Gross morphology of Euphausia superba and its central nervous system.

a) Image of a swimming E. superba, facing with dorsal to the top and anterior to the right. Thick white arrows point to the five pairs of swimming legs (pleopods) on the abdomen. b, c) Lateral (b) and dorsal (c) view of the dissected central nervous system (CNS) of krill. The CNS can be divided into subesophageal (SEG), thoracic (T1-8), and abdominal (A1-A5 + TAG) ganglia. Arrows point to the compound eyes (CE), central brain (CB), and the 1st and the base of the 2nd antennae (AN1 and AN2, respectively). d) Pigment-dispersing hormone-immunoreactivity (PDH-ir) within the CNS of krill. Note that the terminal abdominal ganglion (TAG) was recorded independently of the rest of the CNS due to restrictions in dissections. e) Detailed view of the SEG and the first two thoracic ganglia (T1, T2). PDH-ir cells are labelled by white arrowheads. Fibers connecting the ganglia in the lateral connectives are labelled by blank orange arrowheads, and fibers running through the median neurite bundle by a filled orange arrowhead. f) Representative cell of one of the paired cells in the ventral nerve cord ganglia labelled by anti-PDH (green) and pdh in situ hybridization (magenta). Blank arrowheads label the circumesophageal ganglia. The scalebar represents 3 mm for (b-d), 250 µm for e, and 50 µm for (f). Abbreviations: d, dorsal; v, ventral; a, anterior; p, posterior.

Overview of the PDH-network in the supraesophageal ganglion of krill.

a) Frontal view of one eyestalk and the central brain (CB) showing the position of the five horizontal 200 µm sections depicted in (b-f). b-f) Horizontal vibratome sections of the supraesophageal ganglion (left eyestalk and central brain) stained against β-PDH (orange) with a nuclear counterstaining (Hoechst, gray). The sections show extensive PDH-immunoreactivity from the dorsal (b) to ventral (f) brain and eyestalk. In the eyestalk, two prominent cell clusters (lamina-cluster, lobula-cluster) can be found. g) Reconstruction of major neuropiles found in the supraesophageal ganglion. The eyestalks contain, from lateral to medial, the lamina (La, red), medulla (Me, dark orange), the lobula complex with lobula and lobula plate (Lo, light orange), and the medulla terminalis (TM, light blue) with the hemiellipsoid neuropile (HN, dark blue). The olfactory neuropiles comprise the lateral antennal neuropile (LAN, ochre), the olfactory lobes (OL, yellow), and the antennal neuropile (AnN, green). The color scheme is based on Kenning et al. (2013). Gray arrows indicate continuation of cut connectives/ nerves. Note that, due to the eyestalks’ fragile connection to the central brain, the right eyestalk was rotated horizontally approximately 40° counterclockwise during processing. h) 3D projection of the whole PDH-network spanning the krill supraesophageal ganglion. The neuropiles from (g) are shown in gray for orientation. Scale bars in (a-f) represent 250 µm and 500 µm in (g) and (h). Abbreviations: ES, esophagus; SG, sinus gland; ePP, eyestalk photophore.

Detailed morphology of PDH-positive cell clusters and their projections in the krill eyestalks.

a-d) Maximum intensity projections showing PDH-positive cells and their projections in the lamina-cluster. a-c) Filled arrowheads point to cells distributed along the proximal surface of the lamina, open arrowhead in c) points to projections into the medulla. Single and double asterisks in a) indicate the first and second optic chiasma, respectively. e-k) Maximum intensity projections of PDH-positive cells and their projections in the lobula-cluster. e-g) single arrows point to lobula-subcluster 1 at the posterior side of the optic lobe and filled arrowheads point to cells of lobula-subcluster 2, projecting into the medulla terminalis. Blank arrowheads in e-f) point at single PDH-positive cells between the lobula and medulla terminalis. Double arrowheads in e) and j) point towards the dense fibre knot formed by projections of lobula-subcluster 1 cells, whereas thin lines e) and f) outline their projection lateral of the knot. i-h) Maximum intensity projection of subcluster 2 of the lobula-cluster showing five PDH-positive cells, including one characteristic large cell body (open arrowhead) and four smaller cells (filled arrowheads). k) Projections of lobula-subcluster 1 through the eyestalk towards the central brain (filled arrowheads). Scale bars represent 200 µm in a-c), and 100 µm in d-g), j), and k). Scalebars in h and i) represent 50µm. Abbreviations: La, lamina; Me, medulla; Lo, lobula; TM, medulla terminalis; SG, sinus gland; a, anterior; p, posterior.

Detailed morphology of PDH-positive cells and projections in the krill brain

a-c) Maximum intensity projections of the PDH-positive network in dorsal (a) and medial to ventral regions of the central brain (b-c). a) Filled arrowheads point to PDH-positive cells found dorsal in the medial protocerebrum, while blank arrowheads point to their projections. Asterisks indicate a paired, granular, PDH-ir structure which is targeted by fibres coming from the optic lobes. b) PDH-ir fibres originating from the medulla terminales form dense arborizations in the medial protocerebrum. Filled and blank arrowheads indicate the paths of PDH-ir projections forming dorsal and ventral commissures, respectively. c) Filled arrowheads indicate fibres that enter the central brain through the eyestalks and exit ipsilaterally via the oesophageal connectives. The scalebar in a) represents 100µm, and in b and c) 200µm. Abbreviations: TM, medulla terminalis; a, anterior; p, posterior.

PDH-positive cells co-localize with cry2 and per transcripts in the krill optic lobes.

a-b’’) PDH-positive (green, a, b), as well as cry2- (magenta, a’’) and per-positive (magenta, b’’) cells in the lamina-cluster of the krill optic lobe. a’ and b’) show co-localization of the anti-PDH and in situ hybridization signal in a subset of cry2 (filled arrowheads in a’) and per-positive (filled arrowheads in b’) cells. c-d’’) PDH-positive (green, c, d), as well as cry2 (magenta, c’’) and per-positive (magenta, d’’) cells in the lobula-cluster. The PDH-positive cells co-localize with a subset of the cry2 (filled arrowheads in c’) and per-positive (filled arrowheads in d’) cells, respectively. Blank arrowheads in (a-d’’) show cry2 and per-expressing cells that do not colocalize with anti-PDH. Abbreviations: La, lamina; Me, medulla; Lo, lobula. Scale bars represent 100 µm.

Distribution of cry2 and per transcripts in relation to the PDH-network in the krill brain.

a-h) Stereo microscopic images of 100 µm sections of the krill central brain showing cells expressing cry2 (a-d) and per (e-h) transcripts. From left to right, images show sections progressing from dorsal to ventral regions of the central brain. Areas with high per/ cry2-expression are marked by white arrowheads. i-i’’) PDH-positive neurons in the krill central brain (green arrowheads) do not colocalize with cry2-expressing neurons (blank arrowheads). j-j’’) Similarly, PDH-positive neurons (green arrowheads) do not colocalize with per-expressing neurons (blank arrowheads) as revealed by RNA in situ hybridization. Abbreviations: OL, olfactory lobes; LAN, lateral antennal neuropile; AnN, antennal neuropile. The scale bars represent 200 µm in a-h) and 100 µm in i-j’’).

Location of clock neurons and neuropile regions in the supraesophageal ganglion of krill.

Left hemisphere: Schematic representation of the general location of neurons expressing cry2 and per and or pdh (dotted areas). Cells expressing cry2, per, and pdh are considered clock neurons. In the optic lobe, the location and the number of cry2 and per-expressing neurons suggest colocalization also in PDH-negative neurons, categorizing them as potential clock neurons. However, we could not investigate colocalization via in situ hybridization due to methodological limitations. per and cry2 expression in the central brain was present in numerous cells, but distinct clusters could not be defined. Note that PDH-positive cells in the central brain did not colocalize with per or cry2-expressing cells. Right hemisphere: Schematic representation of the neuropiles identified in the krill brain. Black dashed lines represent major PDH-positive projections. Abbreviations: La, lamina; Me, medulla; Lo, lobula; TM, medulla terminalis; SG, sinus gland; HN, hemiellipsoid neuropile; LAN, lateral antennal neuropile; OL, olfactory lobes; AnN; antennal neuropile; ES, esophagus; CB, central brain.

Primer sequences used to synthesize in situ hybridization probes.

For cry2 in situ hybridizations, a mix of two probes, termed Es_cry2_a and Es_cry2_b, was used.

pdh in situ hybridization colocalizes with PDH-immunoreactivity.

a) pdh in situ hybridization (red) on a horizontal vibratome section labels a big cell cluster below the lamina (La). b-d) single focal planes through the pdh-expressing cell cluster (magenta) from anterior (b) to posterior (d), double labelled with anti-PDH (green), show PDH-immunoreactive (PDH-ir) cells are indeed all expressing pdh. e) A second cluster of pdh-expressing cells is found in the eyestalk, closely associated with the lobula (Lo). f, g) Double labelling with anti-PDH shows colocalization of strongly PDH-ir cells and pdh-expressing cells also in this cluster. Occasionally, we found weak PDH-ir cells (blank arrowheads in (g)) where we could not unambiguously identify colocalization between PDH-ir and pdh-expression. h) pdh in situ hybridization reveals several pdh-expressing cells in the central brain. i, j) Double labelling with anti-PDH shows, like for the other clusters, that strongly immunoreactive cells are also expressing pdh. While we could only reliably identify two cells per hemisphere (i, left; j, right hemisphere) labelled by in situ hybridization using fluorescence microscopy, we found, for the chromogenic labelling, multiple cells that we might not have been able to distinguish from the strong autofluorescent background in fluorescent microscopy (compare h and i/j). Hence, we could not show colocalization for weak PDH-ir cells in the central brain (blank arrowheads in (i, j)), similar to the weakly immunoreactive cells close to the lobula. Scale bars in a, e, h represent 250 µm and 100 µm for the other panels. Abbreviations: Me, medulla, TM, medulla terminalis; a, anterior; p, posterior; l, lateral; m, medial.

Chromogenic RNA in situ hybridization of clock gene mRNA on horizontal vibratome sections of the optic lobe.

Bright field image of the cry2 (a, b) and per-expression (c, d) in the optic lobes of krill, visualized by chromogenic in situ hybridization (violet, arrows). Expression of cry2 and per matches the location of the PDH-positive lamina- and lobula-cluster identified by antibody staining. Scale bars represent 250µm. Abbreviations: La, lamina; Me, medulla; Lo, lobula; TM, medulla terminalis; a, anterior; p, posterior.

Negative controls for RNA in situ hybridization.

a-d’’’) Horizontal vibratome sections of krill optic lobes were used in chromogenic in situ RNA hybridization without RNA probes as a control for unspecific antibody binding or discoloration of the tissue. (a-c’’) sections using BCIP/NBT (violet, a, b, c) co-labelled with anti-PDH (green, a’’, b’’, c’’) do not show any in situ hybridization signal, hence also no colocalization with PDH-positive cells (blank arrowheads, a’, b’, c’). This was true for the lobula-cluster (a-a’’) and for the lamina-cluster (b-c’’). d-d’’’) Vibratome sections of krill optic lobes used in chromogenic in situ RNA hybridization without RNA probes, stained by Fast Red (red/ magenta, d, d’’) and subsequent immunolabeling with anti-PDH (green, d’, d’’’), showed, similar to BCIP/NBT, no signal for the in situ hybridization and hence no colocalization with anti-PDH (blank arrowheads, d’’’). d) brightfield micrograph. d’-d’’’) fluorescent micrographs. Scale bars represent 250 µm.