A three-dimensional immunofluorescence atlas of the brain of the hackled-orb weaver spider, Uloborus diversus

abstract

Spider orb-web building is a captivating, rare example of animal construction, whose neural underpinnings remain undiscovered.

We created a three-dimensional atlas for the hackled-orb weaver, Uloborus diversus , based on immunostaining for the presynaptic component, synapsin, in whole-mounted spider synganglia.

Whereas spider neuroanatomy has thus far been most comprehensively studied in cursorial species, this optically sectioned atlas contributes a continuous, finely resolved model of the central nervous system of an orb-web building spider.

Aligned to this volume, we examined the expression patterns of neuronal populations representing many of the classical neurotransmitters and neuromodulators (GABA, acetylcholine, serotonin, and octopamine/tyramine), as well as a subset of neuropeptides (allatostatin A, crustacean cardioactive peptide (CCAP), FMRFamide, proctolin) – detailing immunoreactivity in an unbiased fashion throughout the synganglion to reveal co-expression in known structures (such as the arcuate body), as well as novel neuropils not readily apparent in prior spider research, including the tonsillar neuropil as well as a potential protocerebral bridge.

These structures provide targets for future functional studies, and taken together, could represent a spider equivalent of the central complex, contributing to behaviors such as web-building.

introduction

Introduction Brain atlases are essential tools for neuroscience in model organisms – ranging from neuropil annotations ( Jundi and Heinze, 2020 ), to neuronal subtype and transcriptional expression pattern atlases ( Zhang et al., 2023 ), to ultrastructural connectivity maps ( Cook et al., 2019 ; Chua et al., 2023 ; Dorkenwald et al., 2024 ; Yim et al., 2024 ; Cook et al., 2025 ; Verasztó et al., 2025 ; White et al., 1986 ; Winding et al., 2023 ).

In recent years, three-dimensional atlases of major neuropil structures have also been created for non-canonical arthropod study species ( Brenneis, 2022 ), including a number of insects ( Adden et al., 2020 ; Althaus et al., 2022 ; Dreyer et al., 2010 ) and spiders ( Steinhoff et al., 2017 ; Steinhoff et al., 2024 ).

The hackled-orb weaver spider, Uloborus diversus ( Eberhard, 1972 ), is an emerging model system for the study of orb-web building in spiders ( Corver et al., 2021 ; Miller et al., 2022 ), whose central nervous system has yet to be investigated.

To date, the majority of studies of the spider central nervous system have been performed in one de facto model species, Cupiennius salei , a cursorial spider which hunts without building webs for prey capture ( Babu and Barth, 1984 ).

While isolated anatomical treatments exist for orb weavers and other web-based spiders ( Long, 2021 ; Hwang et al., 2015 ; Long, 2016 ; Moon and Tillinghast, 2013 ; Park et al., 2013 ; Rivera‐Quiroz and Miller, 2022 ; Becherer and Schmid, 1999 ; Steinhoff et al., 2024 ; Wegerhoff and Breidbach, 1995 ; Weltzien and Barth, 1991 ), the preponderance of C. salei literature is even starker when considering examinations beyond general neuronal stains, where C. salei is essentially the only spider species in which the expression pattern of more than a single neurotransmitter has been broadly mapped ( Becherer and Schmid, 1999 ; Fabian-Fine et al., 1999 ; Fabian-Fine et al., 2015 ; Fabian-Fine et al., 2017 ; Loesel et al., 2011 ; Schmid and Becherer, 1996 ; Schmid and Duncker, 1993 ; Senior et al., 2020 ; Seyfarth et al., 1990 ; Seyfarth et al., 1993 ; Tarr et al., 2019 ).

Furthermore, the current understanding of spider brain anatomy is substantially based on tissue slice analysis, which can provide exceptional detail and avoid damaging superficial brain structures, but has the disadvantage of being often limited in completeness by the planes which authors chose to exhibit.

Given that the substantial behavioral adaptation of web-building may be reflected in the presence of necessary brain structures and distinct underlying neuronal circuitry, an important step in understanding the basis of this behavior is to have a detailed, foundational architecture of a nervous system which generates it.

We created a three-dimensional immunofluorescence atlas of major neurotransmitter and neuromodulator populations for U. diversus , using whole-mounted synganglia.

Using immunostaining against the presynaptic marker, synapsin, we assembled a standard, full volume of the U. diversus synganglion onto which specific neurosignaling molecule expression patterns were aligned.

These include markers for classical neurotransmitters (GABA and acetylcholine), neuromodulators (dopamine, serotonin, and octopamine/tyramine), and several neuropeptides (allatostatin A, proctolin, CCAP, and FMRFamide).

These volumes provide comprehensive and comparable detail throughout the synganglion, in both undifferentiated and established regions – such as the arcuate body, whose layers become distinguishable through the use of neurosignaling molecule co-stains.

We further identify several previously undescribed neuropils in the supraesophageal ganglion, and the neuronal subtype populations whose specific expression demarcates them.

results

Results The central nervous system of spiders is distinctive among arthropods for its compressed nature.

Residing within the prosoma, the central nervous system, or synganglion, as it has been called ( Steinhoff et al., 2017 ), is comprised of two major divisions named in reference to the esophageal passage traveling between them – the subesophageal mass, comprised primarily of motor and sensory interneurons and comparable to the ventral nerve cord in insects, and the supraesophageal mass, containing the higher-order integration centers ( Figure 1A ).⟦>zach claim=gap: @{Residing within the prosoma, the central nervous system, or synganglion, as it has been called ( Steinhoff et al., 2017 ), is comprised of two major divisions named in reference to the esophageal passage traveling between them – the subesophageal mass, comprised primarily of motor and sensory interneurons and comparable to the ventral nerve cord in insects, and the supraesophageal mass, containing the higher-order integration centers ( Figure 1A ).} The division of the synganglion into subesophageal and supraesophageal masses is asserted here but no claim in the tree states the gross organisation of the U. diversus CNS.⟧

The divisions of the synganglion have also been described in regard to the dorsal-most protocerebrum (containing the optic, arcuate, and mushroom body [MB] neuropils), a deutocerebrum (comprised of the cheliceral neuropil [ChN] and esophagus), and the dorsal and posterior fused postoral ganglia (including the leg, pedipalp, and opisthosomal neuropils [OpN]) ( Long, 2021 ; Steinhoff et al., 2024 ).

Consistent with general arthropod nervous system morphology, the neuronal somata are found superficially ( Figure 1B ), while the internal structure of the brain is comprised primarily of neuropil.⟦>zach claim=gap: @{Consistent with general arthropod nervous system morphology, the neuronal somata are found superficially ( Figure 1B ), while the internal structure of the brain is comprised primarily of neuropil.} That somata sit superficially while the interior is neuropil is an anatomical assertion no claim in the tree makes.⟧

Figure 1. Synganglion of Uloborus diversus .⟦>zach claim=42987256-31b8-4f7d-b81f-8f6976a904f0: @{Figure 1. Synganglion of Uloborus diversus .} protocerebral-bridge-layered-transmitter-architecture⟧

( A ) 3D rendering of U. diversus (female) synganglion from averaged α-synapsin volume, composed of 909 z -planes, oblique posterior–lateral (left) and oblique anterior–lateral (right) views.⟦>zach claim=no-assertion: @{( A ) 3D rendering of U. diversus (female) synganglion from averaged α-synapsin volume, composed of 909 z -planes, oblique posterior–lateral (left) and oblique anterior–lateral (right) views.} This only says which rendering and which viewing angles the panel shows, not what was found in them.⟧

( B ) 3D rendering of α-synapsin (green) and DAPI stained (blue) synganglion, posterior, lateral, and anterior views.⟦>zach claim=no-assertion: @{( B ) 3D rendering of α-synapsin (green) and DAPI stained (blue) synganglion, posterior, lateral, and anterior views.} A channel-and-view key for the rendering (synapsin green, DAPI blue), asserting nothing about the brain.⟧

Compass abbreviations: A = anterior, P = posterior, D = dorsal, V = ventral, L = left, R = right.

Figure 1—video 1. α-Synapsin volume.⟦>zach claim=42987256-31b8-4f7d-b81f-8f6976a904f0: @{Figure 1—video 1. α-Synapsin volume.} protocerebral-bridge-layered-transmitter-architecture⟧

Figure 1—video 2. Neurotransmitter volume.⟦>zach claim=42987256-31b8-4f7d-b81f-8f6976a904f0: @{Figure 1—video 2. Neurotransmitter volume.} protocerebral-bridge-layered-transmitter-architecture⟧

Figure 1—video 3. Neuropeptide volume.⟦>zach claim=42987256-31b8-4f7d-b81f-8f6976a904f0: @{Figure 1—video 3. Neuropeptide volume.} protocerebral-bridge-layered-transmitter-architecture⟧

To create a standard, three-dimensional brain atlas framework for U. diversus , we used elastix ( Klein et al., 2010 ; Shamonin, 2014 ) to register and align multiple confocal image volumes of whole-mounted brain immunostains for the presynaptic marker, synapsin (3C11 antibody, DSHB).

By averaging aligned image volumes ( n = 6), we generated a consistent reference volume to use for further registration of more defined antibody targets, while revealing the patterns of neuropil structure of the U. diversus central nervous system, contiguously throughout the sub- and supraesophageal regions ( Figure 1—video 1 : synapsin), which we describe beginning at the ventral end and traveling dorsally.⟦>zach claim=42987256-31b8-4f7d-b81f-8f6976a904f0: @{By averaging aligned image volumes ( n = 6), we generated a consistent reference volume to use for further registration of more defined antibody targets, while revealing the patterns of neuropil structure of the U. diversus central nervous system, contiguously throughout the sub- and supraesophageal regions ( Figure 1—video 1 : synapsin), which we describe beginning at the ventral end and traveling dorsally.} protocerebral-bridge-layered-transmitter-architecture⟧

Using anti-synapsin immunoreactivity as a common reference channel for all immunostains enabled application of the derived transformation matrix to respective co-stains for each brain sample, allowing incorporation of these channels into the standard brain, amounting to a total of eight neurosignaling population immunostains in addition to anti-synapsin and DAPI (nuclear marker) unified in the current atlas.

In this way, even though non-synapsin antibody targets were applied to separate brains, alignment of the synapsin channel to the reference enabled comparative analysis of co-expression of several antibody targets within the same reference volume.

We have collected standard brain-aligned confocal volumes for specific neurotransmitter and neuromodulator expressing populations, including cholinergic (anti-ChAT), serotonergic (anti-5-HT), and octopaminergic/tyraminergic (anti-TDC2) ( Figure 1—video 2 : neurotransmitters and neuromodulators).⟦>zach claim=42987256-31b8-4f7d-b81f-8f6976a904f0: @{We have collected standard brain-aligned confocal volumes for specific neurotransmitter and neuromodulator expressing populations, including cholinergic (anti-ChAT), serotonergic (anti-5-HT), and octopaminergic/tyraminergic (anti-TDC2) ( Figure 1—video 2 : neurotransmitters and neuromodulators).} protocerebral-bridge-layered-transmitter-architecture⟧

Neuropeptidergic expression patterns have been revealed for proctolin, allatostatin A, CCAP, and FMRFamide ( Figure 1—video 3 : neuropeptides) – whose functional significance remains unknown, but which have been previously imaged in spiders ( Loesel et al., 2011 ; Breidbach et al., 1995 ).⟦>zach claim=42987256-31b8-4f7d-b81f-8f6976a904f0: @{Neuropeptidergic expression patterns have been revealed for proctolin, allatostatin A, CCAP, and FMRFamide ( Figure 1—video 3 : neuropeptides) – whose functional significance remains unknown, but which have been previously imaged in spiders ( Loesel et al., 2011 ; Breidbach et al., 1995 ).} protocerebral-bridge-layered-transmitter-architecture⟧

These aligned volumes have been made available via an online repository (see Data availability).

GABAergic expression pattern (anti-GAD) is aligned and reported, but of a more limited utility as the antibody signal penetration is limited to the periphery of the tissue.

Certain target populations, such as dopaminergic neuron expression patterns (anti-tyrosine hydroxylase), are also provided, although we were not able to align the volume to the standard brain due to incompatibility of optimal staining conditions between the required antibodies.

The use of other antisera, such as for β-Tubulin3 and horseradish peroxidase (HRP), is also demonstrated, but without alignment to the standard brain.

Subesophageal (fused postoral ganglia) neuropils Leg neuropils The leg neuropils (LNs) are the first and most apparent neuropil structures to appear in the ventral subesophageal mass and are present throughout most of the horizontal planes of this mass (until ~z400, standard brain) ( Figure 2A ; Figure 2—video 1 ; Figure 2—video 2 ; Figure 2—video 3 ).⟦>zach claim=b7856315-9b56-4c97-97c7-4f3e2e2a785d: @{Subesophageal (fused postoral ganglia) neuropils Leg neuropils The leg neuropils (LNs) are the first and most apparent neuropil structures to appear in the ventral subesophageal mass and are present throughout most of the horizontal planes of this mass (until ~z400, standard brain) ( Figure 2A ; Figure 2—video 1 ; Figure 2—video 2 ; Figure 2—video 3 ).} leg-neuropils-consistent-innervation⟧

Their structure and innervation is generally consistent for all legs, with the only discernible difference being that LN1 are larger than those of the other legs, allowing for innervation patterns to be more easily characterized.

Figure 2. Leg neuropils.⟦>zach claim=b7856315-9b56-4c97-97c7-4f3e2e2a785d: @{Figure 2. Leg neuropils.} leg-neuropils-consistent-innervation⟧

( A ) 3D rendering of leg neuropils as annotated from averaged synapsin volume with posterior oblique, anterior oblique, and dorsal views, left to right.⟦>zach claim=no-assertion: @{( A ) 3D rendering of leg neuropils as annotated from averaged synapsin volume with posterior oblique, anterior oblique, and dorsal views, left to right.} Pure graphical encoding: which volume the rendering came from and the order of the three views.⟧

( B ) Maximum intensity projections of z -planes 160–190 for leg neuropil expression of synapsin (α-synapsin, gray), ( i ) serotonergic (α-5-HT, green), (ii) octopaminergic/tyraminergic (α-TDC2, magenta), (iii) cholinergic (α-ChAT, cyan), (iv) proctolin (α-Proctolin, yellow), ( v ) FMRFamide (α-FMRFamide, red), (vi) cardioactive peptide (α-CCAP, cyan), and (vii) allatostatin A (α-AstA, green) immunoreactivity in the standard brain.⟦>zach claim=no-assertion: @{( B ) Maximum intensity projections of z -planes 160–190 for leg neuropil expression of synapsin (α-synapsin, gray), ( i ) serotonergic (α-5-HT, green), (ii) octopaminergic/tyraminergic (α-TDC2, magenta), (iii) cholinergic (α-ChAT, cyan), (iv) proctolin (α-Proctolin, yellow), ( v ) FMRFamide (α-FMRFamide, red), (vi) cardioactive peptide (α-CCAP, cyan), and (vii) allatostatin A (α-AstA, green) immunoreactivity in the standard brain.} A colour key listing which antiserum each subpanel carries, not a statement of what the staining revealed.⟧

Dotted perimeter marks the boundary of Leg Neuropil 1. ( C ) Maximum intensity projections of z -planes 230–260. Dotted perimeter marks the boundary of Leg Neuropil 1. ( D ) Maximum intensity projection showing dopaminergic (α-TH, green) immunoreactivity, dotted perimeter marks the boundary of Leg Neuropil 1. Dopaminergic immunoreactivity is shown in a separate panel because this volume, unlike the other stains, is not aligned to the standard atlas.⟦>zach claim=gap: @{Dotted perimeter marks the boundary of Leg Neuropil 1. ( C ) Maximum intensity projections of z -planes 230–260. Dotted perimeter marks the boundary of Leg Neuropil 1. ( D ) Maximum intensity projection showing dopaminergic (α-TH, green) immunoreactivity, dotted perimeter marks the boundary of Leg Neuropil 1. Dopaminergic immunoreactivity is shown in a separate panel because this volume, unlike the other stains, is not aligned to the standard atlas.} Beyond the perimeter and projection notes, this records that the dopaminergic volume is not registered to the standard atlas — a methodological limitation no claim in the tree captures.⟧

Compass abbreviations: A = anterior, P = posterior, D = dorsal, V = ventral, L = left, R = right.

Figure 2—video 1. Leg neuropil 3D volume.⟦>zach claim=b7856315-9b56-4c97-97c7-4f3e2e2a785d: @{Figure 2—video 1. Leg neuropil 3D volume.} leg-neuropils-consistent-innervation⟧

Figure 2—video 2. Neurotransmitter immunostains in the leg neuropil.⟦>zach claim=b7856315-9b56-4c97-97c7-4f3e2e2a785d: @{Figure 2—video 2. Neurotransmitter immunostains in the leg neuropil.} leg-neuropils-consistent-innervation⟧

Figure 2—video 3. Neuropeptide immunostains in the leg neuropil.⟦>zach claim=b7856315-9b56-4c97-97c7-4f3e2e2a785d: @{Figure 2—video 3. Neuropeptide immunostains in the leg neuropil.} leg-neuropils-consistent-innervation⟧

Most notably in the neuromeres of Legs I (anterior), the serotonergic (anti-5-HT) innervation in the limb neuroarchitecture appears to be supplied in two roughly equal halves, filling the periphery and leaving an area dark of immunoreactivity within ( Figure 2Bi – dotted boundary).

The anterior half of the innervation appears to be supplied from the medial branch of the ‘dorsal-most tract’ (as referenced by Auletta et al., 2020 ).

Dopaminergic signal (anti-TH) is more expansive, evenly filling each LN with a mesh-like network of TH+ varicosities ( Figure 2D ).⟦>zach claim=a527b1e4-3c93-4579-9e3f-4e079a34a78f: @{Dopaminergic signal (anti-TH) is more expansive, evenly filling each LN with a mesh-like network of TH+ varicosities ( Figure 2D ).} leg-neuropil-serotonin-bilateral-innervation — The claim contrasts the serotonergic halves with exactly this uniform, mesh-like dopaminergic innervation of the leg neuropils.⟧

Unlike the approximately uniform innervation produced by dopaminergic neurons, the pattern in TDC2 staining is notably different.

The anterior side of each neuromere contains a patch of continuous, diffuse, and more lightly stained immunoreactivity, while on each posterior side, there is a swath of brightly reactive, sparse puncta ( Figure 2Bii ).

Cholinergic ChAT immunoreactivity is punctate and broadly filling of the neuropil at the ventral end ( Figure 2Biii ) and more limited to the medial portion on the dorsal end ( Figure 2Ciii ).

AstA immunoreactivity has a distinctive pattern within the leg neuromeres, showing robust varicosities but only in the dorsal–posterior portion of neuropil ( Figure 2Cvii ).

This innervation appears to be supplied from the lateral branches of the centro-lateral tract.

A similar pattern regarding expression only in the posterior aspect of the neuropil is observed for proctolin immunoreactivity ( Figure 2Civ ).

CCAP immunoreactivity within the LNs is predominantly in the posterior halves, where sparse puncta are evenly distributed ( Figure 2B, Cvi ).⟦>zach claim=gap: @{CCAP immunoreactivity within the LNs is predominantly in the posterior halves, where sparse puncta are evenly distributed ( Figure 2B, Cvi ).} No claim in the tree records CCAP immunoreactivity or its posterior bias within the leg neuropils.⟧

FMRFamide immunoreactivity is also evident in the LNs ( Figure 2B, Cv ).⟦>zach claim=gap: @{FMRFamide immunoreactivity is also evident in the LNs ( Figure 2B, Cv ).} FMRFamide immunoreactivity in the leg neuropils is reported here but appears in no claim.⟧

Opisthosomal neuropil The OpN is found at the posterior and dorsal-most extension of the subesophageal mass ( Figure 3A ; Figure 3—videos 1–3 ).⟦>zach claim=gap: @{Opisthosomal neuropil The OpN is found at the posterior and dorsal-most extension of the subesophageal mass ( Figure 3A ; Figure 3—videos 1–3 ).} The position of the opisthosomal neuropil at the posterior-dorsal extent of the subesophageal mass is asserted here and in no claim.⟧

The region of the OpN has also been called the abdominal ganglia ( Foelix, 2025 ), and tapers posteriorly to give way to the cauda equina, a nerve bundle composed of multiple paired tracts which innervate organs within the opisthosoma, such as the gonads, book lungs, and spinnerets ( Gonzalez-Fernandez and Sherman, 1984 ).

Within the OpN, there are a number of antero-posterior longitudinal tracts, while a ladder-like appearance of medio-laterally running tracts in the ⍺-synapsin channel can also be observed ( Figure 3B, C ).⟦>zach claim=gap: @{Within the OpN, there are a number of antero-posterior longitudinal tracts, while a ladder-like appearance of medio-laterally running tracts in the ⍺-synapsin channel can also be observed ( Figure 3B, C ).} The ladder-like and longitudinal tract architecture seen in the synapsin channel itself is unclaimed; the tree's ladder claim is specifically about TDC2.⟧

Posteriorly traveling tracts also diverge laterally to follow the circumference of the OpN ( Figure 3B–D ).⟦>zach claim=gap: @{Posteriorly traveling tracts also diverge laterally to follow the circumference of the OpN ( Figure 3B–D ).} The lateral divergence of posteriorly travelling tracts around the neuropil circumference is described here without a corresponding claim.⟧

Figure 3. Opisthosomal neuropil.⟦>zach claim=no-assertion: @{Figure 3. Opisthosomal neuropil.} A bare figure title.⟧

( A ) 3D rendering of the opisthosomal neuropil as annotated from averaged synapsin volume with posterior oblique, anterior oblique, and dorsal views, left to right.⟦>zach claim=no-assertion: @{( A ) 3D rendering of the opisthosomal neuropil as annotated from averaged synapsin volume with posterior oblique, anterior oblique, and dorsal views, left to right.} Graphical encoding: the source volume and the order of views in the rendering.⟧

( B ) Maximum intensity projections of z -planes 415–425 for opisthosomal neuropil expression of synapsin (α-synapsin, gray), ( i ) serotonergic (α-5-HT, green), (ii) octopaminergic/tyraminergic (α-TDC2, magenta), (iii) cholinergic (α-ChAT, cyan), (iv) proctolin (α-Proctolin, yellow), ( v ) FMRFamide (α-FMRFamide, red), (vi) cardioactive peptide (α-CCAP, cyan), and (vii) allatostatin A (α-AstA, green) immunoreactivity in the standard brain.⟦>zach claim=no-assertion: @{( B ) Maximum intensity projections of z -planes 415–425 for opisthosomal neuropil expression of synapsin (α-synapsin, gray), ( i ) serotonergic (α-5-HT, green), (ii) octopaminergic/tyraminergic (α-TDC2, magenta), (iii) cholinergic (α-ChAT, cyan), (iv) proctolin (α-Proctolin, yellow), ( v ) FMRFamide (α-FMRFamide, red), (vi) cardioactive peptide (α-CCAP, cyan), and (vii) allatostatin A (α-AstA, green) immunoreactivity in the standard brain.} A channel-and-colour key for the subpanels, not a finding.⟧

Arrow in the α-TDC2 subpanel marks tracks along the perimeter of the opisthosomal neuropil, supplied anteriorly (brace).

Arrowheads in α-TDC2 and α-Proctolin subpanels mark a neurite running centrally.

( C ) Maximum intensity projections of z -planes 448–465. Longitudinal and lateral projections form a ladder-like structure in the α-TDC2 subpanel (brace).⟦>zach claim=a7565a82-5c41-40d9-a3d3-5e9194904515: @{( C ) Maximum intensity projections of z -planes 448–465. Longitudinal and lateral projections form a ladder-like structure in the α-TDC2 subpanel (brace).} opisthosomal-neuropil-tdc2-perimeter-ladder — The claim states that TDC2 longitudinal and lateral projections form a ladder-like structure in the opisthosomal neuropil, which is what this panel description reports.⟧

A midline-crossing structure (arrow) is seen in the α-Proctolin subpanel.

( D ) Maximum intensity projection showing dopaminergic (α-TH, green) immunoreactivity in the opisthosomal neuropil.⟦>zach claim=no-assertion: @{( D ) Maximum intensity projection showing dopaminergic (α-TH, green) immunoreactivity in the opisthosomal neuropil.} A panel label naming the channel and its colour, with no described pattern.⟧

Compass abbreviations: A = anterior, P = posterior, D = dorsal, V = ventral, L = left, R = right.

Figure 3—video 1. Opisthosomal 3D volume.⟦>zach claim=no-assertion: @{Figure 3—video 1. Opisthosomal 3D volume.} A bare video title.⟧

Figure 3—video 2. Neurotransmitter immunostains in the opisthosomal neuropil.⟦>zach claim=no-assertion: @{Figure 3—video 2. Neurotransmitter immunostains in the opisthosomal neuropil.} A bare video title.⟧

Figure 3—video 3. Neuropeptide immunostains in the opisthosomal neuropil.⟦>zach claim=no-assertion: @{Figure 3—video 3. Neuropeptide immunostains in the opisthosomal neuropil.} A bare video title.⟧

Particular OpN features are revealed by a number of specific immunostains, including the monoamines octopamine/tyramine (TDC2 immunoreactivity), as well as neuropeptides such as proctolin and allatostatin A. TDC2 immunoreactivity displays an intricate pattern within the OpN.

At the ventral anterior end, two triangular formations of puncta ( Figure 3Bii – brace) abut the input of a string of varicosities on each lateral side, which then become heavier and continue to outline the boundary of the OpN ( Figure 3Bii – arrow).

The lateral perimeter tracts are likewise revealed by ChAT and proctolin immunoreactivity ( Figure 3B, Ciii, iv ) as well as by fibers from dopaminergic populations (anti-TH) ( Figure 3D ).⟦>zach claim=a7565a82-5c41-40d9-a3d3-5e9194904515: @{The lateral perimeter tracts are likewise revealed by ChAT and proctolin immunoreactivity ( Figure 3B, Ciii, iv ) as well as by fibers from dopaminergic populations (anti-TH) ( Figure 3D ).} opisthosomal-neuropil-tdc2-perimeter-ladder — The claim states that this perimeter-tract pattern is also partially revealed by proctolin and dopaminergic innervation, which is the finding reported here.⟧

Within the interior, fibers resembling spokes emanate to a ring-like midline where there appears to be a small decussation and a thicker bridge structure joining lateral segments which travel in the anterior–posterior direction.

The fine neurites projecting to the center of the OpN as seen for TDC2 immunoreactivity are also apparent for proctolin immunoreactivity ( Figure 3Bii, iv – arrowhead).

Proctolin signal also reveals a more posterior and dorsal crossing-over point ( Figure 3Civ – arrow).

Intense boutons line a tract running parallel to the midline, while also giving rise dorsally and laterally to a ladder-like structure of projections in the anterior–posterior direction, which can best be seen with octopaminergic/tyraminergic innervation (TDC2+) ( Figure 3Cii – brace) and dopaminergic signal (TH+) ( Figure 3D ).⟦>zach claim=a7565a82-5c41-40d9-a3d3-5e9194904515: @{Intense boutons line a tract running parallel to the midline, while also giving rise dorsally and laterally to a ladder-like structure of projections in the anterior–posterior direction, which can best be seen with octopaminergic/tyraminergic innervation (TDC2+) ( Figure 3Cii – brace) and dopaminergic signal (TH+) ( Figure 3D ).} opisthosomal-neuropil-tdc2-perimeter-ladder — This is the ladder-like arrangement of longitudinal and lateral projections, best seen with TDC2 and TH, that the claim asserts.⟧

AstA+ immunoreactivity is also found in the OpN, with a more confined medial density in the dorso-posterior section, a pattern generally shared with 5HT and proctolin immunoreactivity ( Figure 3Cvii ).

Ample FMRFamide signal is seen within the opisthosomal neuromere, but its signal is more uniform than the other targets investigated ( Figure 3B, Cv ).⟦>zach claim=gap: @{Ample FMRFamide signal is seen within the opisthosomal neuromere, but its signal is more uniform than the other targets investigated ( Figure 3B, Cv ).} FMRFamide innervation of the opisthosomal neuromere, and its comparative uniformity, is reported here but claimed nowhere.⟧

Pedipalpal neuropil As the LNs begin to diminish in the dorsal region of the subesophageal mass, a smaller antero-lateral synaptic density becomes apparent, representing the pedipalpal neuropil (PdN) ( Figure 4A ; Figure 4—videos 1 and 2 ), which serves the anterior-facing pedipalp appendages.⟦>zach claim=gap: @{Pedipalpal neuropil As the LNs begin to diminish in the dorsal region of the subesophageal mass, a smaller antero-lateral synaptic density becomes apparent, representing the pedipalpal neuropil (PdN) ( Figure 4A ; Figure 4—videos 1 and 2 ), which serves the anterior-facing pedipalp appendages.} The position and identification of the pedipalpal neuropil is an anatomical assertion; the tree's pedipalpal claim covers only which transmitters dominate it.⟧

Immunoreactivity for ChAT (acetylcholine) and TDC2 (octopamine/tyramine) is strongest among the antisera tested for the PdN, showing punctate expression which does not extend to fill the anterior portion of the neuropil ( Figure 4Biii and ii , respectively).

No additional structural features were evident within the PdN.

Little to no appreciable immunoreactivity was seen for allatostatin A or CCAP antisera in our defined bounds of the PdN.

This was also true of proctolin immunoreactivity, although signal is present in an immediately medial, adjacent region ( Figure 4Biv ), which is also highlighted by 5-HT immunoreactivity (see z275 in atlas).

This area appears to be supplied by at least two anteriorly located proctolin+ somata ( Figure 4Biv – arrow).

Figure 4. Pedipalp, Blumenthal, and cheliceral neuropil.⟦>zach claim=no-assertion: @{Figure 4. Pedipalp, Blumenthal, and cheliceral neuropil.} A bare figure title.⟧

( A ) 3D rendering of the pedipalp neuropil as annotated from averaged synapsin volume with posterior oblique and anterior oblique views, left to right, and dorsal view, lower.⟦>zach claim=no-assertion: @{( A ) 3D rendering of the pedipalp neuropil as annotated from averaged synapsin volume with posterior oblique and anterior oblique views, left to right, and dorsal view, lower.} Graphical encoding: the source volume and the arrangement of views.⟧

( B ) Optical slices (z275) from the standard brain, containing a cross-section of the pedipalp neuropil (dashed line boundary), with expression of (i) synapsin (α-synapsin, gray), (ii) octopaminergic/tyraminergic (α-TDC2, magenta), (iii) cholinergic (α-ChAT, cyan), and (iv) proctolin (α-Proctolin, yellow) immunoreactivity.⟦>zach claim=no-assertion: @{( B ) Optical slices (z275) from the standard brain, containing a cross-section of the pedipalp neuropil (dashed line boundary), with expression of (i) synapsin (α-synapsin, gray), (ii) octopaminergic/tyraminergic (α-TDC2, magenta), (iii) cholinergic (α-ChAT, cyan), and (iv) proctolin (α-Proctolin, yellow) immunoreactivity.} A key giving the optical plane, the dashed boundary convention, and the colour of each channel.⟧

Arrow in α-Proctolin shows adjacent proctolin+ somata.

( C ) Optical slices (z360) from the standard brain, with a cropped and enlarged selection showing the Blumenthal neuropil (brace), with expression of (i) synapsin (α-synapsin, gray) and proctolin (α-Proctolin, yellow) immunoreactivity above, and only (ii) proctolin below.⟦>zach claim=no-assertion: @{( C ) Optical slices (z360) from the standard brain, with a cropped and enlarged selection showing the Blumenthal neuropil (brace), with expression of (i) synapsin (α-synapsin, gray) and proctolin (α-Proctolin, yellow) immunoreactivity above, and only (ii) proctolin below.} A key giving the optical plane, the crop, the brace convention, and which channels appear above and below.⟧

A circular region of the Blumenthal neuropil (dashed oval) shows proctolin immunoreactivity.

( D ) 3D rendering of the cheliceral neuropil as annotated from averaged synapsin volume with posterior oblique and anterior oblique views, left to right, and dorsal view, lower.⟦>zach claim=no-assertion: @{( D ) 3D rendering of the cheliceral neuropil as annotated from averaged synapsin volume with posterior oblique and anterior oblique views, left to right, and dorsal view, lower.} Graphical encoding: the source volume and the arrangement of views.⟧

( E ) Optical slices (z400) from the standard brain, containing a cross-section of the cheliceral neuropil (dashed line boundary), with expression of (i) synapsin (α-synapsin, gray), (ii) octopaminergic/tyraminergic (α-TDC2, magenta), (iii) allatostatin A (α-AstA, green), (iv) proctolin (α-Proctolin, yellow), (v) serotonergic (α-5-HT, green), and (vi) cholinergic (α-ChAT, cyan) immunoreactivity.⟦>zach claim=no-assertion: @{( E ) Optical slices (z400) from the standard brain, containing a cross-section of the cheliceral neuropil (dashed line boundary), with expression of (i) synapsin (α-synapsin, gray), (ii) octopaminergic/tyraminergic (α-TDC2, magenta), (iii) allatostatin A (α-AstA, green), (iv) proctolin (α-Proctolin, yellow), (v) serotonergic (α-5-HT, green), and (vi) cholinergic (α-ChAT, cyan) immunoreactivity.} A channel-and-colour key for the cheliceral cross-section subpanels.⟧

Arrows in α-TDC2, α-AstA, and α-5-HT subpanels mark an unidentified medially adjacent region to the cheliceral neuropil which shows pronounced immunoreactivity for these antisera.

Compass abbreviations: A = anterior, P = posterior, D = dorsal, V = ventral, L = left, R = right.

Figure 4—video 1. Pedipalp neuropil 3D volume.⟦>zach claim=no-assertion: @{Figure 4—video 1. Pedipalp neuropil 3D volume.} A bare video title.⟧

Figure 4—video 2. Neurotransmitter and neuropeptide immunostains in the pedipalp neuropil.⟦>zach claim=no-assertion: @{Figure 4—video 2. Neurotransmitter and neuropeptide immunostains in the pedipalp neuropil.} A bare video title.⟧

Figure 4—video 3. Cheliceral 3D volume.⟦>zach claim=no-assertion: @{Figure 4—video 3. Cheliceral 3D volume.} A bare video title.⟧

Figure 4—video 4. Neurotransmitter immunostains in the cheliceral neuropil.⟦>zach claim=no-assertion: @{Figure 4—video 4. Neurotransmitter immunostains in the cheliceral neuropil.} A bare video title.⟧

Figure 4—video 5. Neuropeptide immunostains in the cheliceral neuropil.⟦>zach claim=no-assertion: @{Figure 4—video 5. Neuropeptide immunostains in the cheliceral neuropil.} A bare video title.⟧

Figure 4—video 6. Immunostains for α-synapsin and α-proctolin in the blumenthal neuropil.⟦>zach claim=no-assertion: @{Figure 4—video 6. Immunostains for α-synapsin and α-proctolin in the blumenthal neuropil.} A bare video title.⟧

Blumenthal neuropil An additional subesophageal feature previously identified in C. salei is the Blumenthal neuropil ( Anton and Tichy, 1994 ), which is innervated by afferents from the thermoreceptive and hygroreceptive tarsal organ.

Although we also see a paired, synapsin-dense entity close to the midline ( Figure 4C – brace), in the approximate anterio-ventral subesophageal location as described for C. salei , we cannot be certain that this is the same structure – a question which will benefit from tracing techniques.⟦>zach claim=gap: @{Although we also see a paired, synapsin-dense entity close to the midline ( Figure 4C – brace), in the approximate anterio-ventral subesophageal location as described for C. salei , we cannot be certain that this is the same structure – a question which will benefit from tracing techniques.} The observation of a paired midline synapsin-dense entity near the Blumenthal neuropil position, and the stated uncertainty about its identity, is carried by no claim in the tree.⟧

A circular form of saturated proctolin immunoreactivity is seen at the posterior end of an oval-shaped synapsin density ( Figure 4Ci, ii – dashed perimeter), suggesting that it is a subset of a major tract bundle.

In dorsal planes, this immunoreactivity morphs into laterally moving tracts Figure 4—video 6 , becoming difficult to trace.⟦>zach claim=gap: @{In dorsal planes, this immunoreactivity morphs into laterally moving tracts Figure 4—video 6 , becoming difficult to trace.} The dorsal transition of this immunoreactivity into laterally moving tracts is reported here and claimed nowhere.⟧

Immunoreactivity within this synapsin-dense perimeter ( Figure 4Ci ) is not found in the other neurosignaling molecule stains, even those with otherwise abundant subesophageal expression.

Deutocerebral features Cheliceral neuropil At the level of the esophageal passage, an anterior–lateral neuropil begins, wrapping medially to become the ChN ( Figure 4D ; Figure 4—videos 3–5 ).⟦>zach claim=gap: @{Deutocerebral features Cheliceral neuropil At the level of the esophageal passage, an anterior–lateral neuropil begins, wrapping medially to become the ChN ( Figure 4D ; Figure 4—videos 3–5 ).} The course and position of the cheliceral neuropil is asserted here; the tree's cheliceral claim addresses only its transmitter content.⟧

This neuropil is associated with the fanged appendages known as chelicerae, which are used for prey handling and feeding.

Anterograde tracing of the innervation pattern of lyriform organ and tactile hair mechanosensors of the chelicerae of C. salei found projections to terminate in the ChN as well as dorso-ventrally in the sensory longitudinal tracts of the subesophageal mass ( Gorb et al., 1993 ).

The ChN is most abundantly innervated by serotonergic as well as TDC2+ immunoreactivity ( Figure 4Eiv, v ).

Fine varicosities of 5-HT+ immunoreactivity fill the neuropil, while the substantial TDC2+ immunoreactivity appears as large puncta throughout.

TDC2+ expression is also strong in a region immediately medial to the ChN, where 5HT+ immunoreactivity is also found ( Figure 4Ev – arrows).

While AstA+ signal is marginal within the demarcated ChN, strong immunoreactivity is also evident in the adjacent medial region, which overlaps with TDC2+ and 5HT+ expression ( Figure 4Eiii – arrows).

ChAT (cholinergic immunoreactivity) is also present throughout the ChN, while proctolin immunoreactivity is more minor, in the middle and anterior reaches of the structure ( Figure 4Evi, iv ).

Supraesophageal (Protocerebral) neuropils Ventral features The supraesophageal mass begins dorsally to the closure of the esophageal passage, and in its ventral-most planes, can be subdivided anteriorly and posteriorly into two sectors of slightly differing anti-synapsin immunoreactivity ( Figure 5A–C – dashed perimeter, anti-synapsin).⟦>zach claim=gap: @{Supraesophageal (Protocerebral) neuropils Ventral features The supraesophageal mass begins dorsally to the closure of the esophageal passage, and in its ventral-most planes, can be subdivided anteriorly and posteriorly into two sectors of slightly differing anti-synapsin immunoreactivity ( Figure 5A–C – dashed perimeter, anti-synapsin).} The anterior/posterior subdivision of the ventral supraesophageal mass by differing synapsin density is an unclaimed finding.⟧

We will refer to these regions as the anterior and posterior stalk , in order to enable discussion of smaller features of various specific neurostains found within their perimeter.

Lacking any further knowledge, these names are not currently intended to suggest a cohesive form or function for the structures within their bounds.

Figure 5. Ventral supraesophageal features.⟦>zach claim=no-assertion: @{Figure 5. Ventral supraesophageal features.} A bare figure title.⟧

Posterior and anterior stalk region expression of synapsin (α-synapsin, gray), ( i ) serotonergic (α-5-HT, green), (ii) cholinergic (α-ChAT, cyan), (iii) octopaminergic/tyraminergic (α-TDC2, magenta), (iv) proctolin (α-Proctolin, yellow), ( v ) allatostatin A (α-AstA, green), and (vi) cardioactive peptide (α-CCAP, cyan) immunoreactivity in the standard brain, for: ( A ) z -plane 461. Arrow in the α-TDC2 subpanel marks tracks along perimeter of the opisthosomal neuropil, supplied anteriorly (brace).⟦>zach claim=a7565a82-5c41-40d9-a3d3-5e9194904515: @{Posterior and anterior stalk region expression of synapsin (α-synapsin, gray), ( i ) serotonergic (α-5-HT, green), (ii) cholinergic (α-ChAT, cyan), (iii) octopaminergic/tyraminergic (α-TDC2, magenta), (iv) proctolin (α-Proctolin, yellow), ( v ) allatostatin A (α-AstA, green), and (vi) cardioactive peptide (α-CCAP, cyan) immunoreactivity in the standard brain, for: ( A ) z -plane 461. Arrow in the α-TDC2 subpanel marks tracks along perimeter of the opisthosomal neuropil, supplied anteriorly (brace).} opisthosomal-neuropil-tdc2-perimeter-ladder — The arrow-marked finding here — TDC2 tracks along the opisthosomal perimeter, supplied anteriorly — is what the claim asserts.⟧

Top and bottom arrows in α-Proctolin subpanel represent posterior and anterior bridging immunoreactivity, respectively, in the stomodeal bridge (StB) area.

Arrowhead in α-AstA subpanel marks a faint band of immunoreactivity across the midline.

( B ) z -plane 490. Further ventrally, a bridging structure is also visible on the posterior end of the ventral supraesophageal, as seen in the α-Proctolin subpanel (brace).⟦>zach claim=gap: @{( B ) z -plane 490. Further ventrally, a bridging structure is also visible on the posterior end of the ventral supraesophageal, as seen in the α-Proctolin subpanel (brace).} A proctolin-positive bridging structure at the posterior end of the ventral supraesophageal is reported here and appears in no claim.⟧

Cholinergic immunoreactivity is present in the protocerebral tract (PCT), arrowhead in α-ChAT subpanel.

Bands of α-TDC2 immunoreactivity (arrow) which do not correspond to clear structures in the synapsin channel.

( C ) z -plane 511. Arrowhead in the α-ChAT subpanel marks prominent cholinergic immunoreactivity in the PCT, more clearly visible at this plane.⟦>zach claim=gap: @{( C ) z -plane 511. Arrowhead in the α-ChAT subpanel marks prominent cholinergic immunoreactivity in the PCT, more clearly visible at this plane.} Prominent cholinergic immunoreactivity in the protocerebral tract is unclaimed; the tree's cholinergic layering claim concerns the protocerebral bridge, a different structure.⟧

In the α-AstA subpanel, an arrow shows an oxbow-like structure and pronounced innervation at the posterior midline and central anterior stalk AstA+ innervation (brace).

Arrows in the α-TDC2 subpanel mark centrally located concentrations of TDC2 immunoreactivity anterior and lateral to the PCT.

Compass abbreviations: A = anterior, P = posterior, D = dorsal, V = ventral, L = left, R = right.

Figure 5—video 1. Neurotransmitter Immunostains in the Ventral Supraesophageal Ganglion.⟦>zach claim=no-assertion: @{Figure 5—video 1. Neurotransmitter Immunostains in the Ventral Supraesophageal Ganglion.} A bare video title.⟧

Figure 5—video 2. Neuropeptide immunostains in the ventral supraesophageal ganglion.⟦>zach claim=no-assertion: @{Figure 5—video 2. Neuropeptide immunostains in the ventral supraesophageal ganglion.} A bare video title.⟧

The esophageal passage is bridged at the anterior side by a region named the stomodeal bridge (StB) ( Steinhoff et al., 2017 ; Figure 5A – brace).⟦>zach claim=gap: @{The esophageal passage is bridged at the anterior side by a region named the stomodeal bridge (StB) ( Steinhoff et al., 2017 ; Figure 5A – brace).} The identification of the stomodeal bridge in U. diversus is an anatomical assertion no claim in the tree carries.⟧

A bridge structure also exists at the posterior end, where additional undifferentiated synaptic density is flanking ( Figure 5B – brace with asterisk).⟦>zach claim=gap: @{A bridge structure also exists at the posterior end, where additional undifferentiated synaptic density is flanking ( Figure 5B – brace with asterisk).} A posterior bridge structure with flanking undifferentiated synaptic density is reported here and claimed nowhere.⟧

Within these planes, a protocerebral tract (PCT) is essentially parallel to the ventro-dorsal axis and appears as twin, dense nodes rising in the central burgeoning protocerebrum ( Figure 5B, C ). 5-HT immunoreactivity is prominent in the posterior bridging area dorsal to the esophageal passage ( Figure 5B, Ci , Figure 5—video 1 ), as well as the laterally adjacent tissue, and not as apparent in the anterior StB. 5-HT immunoreactivity is otherwise weak within the stalk regions.⟦>zach claim=gap: @{Within these planes, a protocerebral tract (PCT) is essentially parallel to the ventro-dorsal axis and appears as twin, dense nodes rising in the central burgeoning protocerebrum ( Figure 5B, C ). 5-HT immunoreactivity is prominent in the posterior bridging area dorsal to the esophageal passage ( Figure 5B, Ci , Figure 5—video 1 ), as well as the laterally adjacent tissue, and not as apparent in the anterior StB. 5-HT immunoreactivity is otherwise weak within the stalk regions.} The protocerebral tract's twin dense nodes and the distribution of 5-HT across the posterior bridging area versus the stalk regions are unclaimed findings.⟧

TDC2 immunoreactivity is prominent in the StB and adjacent areas, and at this plane, two lateral bands of immunoreactivity appear which do not correspond to a clear demarcation in the synapsin channel ( Figure 5Biii – arrow, Figure 5—video 1 ).⟦>zach claim=gap: @{TDC2 immunoreactivity is prominent in the StB and adjacent areas, and at this plane, two lateral bands of immunoreactivity appear which do not correspond to a clear demarcation in the synapsin channel ( Figure 5Biii – arrow, Figure 5—video 1 ).} TDC2 prominence in the stomodeal bridge and the two lateral bands with no synapsin counterpart are reported here and appear in no claim.⟧

Dorsally, octopaminergic/tyraminergic (TDC2+) signal is prominent along an antero-lateral stretch marking the boundary of what we define as the posterior stalk, a perimeter also visible in the synapsin channel ( Figure 5Ciii ).

Concentrations of TDC2+ immunoreactivity are also apparent centrally, both anterior and lateral to the PCTs ( Figure 5Ciii – arrows, Figure 5—video 1 ).⟦>zach claim=gap: @{Concentrations of TDC2+ immunoreactivity are also apparent centrally, both anterior and lateral to the PCTs ( Figure 5Ciii – arrows, Figure 5—video 1 ).} Central TDC2 concentrations anterior and lateral to the protocerebral tracts are unclaimed.⟧

Similar to what has been described for M. muscosa as the StB ( Steinhoff et al., 2017 ), the area adjacent to the esophagus on the anterior side has immunoreactivity to allatostatin A, although the actual bridge which crosses the midline is modest, with thin representation in the posterior commissure ( Figure 5Av – arrowhead, Figure 5—video 2 ).⟦>zach claim=gap: @{Similar to what has been described for M. muscosa as the StB ( Steinhoff et al., 2017 ), the area adjacent to the esophagus on the anterior side has immunoreactivity to allatostatin A, although the actual bridge which crosses the midline is modest, with thin representation in the posterior commissure ( Figure 5Av – arrowhead, Figure 5—video 2 ).} Allatostatin A immunoreactivity beside the esophagus and its thin representation in the posterior commissure is carried by no claim.⟧

In U. diversus , strong AstA immunoreactivity is present on the posterior side of where the esophagus closes, in the posterior stalk area ( Figure 5Cv ).

The posterior region adjacent to the midline, previously highlighted with 5HT immunoreactivity, also shows partial AstA+ innervation, displaying a unique oxbow type pattern ( Figure 5Cv – arrow, Figure 5—video 2 ).⟦>zach claim=gap: @{The posterior region adjacent to the midline, previously highlighted with 5HT immunoreactivity, also shows partial AstA+ innervation, displaying a unique oxbow type pattern ( Figure 5Cv – arrow, Figure 5—video 2 ).} The oxbow-shaped AstA innervation of the posterior midline-adjacent region is an unclaimed finding.⟧

There is also a patch of AstA+ immunoreactivity in the central area of the anterior stalk ( Figure 5Cv – brace).

On the posterior edge of the StB, there is a thin Proc+ commissure, while the anterior edge of the StB is highlighted by a bolder vein of varicosities ( Figure 5Aiv – arrows).

Proctolin signal is present adjacent to the midline around the posterior bridging area, shared with 5-HT, AstA, and TDC2+ immunoreactivity, and together with the commissure evident by synapsin staining, forms a circular pattern ( Figure 5Biv – brace).

This circular pattern of immunoreactivity is also visible by 5-HT+ immunoreactivity ( Figure 5Bi ), as well as TDC2+ innervation, though more clearly seen further dorsally ( Figure 5Ciii ) for this channel.

There is also proctolin immunoreactivity seen centrally, medial to where the PCT is ascending ( Figure 5B, Civ ).⟦>zach claim=gap: @{There is also proctolin immunoreactivity seen centrally, medial to where the PCT is ascending ( Figure 5B, Civ ).} Central proctolin immunoreactivity medial to the ascending protocerebral tract is reported here and claimed nowhere.⟧

ChAT+ immunoreactivity is diffusely present throughout the stalk regions ( Figure 5Cii ), and unlike the other antisera, co-stains the posterior aspect of the PCT, which is prominently visible with synapsin immunoreactivity ( Figure 5B, Cii – arrowheads and dashed circles).⟦>zach claim=gap: @{ChAT+ immunoreactivity is diffusely present throughout the stalk regions ( Figure 5Cii ), and unlike the other antisera, co-stains the posterior aspect of the PCT, which is prominently visible with synapsin immunoreactivity ( Figure 5B, Cii – arrowheads and dashed circles).} Diffuse cholinergic signal in the stalk regions and the unique ChAT co-staining of the posterior protocerebral tract is unclaimed.⟧

Hagstone neuropil and mid-supraesophageal features We will define the hagstone neuropil as a central, midline adjacent, paired structure, whose ventral bounds share the same plane as the appearance of a prominent protocerebral commissure (standard brain, ~ z = 540), and which continues dorsally until the formation of the MB bridge (standard brain, ~ z = 615).⟦>zach claim=7498d258-3c0e-4a77-bd98-49b7deff2950: @{Hagstone neuropil and mid-supraesophageal features We will define the hagstone neuropil as a central, midline adjacent, paired structure, whose ventral bounds share the same plane as the appearance of a prominent protocerebral commissure (standard brain, ~ z = 540), and which continues dorsally until the formation of the MB bridge (standard brain, ~ z = 615).} hagstone-neuropil-serotonin-defined — The claim introduces the hagstone neuropil as exactly this centrally located, midline-adjacent paired structure of the supraesophageal ganglion.⟧

This approximately kidney-bean-shaped structure is pierced by a circular spot lacking synapsin immunoreactivity ( Figure 6A, C, D – dashed outline, Figure 6—videos 1–3 ) – which may be indicative of a space occupied by a fiber tract, tracheal passageway, and/or potentially glia.⟦>zach claim=gap: @{This approximately kidney-bean-shaped structure is pierced by a circular spot lacking synapsin immunoreactivity ( Figure 6A, C, D – dashed outline, Figure 6—videos 1–3 ) – which may be indicative of a space occupied by a fiber tract, tracheal passageway, and/or potentially glia.} The kidney-bean shape and the central synapsin-negative void, with its candidate explanations, is a finding no claim records.⟧

Figure 6. Hagstone neuropil and mid-supraesophageal features.⟦>zach claim=no-assertion: @{Figure 6. Hagstone neuropil and mid-supraesophageal features.} A bare figure title.⟧

( A ) 3D rendering of the hagstone neuropil as annotated from averaged synapsin volume with posterior oblique, anterior oblique, and dorsal views, top to bottom.⟦>zach claim=no-assertion: @{( A ) 3D rendering of the hagstone neuropil as annotated from averaged synapsin volume with posterior oblique, anterior oblique, and dorsal views, top to bottom.} Graphical encoding: the source volume and the order of views.⟧

( B–D ) Hagstone neuropil and posterior feature expression of synapsin (α-synapsin, gray), ( i ) serotonergic (α-5-HT, green), (ii) proctolin (α-Proctolin, yellow), (iii) octopaminergic/tyraminergic (α-TDC2, magenta), and (iv) allatostatin A (α-AstA, green) immunoreactivity in the standard brain, fo: ( B ) z -plane 540. A circular tract pattern distinctive to the α-5-HT stain is visible in the posterior mid-supraesophageal (brace, α-5-HT subpanel).⟦>zach claim=7498d258-3c0e-4a77-bd98-49b7deff2950: @{( B–D ) Hagstone neuropil and posterior feature expression of synapsin (α-synapsin, gray), ( i ) serotonergic (α-5-HT, green), (ii) proctolin (α-Proctolin, yellow), (iii) octopaminergic/tyraminergic (α-TDC2, magenta), and (iv) allatostatin A (α-AstA, green) immunoreactivity in the standard brain, fo: ( B ) z -plane 540. A circular tract pattern distinctive to the α-5-HT stain is visible in the posterior mid-supraesophageal (brace, α-5-HT subpanel).} hagstone-neuropil-serotonin-defined — The braced feature described here is the distinctive circular serotonergic tract pattern around the supraesophageal midline that the claim asserts.⟧

( C ) z -planes 565, with the boundary of hagstone neuropil (dashed perimeter).⟦>zach claim=no-assertion: @{( C ) z -planes 565, with the boundary of hagstone neuropil (dashed perimeter).} A plane number and the dashed-perimeter convention, with no finding stated.⟧

An umbrella-like innervation pattern is present posteriorly (brace), in both the α-5-HT and α-TDC2 subpanels.

( D ) z -plane 585, with the boundary of hagstone neuropil (dashed perimeter).⟦>zach claim=no-assertion: @{( D ) z -plane 585, with the boundary of hagstone neuropil (dashed perimeter).} A plane number and the dashed-perimeter convention, with no finding stated.⟧

In the α-Proctolin subpanel, a brace marks a crescent-shaped zone of Proctolin immunoreactivity, which has also been observed in another spider species.

PCT = protocerebral tract, MB = mushroom body.

Compass abbreviations: A = anterior, P = posterior, D = dorsal, V = ventral, L = left, R = right.

Figure 6—video 1. Hagstone 3D volume.⟦>zach claim=no-assertion: @{Figure 6—video 1. Hagstone 3D volume.} A bare video title.⟧

Figure 6—video 2. Neurotransmitter immunostains in the hagstone neuropil.⟦>zach claim=no-assertion: @{Figure 6—video 2. Neurotransmitter immunostains in the hagstone neuropil.} A bare video title.⟧

Figure 6—video 3. Neuropeptide immunostains in the hagstone neuropil.⟦>zach claim=no-assertion: @{Figure 6—video 3. Neuropeptide immunostains in the hagstone neuropil.} A bare video title.⟧

The hagstone neuropil, as well as other prominent features of the mid-central protocerebrum, are most clearly defined by serotonergic immunoreactivity.

Serotonergic fibers form a distinctive circular tract pattern around the midline of the supraesophageal ganglion ( Figure 6Bi – brace), not as clearly seen with any other neuronal-subtype stain.

The semi-circular tracts bend medially ( Figure 6Bi ), before a fusion of seemingly all three directions is seen immediately dorsal ( Figure 6C ).⟦>zach claim=gap: @{The semi-circular tracts bend medially ( Figure 6Bi ), before a fusion of seemingly all three directions is seen immediately dorsal ( Figure 6C ).} The medial bending of the semi-circular tracts and their dorsal fusion is a trajectory detail the claim naming the circular serotonergic tract does not assert.⟧

The hagstone neuropil is essentially completely filled with serotonergic immunoreactivity, matching the outline defined in the anti-synapsin channel ( Figure 6C, Di ).⟦>zach claim=7498d258-3c0e-4a77-bd98-49b7deff2950: @{The hagstone neuropil is essentially completely filled with serotonergic immunoreactivity, matching the outline defined in the anti-synapsin channel ( Figure 6C, Di ).} hagstone-neuropil-serotonin-defined — The claim states that the hagstone neuropil is essentially completely filled with serotonergic immunoreactivity matching its synapsin outline.⟧

On the posterior end is a diffuse, arching band of varicosities, with fiber tracts at the midline, resembling an umbrella-like form ( Figure 6C, Di – brace, anti-5-HT).⟦>zach claim=gap: @{On the posterior end is a diffuse, arching band of varicosities, with fiber tracts at the midline, resembling an umbrella-like form ( Figure 6C, Di – brace, anti-5-HT).} The umbrella-like posterior band of serotonergic varicosities with midline fiber tracts is a distinct feature no claim records.⟧

While the umbrella-like band formation is not distinct enough to be reliably annotated from synapsin immunoreactivity, we find that signal within this formation is also visible in other neuromodulator and neuropeptide immunostains.

TDC2 immunoreactivity is also present in the umbrella-like posterior region innervated by 5-HT ( Figure 6Ciii – brace, Figure 6—video 2 ), and sparser puncta within the bounds of the hagstone neuropil.⟦>zach claim=gap: @{TDC2 immunoreactivity is also present in the umbrella-like posterior region innervated by 5-HT ( Figure 6Ciii – brace, Figure 6—video 2 ), and sparser puncta within the bounds of the hagstone neuropil.} TDC2 innervation of the umbrella-like posterior region and its sparse puncta inside the hagstone neuropil is unclaimed.⟧

Proctolin immunoreactivity is present in the posterior, midline-spanning umbrella structure observed for 5-HT and TDC2, as well as fine varicosities in the hagstone neuropil ( Figure 6Dii ).

This is likewise the case for AstA immunoreactivity in the hagstone neuropil, while posteriorly, there is abundant AstA+ signal which partially is coincident with the umbrella-like band.

Posterior to the cup-shaped synaptic density formed by the MB hafts continuing with the rest of the MB, is a crescent of proctolin immunoreactivity ( Figure 6Cii – brace), which also appears to be present in C. salei ( Becherer and Schmid, 1999 ).

ChAT immunoreactivity, as before, is found broadly, including within the hagstone neuropil, though no greater structure is discernible from this channel in the mid-supra otherwise.

It is difficult to ascertain to what degree the innervation in this region is continuous with that of dorsally located features.

Mushroom bodies The mushroom bodies (MBs) of U. diversus ( Figure 7A, B ; Figure 7—video 1 ) tend to show the most robust synapsin immunoreactivity of all structures in the supraesophageal mass ( Figure 7B , maximum intensity projection), indicating a great degree of synaptic density.⟦>zach claim=80deef22-24b8-4140-8141-276873993f34: @{Mushroom bodies The mushroom bodies (MBs) of U. diversus ( Figure 7A, B ; Figure 7—video 1 ) tend to show the most robust synapsin immunoreactivity of all structures in the supraesophageal mass ( Figure 7B , maximum intensity projection), indicating a great degree of synaptic density.} mushroom-bodies-present-asta-exclusive⟧

While web-building species have been reported to have simplified ( Steinhoff et al., 2024 ) or even entirely absent mushroom bodies ( Hanström, 1928 ; Long, 2016 ; Long, 2021 ; Steinhoff et al., 2024 ) although notable exceptions have also been observed such as D. spinosa and A. trifasciata ( Long, 2016 ), these structures are present in U. diversus and retain the complete form seen in more visually reliant species ( Steinhoff et al., 2017 ; Steinhoff et al., 2024 ), even if they are smaller relative to the protocerebrum as a whole ( Figure 7A–C ).⟦>zach claim=80deef22-24b8-4140-8141-276873993f34: @{While web-building species have been reported to have simplified ( Steinhoff et al., 2024 ) or even entirely absent mushroom bodies ( Hanström, 1928 ; Long, 2016 ; Long, 2021 ; Steinhoff et al., 2024 ) although notable exceptions have also been observed such as D. spinosa and A. trifasciata ( Long, 2016 ), these structures are present in U. diversus and retain the complete form seen in more visually reliant species ( Steinhoff et al., 2017 ; Steinhoff et al., 2024 ), even if they are smaller relative to the protocerebrum as a whole ( Figure 7A–C ).} mushroom-bodies-present-asta-exclusive⟧

Figure 7. Mushroom bodies (MBs).⟦>zach claim=01384854-d4e5-4e8c-bf0b-239409c22675: @{Figure 7. Mushroom bodies (MBs).} globuli-cells-cholinergic-gabaergic⟧

( A ) 3D rendering of MB neuropil as annotated from averaged α-synapsin volume, dorsal (top), and oblique posterior (bottom).⟦>zach claim=80deef22-24b8-4140-8141-276873993f34: @{( A ) 3D rendering of MB neuropil as annotated from averaged α-synapsin volume, dorsal (top), and oblique posterior (bottom).} mushroom-bodies-present-asta-exclusive⟧

( B ) Maximum intensity projection of averaged α-synapsin volume, showing the mushroom bodies to be the most strongly immunoreactive structure in the supraesophageal ganglion.⟦>zach claim=80deef22-24b8-4140-8141-276873993f34: @{( B ) Maximum intensity projection of averaged α-synapsin volume, showing the mushroom bodies to be the most strongly immunoreactive structure in the supraesophageal ganglion.} mushroom-bodies-present-asta-exclusive⟧

( C ) Optical sections of the supraesophageal ganglion from an averaged α-synapsin volume (ventral (top) to dorsal (bottom)).⟦>zach claim=no-assertion: @{( C ) Optical sections of the supraesophageal ganglion from an averaged α-synapsin volume (ventral (top) to dorsal (bottom)).} Graphical encoding: which volume the optical sections come from and their ventral-to-dorsal ordering.⟧

The haft, body, and head regions of the MB are labeled.

( D ) α-βTubulin3 (magenta) immunoreactivity aligned with α-Synapsin volume (gray) (compare to bottom portion of previous subfigure) showing the arching form of the mid-line spanning MB bridge.⟦>zach claim=80deef22-24b8-4140-8141-276873993f34: @{( D ) α-βTubulin3 (magenta) immunoreactivity aligned with α-Synapsin volume (gray) (compare to bottom portion of previous subfigure) showing the arching form of the mid-line spanning MB bridge.} mushroom-bodies-present-asta-exclusive — The claim asserts that the mushroom bodies retain the complete haft-body-head form with the midline-spanning connecting bridge shown here.⟧

( E ) Allatostatin A immunoreactivity (α-AstA, green) present in the MB haft (pink dotted line marking location of α-synapsin immunoreactivity) with arrows pointing to innervation from the posterior side.⟦>zach claim=80deef22-24b8-4140-8141-276873993f34: @{( E ) Allatostatin A immunoreactivity (α-AstA, green) present in the MB haft (pink dotted line marking location of α-synapsin immunoreactivity) with arrows pointing to innervation from the posterior side.} mushroom-bodies-present-asta-exclusive⟧

( F ) α-βTubulin3 (magenta) and α-Synapsin (green) immunoreactivity in the supraesophageal ganglion at the plane where the MB hafts appear (round, intensely immunoreactive).⟦>zach claim=80deef22-24b8-4140-8141-276873993f34: @{( F ) α-βTubulin3 (magenta) and α-Synapsin (green) immunoreactivity in the supraesophageal ganglion at the plane where the MB hafts appear (round, intensely immunoreactive).} mushroom-bodies-present-asta-exclusive — The presence of the mushroom body hafts shown in this plane is part of the complete haft-body-head form the claim asserts.⟧

Arrows mark a fiber tract flanking the haft which could be the origin of the innervation in the preceding subfigure.

( G ) Tripart tract entering at the MB head to fuse with the tract descending through the MB.⟦>zach claim=gap: @{( G ) Tripart tract entering at the MB head to fuse with the tract descending through the MB.} The tripart tract entering the mushroom body head and fusing with the descending tract is a structural finding no claim records.⟧

( H ) Cropped optical section from an individual cholinergic stain (α-ChAT, cyan), of a plane just dorsal to the MB heads, showing putative globuli cells (arrows) within the protrusion of the secondary visual pathway, DAPI stain (red).⟦>zach claim=01384854-d4e5-4e8c-bf0b-239409c22675: @{( H ) Cropped optical section from an individual cholinergic stain (α-ChAT, cyan), of a plane just dorsal to the MB heads, showing putative globuli cells (arrows) within the protrusion of the secondary visual pathway, DAPI stain (red).} globuli-cells-cholinergic-gabaergic⟧

( I ) Cropped optical slice from standard brain (z609) at the level of MB heads, with Synapsin (α-synapsin, gray), GABAergic (α-GAD, red), and merged immunoreactivity, with arrow indicating potential innervation of the globuli cells.

Compass abbreviations: A = anterior, P = posterior, D = dorsal, V = ventral, L = left, R = right.

Figure 7—video 1. Mushroom body 3D volume.⟦>zach claim=01384854-d4e5-4e8c-bf0b-239409c22675: @{Figure 7—video 1. Mushroom body 3D volume.} globuli-cells-cholinergic-gabaergic⟧

U. diversus MBs display a haft, body, and head region, with the two pairs connected by a bridge ( Figure 7A–C ).⟦>zach claim=80deef22-24b8-4140-8141-276873993f34: @{U. diversus MBs display a haft, body, and head region, with the two pairs connected by a bridge ( Figure 7A–C ).} mushroom-bodies-present-asta-exclusive⟧

Synapsin immunoreactivity is modest within the bridge region, whose true thickness is better visualized with staining for βTubulin3 ( Figure 7D ).⟦>zach claim=gap: @{Synapsin immunoreactivity is modest within the bridge region, whose true thickness is better visualized with staining for βTubulin3 ( Figure 7D ).} That the bridge is only modestly synapsin-immunoreactive and its thickness is better seen with βTubulin3 is an unclaimed observation.⟧

Despite the strong synapsin immunoreactivity in the MBs, we surprisingly did not see co-expression with most of our specific neurosignaling molecule antibodies.

This pattern is also reflected in the extant spider literature, with a single study showing immunoreactivity in the mushroom bodies of C. salei for anti-GAD and anti-proctolin staining ( Becherer and Schmid, 1999 ).

A particularly intensely stained MB bridge was also seen in A. sclopetarius using a non-antisera stain of acetylcholinesterase activity ( Meyer and Pospiech, 1977 ).

In our hands, only anti-allatostatin A staining showed co-expression throughout the MB ( Figure 7E ).⟦>zach claim=80deef22-24b8-4140-8141-276873993f34: @{In our hands, only anti-allatostatin A staining showed co-expression throughout the MB ( Figure 7E ).} mushroom-bodies-present-asta-exclusive⟧

Although difficult to trace the source, it appears the hafts are innervated from the posterior side ( Figure 7F – arrows).⟦>zach claim=gap: @{Although difficult to trace the source, it appears the hafts are innervated from the posterior side ( Figure 7F – arrows).} Posterior innervation of the mushroom body hafts is reported here and appears in no claim.⟧

By βTubulin3 immunoreactivity, we observe two tracts which straddle the MB hafts as they descend from the dorsal somata layer ( Figure 7G ).⟦>zach claim=gap: @{By βTubulin3 immunoreactivity, we observe two tracts which straddle the MB hafts as they descend from the dorsal somata layer ( Figure 7G ).} The two βTubulin3 tracts straddling the hafts as they descend from the somata layer are unclaimed.⟧

Finer neurites are not distinguishable in the βTub3 immunoreactivity, but it is plausible that the AstA+ neurites entering the MB hafts might stem from the medial of these two tracts.

Babu and Barth, 1984 described the protocerebro-dorsal tract as providing input to the hafts of the mushroom bodies.

The connection of this tract to the MB hafts is not apparent by synapsin or βTubulin3 immunoreactivity in U. diversus , which was likewise the case with silver staining for P. amentata , M. muscosa , A. bruennichi , and P. tepidariorum ( Steinhoff et al., 2024 ).

The antero-dorsal input to the MB heads, representing the secondary eye pathway ( Strausfeld and Barth, 1993 ), is much more conspicuous and has received considerable treatment within the literature.

The MB heads are sometimes referred to as the third-order visual neuropil in this pathway, with the ample parallel fibers which give this structure its shape arising from globuli cells which cap the MB head.

The globuli cells are not distinguishable from the surrounding nuclei by DAPI signal, but can potentially be discerned through specific neurosignaling molecule immunostains.

We find the cluster of cells closely associated with the MB heads is revealed by ChAT immunoreactivity, and to a lesser extent by GAD immunoreactivity, suggesting they represent cholinergic and GABAergic populations, respectively ( Figure 7H, I – arrow(s)).⟦>zach claim=01384854-d4e5-4e8c-bf0b-239409c22675: @{We find the cluster of cells closely associated with the MB heads is revealed by ChAT immunoreactivity, and to a lesser extent by GAD immunoreactivity, suggesting they represent cholinergic and GABAergic populations, respectively ( Figure 7H, I – arrow(s)).} globuli-cells-cholinergic-gabaergic⟧

Globuli cells in C. salei have previously been shown to be ChAT+ ( Fabian-Fine et al., 2017 ).

By βTubulin3 staining, we also observed a trident of tracts feeding into the dorsal aspect of the MB head ( Figure 7G ).⟦>zach claim=gap: @{By βTubulin3 staining, we also observed a trident of tracts feeding into the dorsal aspect of the MB head ( Figure 7G ).} The trident of tracts feeding the dorsal aspect of the mushroom body head is a structural finding no claim carries.⟧

Visual system U. diversus , like many orb weavers, builds its web at night and can do so in essentially complete darkness in laboratory conditions, suggesting that vision is expendable to much of the spider’s behavioral repertoire ( Eberhard, 1971 ).

Web-building spiders are considered to have poorer vision than spiders which depend on sight to capture prey, which is reflected in their diminished optic neuropils and tract pathways ( Long, 2021 ; Rivera‐Quiroz and Miller, 2022 ; Steinhoff et al., 2024 ).

The number and particularly size and spatial arrangement of spider eyes is variable and characteristic across species, but most commonly, spiders have eight eyes which give rise to two visual system pathways, the principal and secondary eye pathways ( Strausfeld and Barth, 1993 ; Strausfeld and Barth, 1993 ; Long, 2021 ; Steinhoff et al., 2024 ).

The pair of anterior medial eyes is also known as the principal eyes which innervate the principal pathway terminating primarily in the arcuate body.

The remaining pairs are the six secondary eyes, including the anterior lateral eyes, posterior medial eyes, and posterior lateral eyes, whose projections form the secondary eye pathway.

U. diversus has eight eyes which are approximately equal in size.

Relative to cursorial species ( Babu and Barth, 1984 ; Steinhoff et al., 2017 ; Steinhoff et al., 2024 ), in U. diversus the anterior extensions of the protocerebrum containing the first and second-order optic neuropils are considerably thinner and not as extensively fused with the continuous neuropil of the supraesophageal mass, thus being prone to separating during dissection.

Consequently, neither the primary nor secondary visual pathway neuropils appear reliably enough in the anti-synapsin volumes to be apparent in the averaged standard brain representation, but nevertheless, these structures are exhibited in various individual preparations.

The optic neuropils in U. diversus tended to show weaker synapsin immunoreactivity but were clearly seen with antisera to HRP ( Figure 8A ).⟦>zach claim=gap: @{The optic neuropils in U. diversus tended to show weaker synapsin immunoreactivity but were clearly seen with antisera to HRP ( Figure 8A ).} The weak synapsin but clear HRP labelling of the optic neuropils is an unclaimed methodological and anatomical finding.⟧

Figure 8. Visual pathways.⟦>zach claim=no-assertion: @{Figure 8. Visual pathways.} A bare figure title.⟧

( A ) Immunostaining for α-HRP (magenta) for neuropil and use of DAPI (blue) for nuclei, arrows show the primary (1°) and secondary (2°) visual pathway extensions from the bulk of the supraesophageal tissue.⟦>zach claim=no-assertion: @{( A ) Immunostaining for α-HRP (magenta) for neuropil and use of DAPI (blue) for nuclei, arrows show the primary (1°) and secondary (2°) visual pathway extensions from the bulk of the supraesophageal tissue.} A colour key and an arrow convention for the panel.⟧

( B ) 3D renderings of synapsin immunoreactivity in the dorsal supraesophageal ganglion, with tissue of the primary (1°) and secondary (2°) visual pathway visible.⟦>zach claim=no-assertion: @{( B ) 3D renderings of synapsin immunoreactivity in the dorsal supraesophageal ganglion, with tissue of the primary (1°) and secondary (2°) visual pathway visible.} Graphical encoding: what the rendering contains and which region it covers.⟧

Brace shows the path of the secondary pathway input to the mushroom body head.

Compass abbreviations: A = anterior, P = posterior, D = dorsal, V = ventral, L = left, R = right.

As in other species, the secondary pathway is larger ( Figure 8B ), lifting away anteriodorsally from the zone of the MB heads.⟦>zach claim=gap: @{As in other species, the secondary pathway is larger ( Figure 8B ), lifting away anteriodorsally from the zone of the MB heads.} The larger secondary visual pathway and its anteriodorsal departure from the mushroom body heads is reported here and claimed nowhere.⟧

This continuity can be seen from the sliced three-dimensional maximum intensity projection of synapsin ( Figure 8B – brace).⟦>zach claim=no-assertion: @{This continuity can be seen from the sliced three-dimensional maximum intensity projection of synapsin ( Figure 8B – brace).} Narration pointing at where a continuity already stated elsewhere can be seen, rather than a result of its own.⟧

The primary pathway is diminutive in U. diversus and emerges as a bulbous shape at the dorsal-most end of the brain through a field of somata ( Figure 8A ).⟦>zach claim=gap: @{The primary pathway is diminutive in U. diversus and emerges as a bulbous shape at the dorsal-most end of the brain through a field of somata ( Figure 8A ).} The diminutive, bulbous primary visual pathway emerging at the dorsal-most brain is an unclaimed finding.⟧

Previous reports have used GABA ( Becherer and Schmid, 1999 ), histamine ( Schmid and Duncker, 1993 ), dopamine ( Auletta et al., 2020 ), CCAP ( Loesel et al., 2011 ), and FMRFamide ( Becherer and Schmid, 1999 ) to reveal the successive neuropils of the visual pathways.

As noted above, the only features within the optic pathway for which we observed neurosignaling molecule immunoreactivity were the globuli cells with GAD and ChAT staining.

It is possible that targets for which we could not acquire an effective antisera, such as histamine, could be revelatory of the optic lamellae and other visual pathway structures, as they have been for C. salei ( Schmid and Becherer, 1999 ; Schmid and Duncker, 1993 ).

Specific compartments of the pathways, such as the medulla or lamellae, could not be confidently discerned with any preparation.

Tonsillar neuropil Within the historically non-descript central protocerebrum, we observed a synaptically dense neuropil structure in U. diversus .

Beginning in the planes dorsal to the mushroom bodies, this paired structure is positioned directly on either side of the midline and is centrally located.

Each half has an approximately ovoid appearance, particularly at the anterio-dorsal end, while being bridged at the posterior aspect.

Between the two halves, at the midline, is a furrow which is negative for synapsin immunoreactivity, giving this neuropil, in conjunction with the synapsin-negative zone, a likeness to tonsils when viewed from the horizontal optical planes ( Figure 9A, C – α-synapsin, Figure 9—videos 1–3 ) – hence our referral to this structure as the tonsillar neuropil.⟦>zach claim=7fe2b8d9-167a-4743-93af-6795c3c4f34c: @{Between the two halves, at the midline, is a furrow which is negative for synapsin immunoreactivity, giving this neuropil, in conjunction with the synapsin-negative zone, a likeness to tonsils when viewed from the horizontal optical planes ( Figure 9A, C – α-synapsin, Figure 9—videos 1–3 ) – hence our referral to this structure as the tonsillar neuropil.} tonsillar-neuropil-novel-structure — This is the identification and naming of the previously undocumented tonsillar neuropil that the claim asserts.⟧

Figure 9. Tonsillar neuropil.⟦>zach claim=no-assertion: @{Figure 9. Tonsillar neuropil.} A bare figure title.⟧

( A ) 3D rendering of tonsillar neuropil as annotated from averaged synapsin volume with posterior oblique, anterior oblique, and dorsal views, left to right.⟦>zach claim=no-assertion: @{( A ) 3D rendering of tonsillar neuropil as annotated from averaged synapsin volume with posterior oblique, anterior oblique, and dorsal views, left to right.} Graphical encoding: the source volume and the order of views.⟧

( B ) Oblique horizontal optical section of supraesophageal ganglion with α-Synapsin (green) and α-βTubulin3 (magenta) immunoreactivity.⟦>zach claim=no-assertion: @{( B ) Oblique horizontal optical section of supraesophageal ganglion with α-Synapsin (green) and α-βTubulin3 (magenta) immunoreactivity.} A description of the section orientation and the colour of each channel.⟧

The tonsillar neuropil is seen centrally, with the arrow denoting a fiber tract which passes medially across it.

( C ) Ventral (z682) and dorsal (z700) views of the tonsillar neuropil, demarcated by dashed lines, from the standard brain, showing expression of synapsin (gray), (i, vi) serotonergic (α-5-HT, green), (ii, vii) octopaminergic/tyraminergic (α-TDC2, magenta), (iii, viii) proctolin (α-Proctolin, yellow), (iv, ix) allatostatin A (α-AstA, green) and (v, x ) FMRFamide (α-FMRFamide, red) immunoreactivity.⟦>zach claim=no-assertion: @{( C ) Ventral (z682) and dorsal (z700) views of the tonsillar neuropil, demarcated by dashed lines, from the standard brain, showing expression of synapsin (gray), (i, vi) serotonergic (α-5-HT, green), (ii, vii) octopaminergic/tyraminergic (α-TDC2, magenta), (iii, viii) proctolin (α-Proctolin, yellow), (iv, ix) allatostatin A (α-AstA, green) and (v, x ) FMRFamide (α-FMRFamide, red) immunoreactivity.} A key giving the two z-planes and the colour of each antiserum subpanel.⟧

Compass abbreviations: A = anterior, P = posterior, D = dorsal, V = ventral, L = left, R = right.

Figure 9—video 1. Tonsillar neuropil 3D volume.⟦>zach claim=no-assertion: @{Figure 9—video 1. Tonsillar neuropil 3D volume.} A bare video title.⟧

Figure 9—video 2. Neurotransmitter immunostains in the tonsillar neuropil.⟦>zach claim=no-assertion: @{Figure 9—video 2. Neurotransmitter immunostains in the tonsillar neuropil.} A bare video title.⟧

Figure 9—video 3. Neuropeptide immunostains in the tonsillar neuropil.⟦>zach claim=no-assertion: @{Figure 9—video 3. Neuropeptide immunostains in the tonsillar neuropil.} A bare video title.⟧

In individual anti-synapsin stains, a fiber tract traveling laterally adjoins this neuropil in the more dorsal–posterior portions.

By tubulin immunoreactivity, it appears to bifurcate the structure below the bridge in the dorsal portion ( Figure 9B ).⟦>zach claim=gap: @{By tubulin immunoreactivity, it appears to bifurcate the st