNVNLAKEDSVNAKQNDTEASNENVAEEITKPPQKQPDTEEGEITENNEKNSSARSPSKEKQLSANRSRSRERRSHSGDSRGAHSGDKGSPSRLKRSPSRSKKSPSRSKRSVSRNRSSSRHGRDSSRNRRSVSGDKENQLRRKSRSRSSRSRSRSRSRSRSRRPERKHRSRTPRRSRSRERRHERRRSVSSDYDARRRSRRSESIERRREERKRRHAERDEREKSKRSRRDEDDSFKTNKVSAEMEKDKENDNATVTDPKAKITERQRKTVDILTSRTGGAYIPPAKLRMMQAEITDKASAAYQRIAWEALKKSIHGYINKVNVDNIAIITRELLKENIVRGRGLLCRSIIQAQAASPTFTHVYAALVAIINSKFPNIGELLLKRLVIQFKRAFRRNDKTVCLSSSRFIAHLVNQRVAHEILALEILTLLVESPTDDSVEVAIAFLKECGMKLTEVSSKGIGAIFEMLKNILHEGKLDKRVQYMIEVVFQVRKDGFKDHQSVIESLELVEEDDQFTHLLMLDDATSPEDVLNAFKFDEQYEANEEKYKGLSKEILGSDASDSDGSSGSGSDSDSESSDSDEDKNEGDEKPTAGDIIDNTETNLIALRRTIYLTINSSLDYEECAHKLMKMQLKPGQEIELCHMFLDCCAEQRTYEKFYGLLAQRFCNINKSYIEPFEEIFKDTYQTTHRLDTNRLRNVSKFFAHLLFTDAISWDVLDCIKLNEDDTTSSSRIFIKILFQELAEYMGLGQLNKKLKDEVLAESLAGLFPKDNPRNTRFSINFFTSIGLGGLTDELRQFLKNAPKSVPAINAEILANKPVDVSNSSSSSSSSSSSTSSSSSSSSSSSESSASSSSSDSSSDSSSDSEVDKKKRKGKVKKTKKKKTSNKKEKTKKSKSKNKKDAKKKAKKRKSKKGSTSSEDDSSKSESSSSESKSSDSSDSEQSGNDKSKKKTKSKSKTKPNKKSKRSDSEDVENERDIKRKKREDFGNYIHEDRRRDEEYSKNGGRDRKKDNHGNNKEKREIPQRRDSEIERRREEREKRHRERERNFSRSRSRSRGGSGRKDRDRRETNGRSERKDRERDRDSDHRRYRKDYDRSRSRDRNEKREREYSRSKLRNTSKERG

>Mdmd II MNATDAESRKPENKPSSESSSSGSTSGSSDGEVSSKTYFKNNKSKVLSGQREVVLEVVRDLSYTICKEAEEKLVERFPRKDGSNEMLPKEDSINTNHNYTTDSNEHPVELTTKTEECKNTEKTKKKSFVRALSKDKQLSAYRSRSRSTRLSYSGHISRTHSVEKSLSRYKTSVLRNRRTSFGHGRDSSTTKRSVSRDKDNRLRRRIGSSRSHTRSHSRFRRSEKKLPSRSPRRIRSQERRHERRRSMSSDYERIALRRSEPIKRRDKDEFFKNNKKVSGDIKKGKGNDNGTVAELEAKITERQRKSLDILTSRTGGACLTPDKLRMIQAEITDKSSAAYQSIAREALKKYIHGYINKVNVDSVAVITRKLLKDNIVRGRGVLCHSIIQAQATSPTFTHVYAAMVAIINSKFPNIGELLLKRLVIQFKRAFGCNDKTVCLTSSHFIAHLVNQRVAHEILALEILTLLIESPTDDNVEVAITFLKECGMKLTEVSSDRVGGIFELLKNILHQGKLDKRVQYMIKVLFQVRRDGFKDHQSIIESLELVEEYAQFTHLLLLEDVTYPKDILNEFRFDDQYETNEEKYKALSKNILGSHASDSDGSFGSGSNSETALSDCDKGKNEVNDKYTSGDIIDETKPNLIALRRTIYLTLNSCLDYEECAQKLMKMQLKTCQQNEFCQILLDCCAEQRTYEKFYGLLTHRICKMNKSFIEPFKEIFKDICQTTHCLDTNRLRNISKFFAHLLFTDAISWDVLDCIKLTEDEAITSRCIFIKSFFQELVEYMGLYHFNKKLKTEVLAGTLAGLFPKDNPRNIRFSINFFTSIGLGGITNELCQLLKIAPKSAPSSSSSSSLSSELSAPSDDDSSSDSENKKKHKGKNKKMTKKKNPSKKKEKTKKIVGKNKIAAKNKTIKRRTDKDNSSSKDNFLKSESSSNESISLDSLSSELFAPSSYSSSESSNDSESKEKHKGKNKKMTKKKNPSNKREKTKKKLSKNKKAPNKNTKKRMTEKDISSSESSISESKSLNCSASNQNENEKRKKRVTSKSRTKRVKMFKQCQWVDADNQRDIKRKKRAEYRYEPLVYRKRNEECLKKGAPNCRKDNYGNRQNHEISQRHDSEIKRRREERKKRHHEKNHSREYKRSKLGLCQREYFLYMCCQFYYPCTFQCLCQNCHFTFYS

>Mdmd III MNATDAESRKPENKPSSESSSSGSTSGSSDGEVSSKTYFKNNKSKVLSGQREVVLEVVRDLSYTICKEAEEKLVERFPRKDGSNEMLPKEDSINTNHNYTTDSNEHPVELTTKTEECKNTEKTKKKSFVRALSKDKQLSAYRSRSRSTRLSYSGHISRTHSVEKSLSRYKKSVLRNRRTSFGHGRDSSTTKRSVSRDKDNRLRRRIGSSRSHTRSHSRFRRSEKKLPSRSPRRIRSQERRHERRRSMSSDYERIALRRSEPIKRRDKDEFFKNNKKVSGDIKKGKGNDNGTVAELEAKITERQRKSLDILTSRTGGACLTPDKLRMIQAEITDKSSAAYQSIAREALKKYIHGYINKVNVDSVAV

ITRKLLKDNIVRGRGVLCHSIIQAQATSPTFTHVYAAMVAIINSKFPNIGELLLKRLVIQFKRAFGCNDKTVCLTSSHFIAHLVNQRVAHEILALEILTLLIESPTDDNVEVAITFLKECGMKLTEVSSDRVGGIFELLKNILHQGKLDKRVQYMIKVLFQVRRDGFKDHQSIIESLELVEEYAQFTHLLLLEDVTYPKDILNEFKFDDQYETNEEKYKALSKDILGSHASDSDGSFGSGSNSETALSDCDKVKNEVNDKYTSGDIIDETKPNLIALRRTIYLTLNSCLDYEECAQKLMKMQLKTCQQNEFCQILLDCCAEQRTYEKFYGLLTHRICKMNKSFIEPFKEIFKDICQTTHCLDTNRLRNISKFFAHLLFTDAISWDVLDCIKLTEDEAITSRCIFIKSFFQELVEYMGLYHFNKKLKTEVLAGTLAGLFPKDNPRNIRFSINFFTSIGLGGITNELCQLLKIAPKSAPSSSSSSSLSSELSAPSDDDSSSDSENKKKHKGKNKKMTKKKNPSKKKEKTKKFVGKNKIAAKNKTIKRRTDKDNSSSKDNFLKSESSSNESISLDSLSSELFAPSSYSSSESSNDSESKEKHKGKNKKMTKKKNPSNKKEKTKKKLSKNKKAPNKNTKKRMTEKDISSSESSISESKSLNCSASNQNENEKRKKRVTSKSRTKRVKMFKQCQWVDADNQRDIKRKKRAEYRYEPLVYRKRNEECLKKGAPNCRKDNYGNRQNHEISQRHDSEIKRRREERKKRHHEKNHSREYKRSKLGLCQREYFLYMCCQFYYPCTFQCLCQNCHFTFYS

>Mdmd V MNATDAESRKPENKPSSESSSSGSTSGSSDGEVSSKTYFKNNKSKVLSGQREVVLEVVRDLSYTICKEAEEKLVERFPRKDGSNEMLPKEDSINTNHNYTTDSNEHPVELTTKTEECKNTEKTKKKSFVRALSKDKQLSAYRSRSRSTRLSYSGHISRTHSVEKSLSRYKKSVLRSRRTSFGHGRDSSTTKRSVSRDKDNRLRRRIGSSRSHTRSHSRFRRSEKKLPSRSPRRIRSQERRHERRRSMSSDYERIALRRSEPIKRRDKDEFFKNNKKVSGDIKKGKGNDNGTVAELEAKITERQRKSLDILTSRTGGACLTPDKLRMIQAEITDKSSAAYQSIAREALKKYIHGYINKVNVDSVAVITRKLLKDNIVRGRGVLCHSIIQAQATSPTFTHVYAAMVAIINSKFPNIGELLLKRLVIQFKRAFGCNDKTVCLTSSHFIAHLVNQRVAHEILALEILTLLIESPTDDNVEVAITFLKECGMKLTEVSSDRVGGIFELLKNILHQGKLDKRVQYMIKVLFQVRRDGFKDHQSIIESLELVEEYAQFTHLLLLEDVTYPKDILNEFKFDDQYETNEEKYKALSKNILGSHASDSDGSFGSGSNSETALSDCDKGKNEVNDKYTSGDIIDETKPNLIALRRTIYLTLNSCLDYEECAQKLMKMQLKTCQQNEFCQILLDCCAEQRTYEKFYGLLTHRICKMNKSFIEPFKEIFKDICQTTHCLDTNRLRNISKFFAHLLFTDAISWDVLDCIKLTEDEAITSRCIFIKSFFQELVEYMGLYHFNKKLKTEVLAGTLAGLFPKDNPRNIRFSINFFTSIGLGGITNELCQLLKIAPKSAPSSSSSSSLSSELSAPSDDDSSSDSENKKKHKGKNKKMTKKKNPSKKKEKTKKIVGKNKIAAKNKTIKRRTDKDNSSSKDNFLKSESSSNESISLDSLSSELFAPSSYSSSESSNDSESKEKHKGKNKKMTKKKNPSNKREKTKKKLSKNKKAPNKNTKKRMTEKDISSSESSISESKSLNCSASNQNENEKRKKRVTSKSRTKRVKMFKQCQWVDADNQRDIKRKKRAEYRYEPLVYRKRNEECLKKGAPNCRKDNYGNRQNHEISQRHDSEIKRRREERKKRHHEKNHSREYKRSKLGLCQREYFLYMCCQFYYPCTFQCLCQNCHFTFYS

>Mdmd Y MNATDAESRKPENKPSSESSSSGSTSGSSDGEVSSKTYFKNNKSKVLSGQREVVLEVVRDLSYTICKEAEEKLVERFPRKDGSNEMLPKEDSINTNHNYTTDSDEHPVELTTKTEECKNTEKTKKKSFVRALSKDKQLSAYRSRSRSTRLSYSGHISRTHSVEKSLSRYKKSVLRNRRTSFGHGRDSSTTKRSVSRDKDNRLRRRIGSSRSHTRSHSRFRRSEKKLPSRSPRRIRSQERRHERRRSMSSDYERIALRRSEPIKRRDKDEFFKNNKKVSGDIKKGKGNDNGTVAELEAKITERQRKSLDILTSRTGGACLTPDKLRMIQAEITDKSSAAYQSIAREALKKYIHGYINKVNVDSVAVITRKLLKDNIVRGRGVLCHSIIQAQATSPKFTHVYAAMVAIINSKFPNIGELLLKRLVIQFKRAFGCNDKTVCLTSSHFIAHLVNQRVAHEILALEILTLLIESPTDDNVEVAITFLKECGMKLTEVSSDRVGGIFELLKNILHQGKLDKRVQYMIKVLFQVRRDGFKDHQSIIESLELVEEYAQFTHLLLLEDVTYPKDILNEFKFDDQYETNEEKYKALSKNILGSHASDSDGSFGSGSNSETALSDCDKGKNEVNDKYTSGDIIDETKPNLIALRRTIYLTLNSCLDYEECAQKLMKMQLKTCQQNEFCQILLDCCAEQRTYEKFYGLLTHRICKMNKSFIEPFKEIFKDICQTTHCLDTNRLRNISKFFAHLLFTDAISWDVLDCIKLTEDEAITSRCIFIKSFFQELVEYMGLYHFNKKLKTEVLAGTLAGLFPKDNPRNIRFSINFFTSIGLGGITNELCQLLKIAPKSAPSSSSSSSLSSELSAPSDDDSSSDSENKKKHKGKNKKMTKKKNPSKKKEETKKIVGKNKIAAKNKTIKRRTDKDNSSSKDNFLKSESSSNESISLDSLSSELFAPSSYSSSESSNDSESKEKHKGKNKKMTKKKNPSNKREKTKKKLSKNKKAPNKNTKKRMTEKDISSSESSISESKSLNCSASNQNENEKRKKRVTSKSRTKRVKMFKQCQWVDADNQRDIKRKKRAEYRYEPLVYRKRNEEYLKKGGPNCRKDNYGNRQNHEISQRHDSEIKRRREERKKRHHEKNHSREYKRSKLGPCQREYFLYMCCQFYYPCTFQCLCQNCHFTFYS

>Mm_CWC22 isoform X4 [Mus musculus] MKSSVAHMKQSSGHNRRETHSSYRRSSSPEDRYTEQERSPRDRGYSDYSRSDYERSRRGYSYDDSMESRSRDREKRRERERDADHRKRSRKSPSPDRSPARGGGQSSPQEEPTWKKKKDELDPLLTRTGGAYIPPAKLRMMQEQITDKSSLAYQRMSWEALKKSINGLINKVNISNISIIIQELLQENIVRGRGLLSRSVLQAQSASPIFTHVYAALVAIINSKFPQIGELILKRLILNFRKGYRRNDKQLCLTASKFVAHLINQNVAHEVLCLEMLTLLLERPTDDSVEVAIGFLKECGLKLTQVSPRGINAIFERLRNILHESEIDKRVQYMIEVMFAVRKDGFKDHPVILEGLDLVEEDDQFTHMLPLEDDYNPEDVLNVFKMDPNFMENEEKYKAIKKEILDEGDSDSNTDQGAGSSEDEEEEDEEEEGEDEEGGQKVTIHDKTEINLVSFRRTIYLAIQSSLDFEECAHKLLKMEFAESQTKELCNMILDCCAQQRTYEKFFGLLAGRFCMLKKEYMESFESIFKEQYDTIHRLETNKLRNVAKMFAHLLYTDSLPWSVLECIKLSEETTTSSSRIFVK

IFFQELCEYMGLPKLNARLKDETLQPFFEGLLPRDNPRNTRFAINFFTSIGLGGLTDELREHLKNTPKVIVAQKPEAEQKKPALTSSSSESSSASDSSDSESDSSESSSESSSDASDSSSSSSTQSSTSGITAHSAKGTRKKRQGKARGEEVDKLARGHQALERRREGGREDQRHQEGRTERARSERRRAQNSRDADWRDPLAKHIDDRSHENSHSRVGNGREQGSHREPEDRHGEPKKRRERRDSFSENEKQRSRNQDSDNVRRKDRSKSRERSRRHSGHKGDDARCQNSAERRWEKPGRRPEQSRESKRSQDRRREKSPTTQK

>Nv_NCM [Nasonia vitripennis] MDSEEIKLRRKSSSSDNDDVKSKRDKKSKVKRDRSRDKDRSKSRSRNRERSRSKNRDRSKSKNRERSRSKNRDRSKSKNRERSRSRNRERSKSRNRDRSRSRNRERSRSRHRERSRSKNRDRSIGRYRDRSRSRDRKRTRSRNRNQSRSRDYKRSRDDDKSHKYESYWSKEKELEKSESRHGEVSSSYYGTELKKSDSSNGQSIKRNNDKDEEKEKVEEKPENPQVIPARQKKTVDLLTSKTGGAYIPPARLRLMQAEISDKSGPAYQRIAWEALKKSIHGFINKVNTSNIGPITRDLLKENIVRGRGLLARSIIQAQAASLTFTNVYAALVAVINSQFPNIGELLLTRLALQFRRAYKRNDKTMCISSAIFIAHLVNQGVAHEILILEILTLLVHNPTDDSIEVAIAVLKECGMKLTQVCRKGIEAIFEMLRNILHEGNLDKRVQYMIEVMFQIRKDGFKDFESVPKDLDLVEEEDQLTHLIELDQPLDDQNILNAFKFDPEYTANEDKYKELTKVILNSDESGSDDEEGSDDGDDDEDSDKDSESDKVADNGVIVDNTETNLVALRRTIYLTIHSSLDFEECAHKLMKMQLKPGQEIELCNMFLDCCAEMRTYEKFFGLLAGRFCAINKIYVQPFETIFKDSYDTIHRLDTNRLRNVAKFFAHLLMTDSISWAVLSNIKLNEEDTTSSSRVFIKILFQELSEYMGLPKLNKKLKDEDLQESLNGLFPKDEAKNTRFAINFFTSIGLGGLTDDLREHLKNRPKPVATEPILSIKKEKSSSSAGTSSSSSSSSSSSSSSSSSSSTESSSSSDSSEDERRRRKKKKKKLVNKKKTVKRREEKDKMEKKEKKEKKDKKEKKDKKEKKEKKTQ

>Sc_CWC22 [Saccharomyces cerevisiae YJM627] MSTATIQDEDIKFQRENWEMIRSYVSPIISNLTMDNLQESHRDLFQVNILIGRNIICKNVVDFTLNKQNGRLIPALSALIALLNSDIPDIGETLAKELMLMFVQQFNRKDYVSCGNILQCLSILFLYDVIHEIVILQILLLLLEKNSLRLVIAVMKICGWKLALVSKKTHDMIWEKLRYILQTQELSSTLRESLETLFEIRQKDYKSGSQGLFILDPTSYTVHTHSYIVSDEDEANKELGNFEKCENFNELTMAFDTLRQKLLINNTSDTNEGSNSQLQIYDMTSTNDVEFKKKIYLVLKSSLSGDEAAHKLLKLKIANNLKKSVVDIIIKSSLQESTFSKFYSILSERMITFHRSWQTAYNETFEQNYTQDIEDYETDQLRILGKFWGHLISYEFLPMDCLKIIKLTEEESCPQGRIFIKFLFQELVNELGLDELQLRLNPSKLDGMFPLEGDAEHIRYSINFFTAIGLGLLTEDMRSRLTIIQEVEDAEEEEKKLREEEELEKLRKKARESQPTQGPKIHESRLFLQKDTRENSRSRSPFTVETRKRARSRTPPRGSRNHRNRSRTPPRRPKNHRNRSRTPPARRQRHR

>Sp_CWF22 [Schizosaccharomyces pombe] MEKEDKSFGIGMLDYNRENPESSGHSRVAVIRKQKTEQENNNLSWEDRHYIPDLHKSNKIKTPTSLADEKKSHNELDPKAQIKKLMETRSGGTYIPPAKLKALQAQLTDVNTPEYQRMQWEALKKSINGLINKVNKSNIRDIIPELFQENIIRGRALYCRSIMKAQAASLPFTPIYAAMTAVINTKFPQIGELLLTRLIVQFRKSFQRNDKSMCISSSSFIAHLINQKIAHEIVGLQILAVLLERPTNDSIEIAVMLLREIGAYLAEVSTRAYNGVFERFRTILHEGQLERRTQFIIEVLFQTRKDKFKNNPTIPQELDLVEEEDQITHYISLDDNLDVQESLGIFHYDPDYEENEKKYDAIKHEILGEEDDDENEEDEEDSEETSESEEDESVNDEKPQVIDQTNASLVNLRKSIYLTIMSSVDFEECCHKLLKIDLPEGQEIELCNMVIECNSQERTYAKFYGLIGERFCKLSRTWRSTYEQCFKNYYETIHRYETNRLRNIALFFANLLSTDSIGWEVYDCVRLTEDDTTASSRIFLKIMFQEIVEALGLKSLVERLHDPNLVPYLHGLFPVDEARNVRFSINYFTSIGLGALTEEMREYLLTMPKSEPKEQDSEGYSSGSETGSTYSSSYSSTYSRGRSYSRSTRSYSKSRSYSRSRSVTPINNINHKKYIRKDRELSPRGRERSSNRNSYSDLSRSSSLSRGRSRSYTPEGRLIESEDKGYRSRSSSPASRKYRSRQRYRRSYAGSTSRGRSFSRSPSYRRRLSMSCSVSYSRSPSPARPISRSPARNKRSYDSLSYNRQYSPKVSRKRSNESRSPSPYTLRKQKTYHLNKRNEDFVVKMDKKGSESPVILAPWLREVDDGELYKDRNSESDSVRKQPRAD

>Stc_NCM [Stomoxys calcitrans] MPGNSEGKSVSTRSEANTGDQIVVSGKTGDEEMPTNQEEKNSGVERREDCKKSKKEKEKNISKSKNTIEEKEDSGSESSSSSSDSASSSSSGSSDGEVSSSSESSSDSDEADAKDEAQNSPKDGQLPERKKNENEIKNHRLAKDSKENVEKNVASKEDNEHKEQNGNEQQINNQEDVVNKSPVGSKIKETEEGEIEEEDKQQGSNEKITQTNVEINTKRSVSKSRSPSGEKELHRDQKNQSLGKSSRSSSKSPKERNSKQAIPSDSRHQSPQKQRERKQRSKSISRSRSYSRGRSRSRSRSRQTERKRRSRSSRRSHSGEKRSNKRDERRRSPSSDYEKRSSARSASLEKRREERKRRHAERDEREKTDRYKQNTDDSSKANNGNTNKSLKDTENDNSTVTDPKAKITERQRKTVDILTSRTGGAYIPPAKLRMMQAEITDKSSEGYQRIAWEALKKSIHGYINKVNVDNIAIITRELLKENIVRGRGLLCRSIIQAQAASPTFTHVYAALVAIINSKFPNIGELLLKRLVIQFKRAFRRNDKAVCLSSSRFIAHLVNQRVAHEILALEILTLLVESPTDDSVEVAIAFLKECGMKLTEVSSKGIGAIFEMLKNILHEGKLDKRVQYMIEVVFQVRKDGFKDHQSVIESLELVEEDDQFTHLLMLDDATTPEDVLNAFKFDEQYEENEEKYKGLSKEILGSDASDSGDSDDSGSGSDSDSSSGSEDNDKNEDKPTAGDIIDNTETNLIALRRTIYLTINSSLDYEECAHKLMKMQLKPGQEIELCHMFLDCCAEQRTYEKFYGLLAQRFCAINKSYIEPFEEIFKDTYQTTHRLDTNRLRNVSKFFAHLLFTDAISWDVFDCIRLNEDDTTSSSRIFIKILFQELAEYMGLSQLNKKLKDEVLAESLSGLFPKDNPRNTRFSINFFTSIGLGGLTDELRQFLKNAPKSV

PAINAEILANKPVNCSNSSSSSSSSSSSSSSSSSSSTSESSDSSSESSSDSEAPKKKRKGKKQKSKKLSTKNKEKAKKSKRGNKENTKNAKKSKKSKKVETSSSSSASSSDTEKSASSSCESASEKKHSKRKNMKSPPKEKNSKKHQRSEADEDSDHQYKPKKRVHREETESLMEKKRSEYVDNKQRGDREPEKRTVPQQRRNSEIERRREEREKRHRERENKRSRSPSPARRTSREERDRRGDDRREKGRDLDRRRIRDYVDRDRYKGRRSRSYDRSEKREREYSRSKLRNTSKERG

>Tc_NCM [Tribolium castaneum] MTTDTETSPQPKPKRKSRSKSPESRKKRRDDSPHKDSRKRSRRSRSRDRRKSRKYHDLDRPLEEYPRYYGEDQEHKKNSDRYWTKYPRKDDHQVGSRYYGGEPDDKDKESEKETDKSVIAPRERKTVDLLTRTGGAYIPPAKLRMMQAEITDKSSAAYQRIAWEALKKSIHGFINKVNTSNIGIISRELLHENVVRGRGLLCKSIIQAQAASPTFTNVYAALVAVINSKFPNIGELLLKRLVLQFKRGFKQNNKIICISATTFVAHLVNQRVAHEILALEILTLLVETPTDDSVEVAISFLKECGQKLTEVSSKGINAIFEMLRNILHEGKLEPRIQYMIEVMFQIRKDGFKDHAAVIEELDLVDEEHQFTHLITLDDVKQSNAEDILNVFKFDDNYEENEGKYKTLSKEILDMDSESGSGSEEESGDEEDDEDEDEEEEEGAQKNESNIIDNTETNLVALRRTIYLTIQSSLDFEESAHKLMKMELKPGQEIELCHMFLDCCAEQRTYEKFYGLLAQRFCQVNQIYVEPFQQIFKDTYATTHRLDTNKLRNVSKFFAHLLFTDAIGWEVLEIMKLNEEDTNSSNRIFIKILFQELAEYMGLEKLNARLKDATLQPHFEGLFPRDDPKNTRFAINFFTSIGLGGLTAELREHLKTAPKVNPLTITEKSSDSSSSSTSSSSSSSSDSSDSDSSSSEDEKPRKKKEEKKRKKPTKEASDDDKKRKTDARKHRHNERRSRDRDDRRRRRSPERKHKSRR

>Xl_CWC22 [Xenopus laevis] MKSSVAQVRGSNYDKVDTRSSSERSSSPEDSNEERSPSPRDRGYSNRSRDYSDRDRYEDRSRSGRYDRSDESRRRERERSTSPRDRGYTDRRRGYSDRDGYGNDRSRNGRYDRSEDNREREKRQNYQDRDYEKRSPPARRRSPPARRSEEQTEEQNQTEPPVKKKKEELDPILTRTGGAYIPPARLRMMQEQITDKSSMAYQRMSWEALKKSINGLVNKVNVSNIGNIIQELLQENIVRGRGLLARSVLQAQSASPIFTHVYAALVSIINSKFPHIGELILKRLILNFRKGYRRNDKQLCLTSSKFVAHLINQNVAHEVLALEMLTLLLERPNDDSVEVAIGFLKESGLKLTQVTPRGINAIFERLRNILHESEIDKRVQYMIEVMFAVRKDGFKDHPVIPEGLDLVEEEDQFTHMLPLEDDYNQEDVLNVFKMDPDFLENEEKYKAIKKEILDEGDSDSEGDANEGSEDESEEEEEDGQEAGTEGEKMTIHDKTEVNLVAFRRTIYLAIQSSLDFEECAHKLIKMDFPESQTKELCNMILDCCAQQRTYEKFFGLLAGRFCLLKKEYLEAFENIFKEQFETIHRLETNKLRNVAKMFAHLLYTDSLPWSVLECMNLSEETTTSSSRIFVKIFFQELCEYMGLPKLNARLKDVTLQPFFQGLLPMDNPKNTRFAINFFTSIGLGGLTDELREHLKNAPKMIMTQKQNVESSDSSSDSSSGSESSSDSDSSSSSSSESSSSSSDSDSSRRKKHSQKKKKSREAHKAASKKQAPDDRRKEAPKHKHQKEKYDDKQSRKSKRDSKN

References and Notes 1. J. B. Pease, M. W. Hahn, Sex chromosomes evolved from independent ancestral linkage

groups in winged insects. Mol. Biol. Evol. 29, 1645–1653 (2012). doi:10.1093/molbev/mss010 Medline

2. D. Bachtrog, J. E. Mank, C. L. Peichel, M. Kirkpatrick, S. P. Otto, T. L. Ashman, M. W. Hahn, J. Kitano, I. Mayrose, R. Ming, N. Perrin, L. Ross, N. Valenzuela, J. C. Vamosi; Tree of Sex Consortium, Sex determination: Why so many ways of doing it? PLOS Biol. 12, e1001899 (2014). doi:10.1371/journal.pbio.1001899 Medline

3. B. Vicoso, D. Bachtrog, Numerous transitions of sex chromosomes in Diptera. PLOS Biol. 13, e1002078 (2015). doi:10.1371/journal.pbio.1002078 Medline

4. J. W. Erickson, J. J. Quintero, Indirect effects of ploidy suggest X chromosome dose, not the X:A ratio, signals sex in Drosophila. PLOS Biol. 5, e332 (2007). doi:10.1371/journal.pbio.0050332 Medline

5. L. Sánchez, Sex-determining mechanisms in insects. Int. J. Dev. Biol. 52, 837–856 (2008). doi:10.1387/ijdb.072396ls Medline

6. E. C. Verhulst, L. van de Zande, L. W. Beukeboom, Insect sex determination: It all evolves around transformer. Curr. Opin. Genet. Dev. 20, 376–383 (2010). doi:10.1016/j.gde.2010.05.001 Medline

7. D. Bopp, G. Saccone, M. Beye, Sex determination in insects: Variations on a common theme. Sex Dev. 8, 20–28 (2014). doi:10.1159/000356458 Medline

8. M. G. Franco, P. G. Rubini, M. Vecchi, Sex-determinants and their distribution in various populations of Musca domestica L. of Western Europe. Genet. Res. 40, 279–293 (1982). doi:10.1017/S0016672300019157 Medline

9. A. Dübendorfer, M. Hediger, G. Burghardt, D. Bopp, Musca domestica, a window on the evolution of sex-determining mechanisms in insects. Int. J. Dev. Biol. 46, 75–79 (2002). Medline

10. R. L. Hamm, R. P. Meisel, J. G. Scott, The evolving puzzle of autosomal versus Y-linked male determination in Musca domestica. G3 5, 371–384 (2015).

11. M. Hediger, C. Henggeler, N. Meier, R. Perez, G. Saccone, D. Bopp, Molecular characterization of the key switch F provides a basis for understanding the rapid divergence of the sex-determining pathway in the housefly. Genetics 184, 155–170 (2010). doi:10.1534/genetics.109.109249 Medline

12. T. Hiroyoshi, Sex-limited inheritance and abnormal sex ratio in strains of the housefly. Genetics 50, 373–385 (1964). Medline

13. I. Denholm, M. G. Franco, P. G. Rubini, M. Vecchi, Identification of a male determinant on the X chromosome of the housefly (Musca domestica L.) population in South-East England. Genet. Res. 42, 311–322 (1983). doi:10.1017/S0016672300021790

14. M. Kozielska, B. Feldmeyer, I. Pen, F. J. Weissing, L. W. Beukeboom, Are autosomal sex-determining factors of the housefly (Musca domestica) spreading north? Genet. Res. 90, 157–165 (2008). doi:10.1017/S001667230700907X Medline

15. J. G. Scott, W. C. Warren, L. W. Beukeboom, D. Bopp, A. G. Clark, S. D. Giers, M. Hediger, A. K. Jones, S. Kasai, C. A. Leichter, M. Li, R. P. Meisel, P. Minx, T. D. Murphy, D. R. Nelson, W. R. Reid, F. D. Rinkevich, H. M. Robertson, T. B. Sackton, D. B. Sattelle, F. Thibaud-Nissen, C. Tomlinson, L. van de Zande, K. K. Walden, R. K. Wilson, N. Liu, Genome of the house fly, Musca domestica L., a global vector of diseases with adaptations to a septic environment. Genome Biol. 15, 466 (2014). doi:10.1186/s13059-014-0466-3 Medline

16. A. L. Steckelberg, V. Boehm, A. M. Gromadzka, N. H. Gehring, CWC22 connects pre-mRNA splicing and exon junction complex assembly. Cell Rep. 2, 454–461 (2012). doi:10.1016/j.celrep.2012.08.017 Medline

17. A. L. Steckelberg, J. Altmueller, C. Dieterich, N. H. Gehring, CWC22-dependent pre-mRNA splicing and eIF4A3 binding enables global deposition of exon junction complexes. Nucleic Acids Res. 43, 4687–4700 (2015). doi:10.1093/nar/gkv320 Medline

18. J. Saulière, N. Haque, S. Harms, I. Barbosa, M. Blanchette, H. Le Hir, The exon junction complex differentially marks spliced junctions. Nat. Struct. Mol. Biol. 17, 1269–1271 (2010). doi:10.1038/nsmb.1890 Medline

19. Z. Wang, V. Murigneux, H. Le Hir, Transcriptome-wide modulation of splicing by the exon junction complex. Genome Biol. 15, 551 (2014). doi:10.1186/s13059-014-0551-7 Medline

20. A. B. Hall, S. Basu, X. Jiang, Y. Qi, V. A. Timoshevskiy, J. K. Biedler, M. V. Sharakhova, R. Elahi, M. A. Anderson, X. G. Chen, I. V. Sharakhov, Z. N. Adelman, Z. Tu, A male-determining factor in the mosquito Aedes aegypti. Science 348, 1268–1270 (2015). doi:10.1126/science.aaa2850 Medline

21. E. Krzywinska, N. J. Dennison, G. J. Lycett, J. Krzywinski, A maleness gene in the malaria mosquito Anopheles gambiae. Science 353, 67–69 (2016). doi:10.1126/science.aaf5605 Medline

22. R. Schmidt, M. Hediger, R. Nöthiger, A. Dübendorfer, The mutation masculinizer (man) defines a sex-determining gene with maternal and zygotic functions in Musca domestica L. Genetics 145, 173–183 (1997a). Medline

23. M. G. Grabherr, B. J. Haas, M. Yassour, J. Z. Levin, D. A. Thompson, I. Amit, X. Adiconis, L. Fan, R. Raychowdhury, Q. Zeng, Z. Chen, E. Mauceli, N. Hacohen, A. Gnirke, N. Rhind, F. di Palma, B. W. Birren, C. Nusbaum, K. Lindblad-Toh, N. Friedman, A. Regev, Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat. Biotechnol. 29, 644–652 (2011). doi:10.1038/nbt.1883 Medline

24. B. Li, C. N. Dewey, RSEM: Accurate transcript quantification from RNA-Seq data with or without a reference genome. BMC Bioinformatics 12, 323 (2011). doi:10.1186/1471-2105-12-323 Medline

25. M. D. Robinson, D. J. McCarthy, G. K. Smyth, edgeR: A Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139–140 (2010). doi:10.1093/bioinformatics/btp616 Medline

26. D. J. McCarthy, Y. Chen, G. K. Smyth, Differential expression analysis of multifactor RNA-Seq experiments with respect to biological variation. Nucleic Acids Res. 40, 4288–4297 (2012). doi:10.1093/nar/gks042 Medline

27. T. D. Wu, C. K. Watanabe, GMAP: A genomic mapping and alignment program for mRNA and EST sequences. Bioinformatics 21, 1859–1875 (2005). doi:10.1093/bioinformatics/bti310 Medline

28. M. Johnson, I. Zaretskaya, Y. Raytselis, Y. Merezhuk, S. McGinnis, T. L. Madden, NCBI BLAST: A better web interface. Nucleic Acids Res. 36, W5–W9 (2008). doi:10.1093/nar/gkn201 Medline

29. W. G. Kelly, S. Xu, M. K. Montgomery, A. Fire, Distinct requirements for somatic and germline expression of a generally expressed Caernorhabditis elegans gene. Genetics 146, 227–238 (1997). Medline

30. C. M. A. Coelho, B. Kolevski, C. D. Walker, I. Lavagi, T. Shaw, A. Ebert, S. J. Leevers, S. J. Marygold, A genetic screen for dominant modifiers of a small-wing phenotype in Drosophila melanogaster identifies proteins involved in splicing and translation. Genetics 171, 597–614 (2005). doi:10.1534/genetics.105.045021 Medline

31. R. C. Edgar, MUSCLE: Multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 32, 1792–1797 (2004). doi:10.1093/nar/gkh340 Medline

32. R. C. Edgar, MUSCLE: A multiple sequence alignment method with reduced time and space complexity. BMC Bioinformatics 5, 113 (2004). doi:10.1186/1471-2105-5-113 Medline

33. F. Ronquist, J. P. Huelsenbeck, MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19, 1572–1574 (2003). doi:10.1093/bioinformatics/btg180 Medline

34. M. Zhong, X. Wang, J. Wen, J. Cai, C. Wu, S. M. Aly, Selection of reference genes for quantitative gene expression studies in the house fly (Musca domestica L.) using reverse transcription quantitative real-time PCR. Acta Biochim. Biophys. Sin. (Shanghai) 45, 1069–1073 (2013). doi:10.1093/abbs/gmt111 Medline

35. C. Ramakers, J. M. Ruijter, R. H. L. Deprez, A. F. M. Moorman, Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data. Neurosci. Lett. 339, 62–66 (2003). doi:10.1016/S0304-3940(02)01423-4 Medline

36. A. Meccariello, S. Monti, A. Romanelli, R. Colonna, P. Primo, M. Grazia Inghilterra, G. Del Corsano, A. Ramaglia, G. Iazzetti, A. Chiarore, F. Patti, S. Dilara Heinze, M. Salvemini, H. Lindsay, E. Chiavacci, A. Burger, M. Robinson, C. Mosimann, D. Bopp, G. Saccone, Highly efficient DNA-free gene disruption in the agricultural pest Ceratitis capitata by CRISPR-Cas9 RNPs. BioRxiv 127506 [Preprint]. 18 April 2017. https://dx.doi.org/10.1101/127506.

37. A. Burger, H. Lindsay, A. Felker, C. Hess, C. Anders, E. Chiavacci, J. Zaugg, L. M. Weber, R. Catena, M. Jinek, M. D. Robinson, C. Mosimann, Maximizing mutagenesis with solubilized CRISPR-Cas9 ribonucleoprotein complexes. Development 143, 2025–2037 (2016). doi:10.1242/dev.134809 Medline

38. G. A. B. Marais, P. R. A. Campos, I. Gordo, Can intra-Y gene conversion oppose the degeneration of the human Y chromosome? A simulation study. Genome Biol. Evol. 2, 347–357 (2010). doi:10.1093/gbe/evq026 Medline

39. T. Connallon, A. G. Clark, Gene duplication, gene conversion and the evolution of the Y chromosome. Genetics 186, 277–286 (2010). doi:10.1534/genetics.110.116756 Medline