The subimago explained. The regulation of metamorphosis in Ephemeroptera

Orathai Kamsoi, Alba Ventos-Alfonso, Isabel Almudi, Fernando Casares, Xavier Belles

SUPPORTING INFORMATION

Material and Methods

Figure S1. Alignment of the Broad complex Zinc fingers of , with those of Blattella germanica, Drosophila melanogaster, danica and Ischnura elegans.

Figure S2. Phylogenetic relationships of the Broad complex Zinc fingers of Cloeon dipterum, Blattella germanica, Drosophila melanogaster, and Ischnura elegans.

Table S1. Primers used to measure expression levels of selected Cloeon dipterum genes by qRT-PCR.

References cited

MATERIALS AND METHODS

Cloeon dipterum rearing in the laboratory

C. dipterum used in the experiments were obtained from a colony starting from gravid females by forced mating in the laboratory at Centro Andaluz de Biología del Desarrollo (Sevilla, Spain). The newly hatched nymphs were reared in unchlorinated and oxygenated water at 22 ± 1ºC, and under 12:12 h (light: dark) photoperiod, feeding them on filamentous algae (Chara sp.). A detailed description of the rearing methods through the entire life cycle is provided by Almudi et al. (1).

RNA extraction and retrotranscription to cDNA

Total RNA was extracted from whole bodies, body parts, or particular tissues of C. dipterum nymphs, subimago and adult, using the Gen Elute Mammalian Total RNA kit (Sigma-Aldrich), according to the manufacturer’s instructions. RNA quantity and quality were estimated by spectrophotometric absorption at 260 nm in a Nanodrop Spectrophotometer ND-1000® (NanoDrop Technologies). A sample of 400 ng from each RNA extraction was then treated with DNase (Promega) and reverse transcribed with first Strand cDNA Synthesis Kit (Roche) and random hexamer primers (Roche).

Determination of mRNA levels by quantitative real-time PCR

Measurements with qRT-PCR were carried out in an iQ5 Real-Time PCR Detection System (Bio-Lab Laboratories), using SYBR®Green (iTaqTM Universal SYBR® Green Supermix; Applied Biosystems). Reactions were carried out in triplicate, and a template-free control was included in all batches. Primers used to measure the transcripts of interest are detailed in Table S1. The efficiency of each set of primers was validated by constructing a standard curve through three serial dilutions. Levels of mRNA were quantified relative to CdActin-5c mRNA (Table S1). Results are given as copies of the examined mRNA per 1000 copies of CdActin-5c mRNA.

Statistical analyses of qRT-PCR measurements

In all qRT-PCR experiments, to test the statistical significance differences between control and treated samples it has been used the Relative Expression Software Tool (REST), which evaluates the significance of the derived results by Pair-wise Fixed Reallocation Randomization Test (2).

Treatments with methoprene

To study the effect of juvenile hormone on metamorphosis, freshly ecdysed last instar nymphs of C. dipterum (less than 5 h after shedding off the exuvia) were treated with methoprene (Sigma-Aldrich) at a dose of 50 µg. Nymphs were immobilized on ice, and a volume of 0.25 µl of an acetone solution of methoprene (200 µg/µl) was topically applied on the mesonotum with a 5 µl Hamilton microsyringe. Controls received 0.25 µl of acetone.

Morphological studies and imaging

The nymphs, subimago and adults were examinations and photographs using a stereomicroscope Zeiss DiscoveryV8 and a bright field microscope Carl Zeiss- AXIO IMAGER.Z1. Ephemera Z3 C P Y C R R M F S C Y Y S L K R H F Q D R H E K S N - M L Y M C E F C S R S Y R T K N S L T T H K S L Q H Cloeon Z3 ------M F S C Y Y S L K R H F Q D R H E K S N - T L Y T C E F C S R S Y R T K N S L T T H K S L Q H Blattella Z3 C P Y C R R T F S C Y Y S L K R H F Q D K H E R S D - T L Y V C E F C S R R Y R T K N S L T T H K S L Q H Drosophila Z3 C P Y C R R T F S C Y Y S L K R H F Q D K H E Q S D - T L Y V C E F C H R R Y R T K N S L T T H K S L Q H Ischnura Z3 C P Y C H R N F S C Y Y S L K R H F K D R H Q P S D - T L H R C E F C H R L Y R T K N S L T T H K S L Q H Ischnura Z5b C Y L C N K S F T R I W S L N R H M A D T H C N - V V R S F E C E V C H R V Y R S K N S L V S H R S Q Y H Blattella Z5 C P L C R K S F T R A W S L Q R H M A D T H F Y - V P Q S F E C D V C G R S Y R S R N S L V S H K S Q Y H Ischnura Z5a C K L C H K S F S R L W S L Q R H V E D V H G K R E G R A F V C N L C F R H Y G T R S S L I S H R S Q Y H Ephemera Z2 C L L C S K V L C S K A S L K R H I A D K H E E K Q - E E F R C V I C E R T Y C S R N S L M T H I Y T Y H Cloeon Z2 C L L C S K V L C S K A S L K R H I A D K H E E K Q - E E F R C V I C E R T Y C S R N S - - A H - - - - - Blattella Z2 C Q L C G K V L C S K A S L K R H V A D K H A E R Q - E E Y R C I I C E R V Y C S R N S L M T H I Y T Y H Drosophila Z2 C Q L C G K L L C S K A S L K R H I A D K H A V R Q - E E Y R C A I C E R V Y C S R N S L M T H I Y T Y H Ischnura Z2 C H L C N K A L C S R S S L R R H M E D K H V I T G - T E F R C I P C N R A Y S S R N S L M K H R Y T Y H Ephemera Z1 - - - C G K N L T S P Q R L R R H I Q N V H A K P V - K P P V C N I C N K V Y S T L N S L R N H K S I Y H Cloeon Z1 C E P C G K N L T S P Q R L R R H I Q N V H A K P V - K P P V C N I C N K V Y S T L N S L R N H K S I Y H Ischnura Z1 C E P C G K N L T S P Q R L R R H I Q N V H A N P T - R A P V C N I C N K V Y S T L N S L R N H K S I Y H Blattella Z1 C E P C G K N L T S L Q R L R R H I Q N V H T H P S - K T P V C N I C N K V Y S T L N S L R N H K S I Y H Drosophila Z1 C N P C N K N L S S L T R L K R H I Q N V H M R P T - K E P V C N I C K R V Y S S L N S L R N H K S I Y H Blattella Z4 C D V C G K L L S T K L T L K R H K E Q Q H L Q P L - H N A V C N L C N K V F R T V N S L N N H R S I Y H Drosophila Z4 C D V C G K L L S T N V T L K R H K E Q Q H L Q P L - N N A V C N L C H K V F R T L N S L N N H K S I Y H Ephemera Z4 C E V C G K V L G T K L T L K R H K E Q Q H L Q P L - H S A V C P V C F K V F R T L N S L N N H R S I Y H Ischnura Z4 C N V C G K V L A S A A T L R R H K E Q Q H E Q P L - H A A V C P V C H K V F R T I N S L H N H R S V Y H Blattella Z6 C E E C G K V L R S P I T L K R H V L D L H R E Q T - E R F W C N V C Q K C Y R T K N S L V V H L C K Y H

Figure S1. Alignment of the Broad complex Zinc fingers of Cloeon dipterum, with those of the neopteran especies Blattella germanica and Drosophila melanogaster, characterized by Piulachs et al. (3), and Di Bello et al. (4), respectively. Also included are those that we identified in the paleopteran species Ephemera danica and Ischnura elegans.The accession numbers of the Broad complex Zinc finger isoforms of B. germanica are: FN651774 (Z1), FN651775 (Z2), FN651776 (Z3), FN651777 (Z4), FN651778 (Z5), and FN651779 (Z6). Those of D. melanogaster are: CAA38474 (Z1), CAA38476 (Z2), CAA38475 (Z3), and AAB09760 (Z4). The Broad complex Zinc fingers of C. dipterum, E. danica and I. elegans were manually annotated from the respective genome projects PRJEB34721, PRJNA171755 and PRJNA353476. The alignment was carried out with Clustal X (5). Drosophila Z1 Blattella Z1 75 Ischnura Z1 Z1 Cloeon Z1 73 85 Ephemera Z1 Blattella Z4 Ephemera Z4 93 Z4 53 Ischnura Z4 Drosophila Z4 87 Cloeon Z2 Ephemera Z2 80 Ischnura Z2 Z2 Drosophila Z2 Blattella Z2 Blattella Z6 Z6 56 Blattella Z5 85 53 Ischnura Z5b Z5 Ischnura Z5a

83 Cloeon Z3 73 56 Ephemera Z3 100 Blattella Z3 Z3 0.2 Ischnura Z3 Drosophila Z3

Figure S2. Phylogenetic relationships of the Broad complex Zinc fingers of Cloeon dipterum, Blattella germanica, Drosophila melanogaster, Ephemera danica and Ischnura elegans, according to the alignment and data showed in Figure S1. The phylogenetic reconstruction was carried out with PHYML 3.0 (6) based on the maximum-likelihood principle with the amino acid substitution model, four substitution rate categories, and a gamma shape parameter of 1.444. The data was bootstrapped for 100 replicates. Bootstrap values higher than 50 are indicated in the corresponding nodes. The sequences corresponding to Cloeon dipterum are indicated in bold. Scale bar indicates the number of substitutions per site.

Table S1. Primers used to measure expression levels of selected Cloeon dipterum genes by qRT-PCR

Gene Forward primer Reverse primer Actin 5C (Act5C) AGAAGTTGCTGCCCTCGTT GACCATCACACCCTGATGC Broad complex (Br-C core) AGGACTTCGTGGATGTGACC TGCACATGGGGTACTCTTGA Broad complex Z1 (Br-C Z1 GCTTGGTTATCGATGCGAAC GTTTCGCGTGCACGTTCT Broad complex Z2 (Br-C Z2) TCGAAGGCTAGTCTCAAACGA CGTTCGCAAATGACACATCT Broad complex Z3 (Br-C Z3) CTGTCGACGCATGTTCAGTT CGTGTACAGCGTGTTGGACT Ecdysone-induced protein 93F CTACGATCGTGACAGCCTGA CGCTCCTTGACTTTGTACTCG (Eip93F, E93 or mblk-1) Hormone receptor 3 (HR3) GCAACAAGAACTGCGTCGT GCTGCTTTTTGGACATTCGT Krüppel homolog 1 (Kr-h1) TGCGAGTACTGCCACAAGTC CATTTGTACGGTCGCTCCTT

Note: Genes manually annotated in Cloeon dipterum genome project accessions PRJEB34721. The sequence reads and the genome assembly have been deposited in the European Nucleotide Archive (ENA).

References cited in the supporting information

1. Almudi I, Martín-Blanco CA, García-Fernandez IM, López-Catalina A, Davie K, Aerts S, Casares F (2019) Establishment of the mayfly Cloeon dipterum as a new model system to investigate evolution. Evodevo 10 (1): 6. 2. Pfaffl MW, Horgan GW, Dempfle L (2002) Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res 30 (9): e36. 3. Piulachs M-D, Pagone V, Belles X (2010) Key roles of the Broad-Complex gene in insect embryogenesis. Insect Biochem Mol Biol 40 (6): 468–475. 4. DiBello PR, Withers DA, Bayer CA, Fristrom JW, Guild GM (1991) The Drosophila broad-complex encodes a family of related proteins containing zinc fingers. Genetics 129: 385–397. 5. Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23 (21): 2947–2948. 6. Guindon S, Gascuel O (2003) A simple, fast and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol 52: 696– 704.