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The Mayfly Subimago Explained. the Regulation of Metamorphosis in Ephemeroptera

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The Mayfly Subimago Explained. the Regulation of Metamorphosis in Ephemeroptera The mayfly 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 Cloeon dipterum, with those of Blattella germanica, Drosophila melanogaster, Ephemera danica and Ischnura elegans. Figure S2. Phylogenetic relationships of the Broad complex Zinc fingers of Cloeon dipterum, Blattella germanica, Drosophila melanogaster, Ephemera danica 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 mayflies 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.
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