SUPPLEMENTARY ONLINE MATERIAL

IS THE SEX COMMUNICATION OF TWO PYRALID MOTHS, PLODIA

INTERPUNCTELLA AND EPHESTIA KUEHNIELLA, UNDER CIRCADIAN CLOCK REGULATION?

RADKA ZÁVODSKÁ, SILVIE FEXOVÁ, GERMUND VON WOWERN, GUI-BIAO HAN,

DAVID DOLEZEL, AND IVO SAUMANN

Supplementary text

Immunocytochemistry

Complete heads of both species were dissected at specific timepoints and immediately fixed at

4ºC overnight in a modified Bouin-Hollande solution (0.7% mercuric chloride, no acetic acid).

The samples were then washed in 70% ethanol and treated by a series of dehydrating ethanol

solutions, ending with chloroform before transfer to melted paraplast. After incubation at

58ºC overnight in a vacuum oven, the paraplast samples were let to cool to room temperature

(RT) and sectioned. 7µm thick sections were attached to microscope slides and dried on a hot

plate (ca 42ºC) for at least 48h. After deparaffinization in xylene, the sections were rehydrated

through a series of ethanol: water solutions, briefly incubated in Lugol's iodine followed by

7.5% sodium thiosulfate to remove residual heavy metal ions, and subsequently washed in

distilled water and phosphate-buffered saline (0.3% Tween-20 [PBST]). Blocking with

normal goat serum (10% in PBST, 30 min at RT) was followed by incubation with a primary

antibody overnight at 4ºC (all primary antibodies were diluted 1: 1000 in PBST). After

rinsing with PBST (3×10 min at RT), samples were incubated for 1h at RT with goat anti-

rabbit IgG-horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch,

1:1000 in PBST). The slides were then washed in PBST (3×10 min at RT), followed by a final

wash in 0.05M Tris-HCl buffer, pH 7.4, for 10min at RT. Staining was developed with

hydrogen peroxide (0.005%) and 3,3'-diaminobenzidine tetrahydrochloride (DAB; 0.25 mM)

as reaction substrates in 0.05M Tris-HCl, pH 7.4. After staining, the sections were washed in

distilled water, dehydrated in an ethanol series and mounted in DPX mounting medium.

The positions of immunopositive neurons and their fibers were reconstructed from the

series of frontal sections examined under the Zeiss Axioplane 2 or Olympus BX41microscope

equipped with the Nomarski (DIC) optics and CCD camera. Distribution of

immunoreactivities in successive sections was drawn over one another to a diagram

illustrating the topology of PBAN- ir, Crz-ir, and PDF-ir.

Data collection and assessment of immunostaining intensity. Heads of adults were collected at

ZT 4 (the mid- photophase) and ZT 16 (the mid- scotophase) for Crz- ir and PDF- ir study. At

least 4 females and 2 males at each time point were tested. To measure the effect of circadian

time on PBAN- ir animals were dissected at 4 hrs intervals through one diurnal cycle under

LD 12: 12 conditions. 5 females and 5 males were fixed at each chosen time (ZT 0, ZT 4, ZT

8, ZT 12, ZT 16, ZT 20, ZT 24). Dim red light (660- 670 nm wavelengths) was used for

dissecting brains during scotophase.

The positions of immunopositive neurons and their fibers were reconstructed from the

series of frontal sections examined under the Zeiss Axioplane 2 or Olympus BX41microscope

equipped with the Nomarski (DIC) optics and CCD camera. Distribution of

immunoreactivities in successive sections was drawn over one another to a diagram

illustrating the topology of PBAN- ir, Crz-ir, and PDF-ir. All preparations were processed

simultaneously and the entire immunohistochemical procedure, from tissue dissection to

staining evaluation, was standardized for all samples in respect to time, temperature, and other

conditions. The intensity of staining was scored subjectively with a 4-point scale ranging from

no reactivity (-) to the weak (+), distinct (++) and strong reactivity (+++).

PBAN- ir at different circadian times

No fluctuations were revealed in the number of cells in P. interpunctella (Fig. S3) as well

as in E. kuehniella (Fig. S4). No significant differences in the staining intensity of PBAN

antigen were found either in perikarya or fibers. The immunostaining was assessed as distinct

(++) or strong (+++) in the brain, the SOG, the CC, and the FG at each examined time point

(Table S1). Nuclear distribution of PBAN-ir material was not observed neither in P.

interpunctella nor E. kuehniella during photophase and scotophase at any ZT. No differences

in the staining pattern were found between sexes.

A Supplementary table

Table S1. Numbers of PBAN-ir, Crz-ir, PDF- ir cells and intensity of immunostaining in the

brain, suboesophageal ganglion and neurohemal organs of P.interpunctella and E. kuehniella.

Localization P. interpunctella E. kuehniella _____________________________ ________________________________ PBAN-ir CRZ-ir PDF-ir PBAN-ir CRZ-ir PDF-ir __________________________________________________________________________________________ SLP medial 4 ++ 3 +++ 2 +++ 5 &5 ++ 2 +++ - lateral 4 +++ 3 +++ 3 +++ 4 ++ 2 +++ 4 ++ horn 2 ++ 3 ++ - 4 ++ 2 +++ - IMP caudal - - - - - 3 + VLP caudal 2 ++ - - - - - PI frontal 6 ++ - 6 ++ 8 ++ - 5 +++ Tr caudal 10 ++ 4 ++ - 8 - - OL Cells - - 4 ++ - - 4 ++ Fibers - - + - ++ ++ SOG Mdb-m 2 +++ - - 2 +++ - - Mxl-m 3 +++ - - 3 +++ - 1 ++ Mxl-l 1 +++ - 1 + 2 +++ 1 ++ - Lb-m 3 +++ - 2 ++ 2 +++ - 1 +++ FG Cells 3 +++ - - 2 ++ - - Fibers + + - - ++ - +++ CC Cells - 4 +++ - - 4 +++ - Fibers +++ +++ + ++ +++ +++ +++ NCA ++ - - + ++ - CA - - - - - -

Immunoreactivity was quantified as absent (-), weak (+), distinct (++) and strong (+++). In

both examined species no differences in distribution and intensity of PDF-ir, Crz-ir and

PBAN-ir were revealed with respect to both the sex and the time points (ZT 4, 16) of the LD

cycle. Cell numbers are given for one of the bilateral clusters (SLP superior lateral

protocerebrum, IMP inferior medial protocerebrum, VLP ventro- lateral protocerebrum, PI

pars intercerebralis, Tr tritocerebrum, OL optic lobe, Pfv proximal fronto- ventral cluster,

SOG suboesophageal ganglion, Mdb, Mxl, Lb mandibular, maxillary and labial neuromere,

respectively, m-medial, l-lateral, FG frontal ganglion, CC corpora cardiaca, NCA nervus

corporis allati, CA corpora allata).

Supplementary Figures (S1-S4)

Fig. S1: Examples of locomotor behavior of individual E. kuehniella and. P. interpunctella

males. The double-plotted actogram (left) shows rhythmic behavior in LD and DD (the arrow

indicates transition to constant darkness). The Periodogram (right) indicates the free running

period in DD [The Chi-square significance line crossing the periodogram corresponds to α =

0.05].

Fig. S2: Locomotor activity of 30 E. kuehniella and 25 P. interpunctella males as was

recorded in 3 days of LD 12: 12 followed by 4 days in constamt light (constant temperature

25oC).

Fig. S3: PBAN- ir perikarya in the brain- SOG complex and neurohemal organs of the

female P. interpunctella. Samples were collected for the examinations in 4 hs intervals from

the LD (12 hs L: 12 hrs D) photoperiodic regime. Zeitgeber time was counted from the lights

on (ZT 0, i.e. 0 hr). (A) Immunopositive cels in the pars intercerebralis .(B) Perikarya in the

superior lateral protocerebrum. (C) A group of small cells in the trirocerebrum. (D) Strongly

stained cells in the area of mandibular (Mdb), maxillar (Mxl) and labial (Lb) neuromere in the

suboesophageal ganglion. (E) Extensive arborization of PBAN- ir axons in the corpora

cardiaca. (F) Immunopositive cells in the frontal ganglion.

Abbreviations: pars intercerebralis (PI), protocerebrum (Pr), tritocerebrum (Tr), oesophageal

foramen (oes for), suboesophageal ganglion (SOG), frontal ganglion (FG), corpora cardiaca

(CC), corpora allata (CA), mandibular neromere (Mdb), maxillary neuromere (Mxl), labial

neuromere (Lb), nervi corporis cardiaci (NCC), nervus recurrens (RN). Bar = 50 ųm to all

micrographs.

Fig. S4: PBAN- ir perikarya in the brain- SOG complex and neurohemal organs of the female

E. kuehniella. Samples were collected for the examinations in 4 hs intervals from the LD (12

hs L: 12 hrs D) photoperiodic regime. Zeitgeber time was counted from the lights on (ZT 0,

i.e. 0 hr). (A) Immunopositive cels in the pars intercerebralis. (B) PBAN-ir neurons in the

superior lateral protocerebrum. (C) A group of small cells in the tritocerebrum. (D) Strongly

stained cells in the area of mandibular (Mdb), maxillar (Mxl) and labial (Lb) neuromere in the

suboesophageal ganglion. (E) Fiber arborization in the corpora cardiaca. (F) Immunopositive

cells in the frontal ganglion.

Abbreviations: pars intercerebralis (PI), protocerebrum (Pr), tritocerebrum (Tr), oesophageal

foramen (oes for), suboesophageal ganglion (SOG), frontal ganglion (FG), corpora cardiaca

(CC), corpora allata (CA), mandibular neromere (Mdb), maxillary neuromere (Mxl), labial

neuromere (Lb), nervus recurrens (RN). Bar = 50 ųm to all micrographs.