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

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). 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.

Ventral supraesophageal features.

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). 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). 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. 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.

The esophageal passage is bridged at the anterior side by a region named the stomodeal bridge (StB) (Steinhoff et al., 2017; Figure 5A – brace). A bridge structure also exists at the posterior end, where additional undifferentiated synaptic density is flanking (Figure 5B – brace with asterisk). 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. 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).

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).

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).

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). 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).

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).