Fig 1.
Phylogenetic tree of the investigated Coleopteran species.
The phylogenetic tree providing information on the design of the antennal lobes, lifestyles (information on habitat and major nutrition) and relative neuropil volumes. Families in which only a single species was examined are: 1 = Pyrochroidae; 2 = Mordellidae; 3 = Lymexylidae; 4 = Nitidulidae; 5 = Lampyridae; 6 = Elateridae; 7 = Ptinidae; 8 = Dermestidae; 9 = Bolboceratidae; 10 = Geotrupidae; 11 = Gyrinidae. Icons to the right of the family names show whether AL substructures could be observed and whether these are immunoreactive to tachykinin-related peptide (TKRP) or if a microglomerular organization could be observed (see legend at the bottom; *: no immunostainings against TKRP are available). Data on lifestyle of the animals extracted from: [98–102].
Fig 2.
Coleopteran antennal lobes (ALs) labeled with various markers (immunostainings against synapsin [Syn, green] and Tachykinin-related peptide [TKRP, magenta] as well as phalloidin labeling [Phal, grey]). Boxes in the lower left corner show details of single glomeruli, marked in the overview images. Arrowheads show glomerular substructures (F, G, J—L, N—P, S, and T), arrows show homogenously stained glomeruli (L, P).
Fig 3.
Antennal backfills and TKRP immunostaining.
Antennal lobes (ALs) of Pachnoda ephippiata (Scarabaeidae) (A, A', and A'') and of Apis mellifera (B, B', and B''). Antennal backfills in P. ephippiata (A, A', A'') demonstrate that olfactory sensory neurons of the antenna (green) do not innervate the glomerular substructures, while the tachykinin related peptide immunoreactive (TKRP-ir) local neurons (LNs) (magenta) innervate mostly exclusive the glomerular substructures, save for several varicosities outside the substructures (A', A'' arrowheads). A' and A'' represent the labeling shown in the inset in A separated in the single channels. In A. mellifera (B, B', and B'') TKRP immunoreactivity in the glomerular core areas seems to stem primarily from LNs (magenta; arrowhead), while the whole glomeruli labeled with the synapsin antibody (Syn, green). B' and B'' represent the two separated labels shown in the inset in B.
Fig 4.
Antennal lobes and mushroom bodies of different Coleoptera.
Antennal lobes (ALs) (A-G, J, L) and mushroom bodies (MBs) (H-I'', K, M) of different Coleoptera stained with antibodies against synapsin (Syn) and against tachykinin related peptides (TKRP) or labeled with DAPI or phalloidin (Phal). Boxes in the upper left show details of single glomeruli (A-C, E, G, J, L) or part of the MB (I) marked in the overview images, or they show the superstition between the AL and the lobus glomerulatus (LG) in the ladybug Harmonia axyridis (G). Arrowheads showing single glomeruli (C-G). Notice the trichotomy of the MB peduncle (PED) (square brackets in H and H'' and arrowheads in M) of the aquatic beetles Ilybius fuliginosus and Gyrinus substriatus. The calyx (CA) is absent in the ladybug Coccinella hieroglyphica as seen in the staining with Phal (I) and in the 3D-reconstruction and 3D-projektion (volume rendering) of Syn and DAPI (I'-I‴). In both cases, no calyx is visible between PED and Kenyon cells (KC). Orientation bars in I’ to I”‘: D = dorsal, P = posterior, L = lateral.
Fig 5.
Considerations to the evolutionary origin the glomerular substructures.
In principal, substructures could originate from the fusion of two (or more) glomeruli, each consisting of one cortex and one core, resulting in a glomerulus with one cortex and two cores / substructures (A). Substructures could also originate from a glomerulus with one cortex and one core and a subsequent division of the single core in multiple cores / substructures (B). C to F showing the possible principal innervation pattern as consequences of the two models (A and B) for olfactory sensory neurons (OSNs) (C and D) for local interneurons (LNs) respectively projection neurons (PNs) (E and F) (see text).
Fig 6.
Glomerular substructures of hemimetabolous insects.
Two Gryllidae Acheta domestica (A) and Gryllusassimilis (B) and two Hemiptera Graphosoma lineatum (C) and Gonocerus acuteangulatus (D). Boxes in the upper left of each image show a magnified view, respectively the two separated channels of the inset withinthe image. In A. domestica (A) and G. assimilis (B) staining with phalloidin (Phal) revealed glomerular substructures (arrowheads), resembling the situation in Gryllus bimaculatus [51,88]. Staining with an antibody against tachykinin related peptide (TKRP) (magenta) and synapsin (Syn) (green) revealed irregular shaped and interconnected TKRP immunoreactive substructuring within the glomeruli of two Hemiptera species: G. lineatum (about 205 glomeruli) (C) and G. acuteangulatus (about 185 glomeruli) (D).