Gross anatomy of the visual processing centers of Hieroglyphus banian

doi:10.21203/rs.3.rs-4859198/v1

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Gross anatomy of the visual processing centers of Hieroglyphus banian

https://doi.org/10.21203/rs.3.rs-4859198/v1

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Hieroglyphus banian (H. banian) is a grasshopper, endemic to South Asia. The optic lobe has been characterized to a great extent in orthoptera, predominantly using Locust species like Schistocerca gregaria, Schistocerca americana, and Locusta migratoria, which are closely related to each other. In this work, we characterize the anatomical features of the optic lobe and associated pathway in the grasshopper species H. banian using tract-tracing immunohistochemistry and intracellular fills. All the areas of the visual pathway that have been reported in the other orthoptera species could be identified in H. banian. These species exhibited similar structure and connectivity as visible in our immunohistochemistry and tract tracing results. However, two new centers in the protocerebrum with prominent innervations from the optic lobe could be identified.

Insect visual system

Optic lobe

Visual pathway

Lobula

Visual systems across different organisms exhibit similarity in structure and function either by convergent evolution or conservation of traits. The visual sensory system first appeared in marine arthropods (Trilobites) around 541 mya during the early Cambrian era (Martinsson, 1975; Clarkson et al., 2006; Honkanen et al., 2019). Although the complexity of the brain and the complexity of visual processing vary across taxonomic categories, the visual systems of insects have been used extensively to understand the principles of visual information processing. Various theories of components of visual processing have been tested on insect systems (Haag et al., 2017; Honkanen et al., 2019; Turner-Evans et al., 2020; Dombrovski et al., 2023).

Most organisms, ranging from insects to higher vertebrates like birds and mammals, must process visual information and often store it to sustain their life. This visual information includes shape, color, motion, depth, and polarization often involving delineable pathways and processing centers. To understand how insects process visual information, we should first know which neuropils are involved in visual processing and the connectivity between them (Sanes and Zipursky, 2010; Joly et al., 2016). Many insect species in the order Diptera, Orthoptera, Lepidoptera, and Hymenoptera have been well-studied to understand their vision (Fischbach and Dittrich, 1989; Bausenwein et al., 1992; Homberg et al., 2003; Heinze and Reppert, 2012; Habenstein et al., 2020).

Ommatidia is the basic unit of the compound eyes. The size and number of ommatidia vary from species to species and even within the different sexes of the same species. Beneath the faceted eye of insects, the optic lobe is present as 3 or 4 nested neuropils. They are the lamina, medulla, and lobula complex in that order from distal to the proximal optic lobe with respect to the medial brain. The lamina and medulla are distinct neuropils with layered structures, conserved across different insect species. The lamina receives input from the receptor neurons in the retina, and the medulla receives input from both the retina and lamina. The number of layers of lamina and medulla differ across species evidenced by staining for different markers (Gokan and Meyer-Rochow, 1990; Heinze and Reppert, 2012; Rosner et al., 2017). There is a chiasma that crosses over between lamina and medulla (Fischbach and Dittrich, 1989; Rosner et al., 2017). The lobula complex is situated proximal to the central brain. In Diptera and other orders, there is a chiasma between the medulla and some of the lobula neuropils (Fischbach and Dittrich, 1989; Rosner et al., 2017).

The structure of the lobula complex differs vastly from species to species, in the number of neuropils and their arrangement. Lobula is made up of a single neuropil in bees (Gowda and Gronenberg, 2019) and ants (Habenstein et al., 2020), two neuropils lobula and lobula plate in flies (Fischbach and Dittrich, 1989), butterflies (Heinze and Reppert, 2012) and in dung beetles (Gokan and Meyer-Rochow, 1990). Lobula complex in the locust (Ito et al., 2014), dragonfly (Fabian et al., 2020), mantis, and cockroach (Rosner et al., 2017) have multiple neuropils. The identifiable sub neuropils are the outer lobula, inner lobula, anterior lobula, and dorsal lobula. All these neuropils and sub-neuropils are situated close to each other within the optic lobe, with lenses at the distal end and the central brain at the proximal end. Several tracts leave the optic lobe and terminate in various centers in the protocerebrum, namely the optic tubercles and accessory lobe of the central complex that are by and large conserved in the insects (Homberg et al., 2003; Mota et al., 2011).

In this work, we characterize the visual system of H. banian in terms of the neuropils involved in vision and the connections between them. We identify all the reported neuropils of the optic lobe as well as other neuropils of the visual pathway, anterior optic tubercle (AOTu), posterior optic tubercle (POTu), and lateral accessory lobe (LAL) by tract tracing and immunohistochemistry. We thus show that all the neuropils of the visual pathway in H. banian are similar to locust species. We identify two new centers receiving inputs directly from the optic lobe.

Animals

Both male and female adult grasshoppers (H. banian) were used in the experiments. Animals were collected from the insect room where they were reared up to adulthood from the eggs that were collected from the previous year's adults as well as those caught from the field and added to the culture. The insect room is maintained at 70% relative humidity, 26–30°C temperature, and 14L:10D photoperiod at the Center for Neural and Cognitive Sciences, University of Hyderabad.

Animal preparation

Legs and wings of the grasshopper were removed the previous night to avoid unnecessary movement and leakage of saline while dissection and recording. The following day the animal was restrained on a platform and a wax cup was made to hold the insect ringer’s saline in such a way that the left eye was uncovered and could directly see the display. Throughout the dissection and recording, the brain was perfused with insect ringer’s saline (140 mM NaCl, 5 mM CaCl₂, 5 mM KCl, 4 mM NaHCO₃, 6.3 mM HEPES, 1 mM MgCl₂ adjusted to pH 7.1; Laurent and Naraghi 1994). After opening the head capsule & removing the fat bodies, the gut is scissored from the front below the brain and removed via a cut in the dorsal-posterior abdomen. The muscles under both the optic lobes were removed to reduce brain movement. A platform is placed below the brain for stability while performing the electrophysiological recordings. The left optic lobe and brain are desheathed with protease (P-5147, Sigma) for accessing the neurons

Ommatidia counting

The animal was restrained on a platform using tape, and a layer of nail polish was applied over the eye surface and let it stand to dry for 30 min-1 hour. Then the cast was peeled and spread out flat on a plane glass slide. Cuts were made in the cast where necessary to flatten it on the slide. A coverslip was kept over it and photographs of the whole cast were taken with overlapping regions under 10x air objective of the Eclipse FN1 Nikon microscope using a Qimaging Retiga R1 CCD camera (1360 x 1024). The images of the different overlapping sections were merged to form the image of the whole cast using a pairwise stitching plugin tool in ImageJ. After creating the image of the whole cast, ommatidia were counted using the multi-point tool in ImageJ.

Bulk tracing

To identify the connected areas within the optic lobes and regions of the optic lobe with the rest of the brain, dextran conjugated dyes were injected as crystals in the area of interest in live animals. Dextran conjugated dyes used were either Dextran, Biotin (D-7135, Invitrogen), Dextran, Tetramethylrhodamine (DTMR) (D3308, Invitrogen), Dextran, Texas red-X (D3328, Invitrogen ) or Dextran, Alexa Fluor™ 488 (D34682, Invitrogen). After applying the dye, animals were continuously perfused with insect ringer’s saline for 10–12 hours to keep the brain alive. Then brains were dissected out and fixed in paraformaldehyde (PFA) to carry out further immunohisto-chemistry procedures.

Immunohistochemistry

All intracellular filled and bulk tracing excised tissues were fixed in 4% formaldehyde solution (FA, LOT Z0760296) in 1X phosphate-buffered saline (PBS, RM7385, Himedia) for 4–6 hrs, then washed 3 times for t20 min each with 1X PBS. Tissues stained with 2% Neurobiotin (SP-1120, Vector Labs) or Biocytin (B4261, Sigma) are permeabilized in 3% Triton X-100 (T8787, Sigma) in 1X PBS (PBST) buffer for 1 hour and then incubated at 1:1000 dilutions with streptavidin-conjugated Alexa Fluor™ 633 (S21375, Invitrogen) or Alexa Fluor™ 488 (S11223, Invitrogen) and left for 5 days with intermittent mixing. After that they are washed 3 times for 20 min in PBST and then in 1X PBS then tissues are dehydrated in ascending alcohol series 20 min each (30-50-70-80-90-100%). Alexa Fluor™ 633 Hydrazide (A30634, Invitrogen) or Alexa Fluor™ 568 Hydrazide (A10437, Invitrogen) stained tissues were washed (3 x 20 min) in PBS and then washed in ascending alcohol concentrations. Then tissues were cleared and mounted in methyl salicylate (M6752, Sigma Aldrich).

For identifying the neuropils of the brain, fixed and washed tissues were permeabilized with PBST (T8787, Sigma) for an hour and then transferred to 10% normal goat serum in PBST for blocking. They were then incubated with primary antibody nc82 (1:1000 dilutions) (DSHB, Iowa city, IA; donated by E. Buchner), a monoclonal antibody that visualizes the neuropils based on the density of the active zone protein bruchpilot (Wagh et.al, 2006) or with anti-GABA (A2052, Sigma-Aldrich) (1:1000 dilutions) for 5 days with intermittent shaking then tissues are washed in PBST and PBS for 3 x 20 min then again permeabilized with PBST for an hour and then incubated with either of the IgG secondary antibody goat-anti mouse Alexa Fluor™ 488 (A11029, Invitrogen) or Alexa Fluor™ 561 (A11031, Invitrogen) or Alexa Fluor™ 633 (A21052, Invitrogen) against nc82 at 1:1000 dilutions or goat anti-rabbit Alexa Fluor™ 488 (A11008, Invitrogen) or Alexa Fluor™ 568 (A11011, Invitrogen) or Alexa Fluor™ 633 (A21070, Invitrogen) against anti-GABA at 1:1000 dilutions for 5 more days with intermittent mixing. Then tissues are washed in PBST and PBS for 3 x 20 min each. Then it was dehydrated in an ascending alcohol series and cleared and mounted in methyl salicylate.

Confocal microscopy and image processing

Cleared whole-mount tissues were scanned with confocal laser scanning microscopy (Leica TCS SP8 STED) using a 10X or 20X objective lens. All the visualization and processing were carried out in ImageJ (version no: 1.53t). In all our figures, dorsoventral axis is from the calyx of the mushroom body to the antennal lobe and the anterior posterior axis is along the vertical lobe to posterior slope axis.

3D reconstructions of neuropils are based on nc82 staining and auto-fluorescence in the 488 excitation. The ‘Segmentation editor’ plugin of ImageJ was used for segmenting the regions and the ‘Show colored surface’ plugin was used to visualize the 3D surface.

Detecting the layered structure of the medulla: Six ROIs were chosen from the medulla with one edge that is approximately 300 units wide aligned along the distal proximal axis that is approximately 300 units wide and 45 units perpendicular to it. They were all scaled to the same number of pixels and averages of the pixel values at each horizontal location in the distal to proximal axis were calculated, along the dorsal to ventral axis to obtain the mean intensities.

Intracellular dye injection

Intracellular fills were carried out in the left optic lobe of the animal. Impalement was carried out using filamented borosilicate sharp electrodes (1 mm OD, 0.5 mm ID, catalog no: BF-100‐50‐10, impedance 80–150 MΩ after filling the electrode with intracellular dye and 200 mM KCl) pulled using Sutter P-97 electrode puller (Sutter Instrument Co., Novato, CA, USA). The neurons were filled iontophoretically with one of the following intracellular dyes, 2% Neurobiotin (SP-1120, Vector Labs) / Alexa Flour™ 633 Hydrazide ( A30634, Invitrogen) / Alexa Fluor™ 568 Hydrazide ( A10437, Invitrogen) / Biocytin (B4261, Sigma) by injecting 1–4 nA positive current for Neurobiotin and Biocytin and negative current for hydrazide dyes at 2 Hz pulse for 15–30 min.

Number of Ommatidia

An image of the whole cast of the eye (Fig. 1a) was used to evaluate the number of the ommatidia. We evaluated the number of ommatidia in a total of 8 animals (Table S1). In females, the ommatidia number varied from 5679 to 8277 including the ommatidia in the dorsal rim area (DRA) which had approximately 210 of them. In males, the number of ommatidia varied from 5248 to 6880, including the 177 ommatidia in the dorsal rim area. In both males and females, the size of the ommatidia in the dorsal rim area is much smaller than the ommatidia in the other part of the eye.

Neuropils visualized by immunostaining with nc82 and anti-GABA antibodies

Immunohistochemistry with nc82 (against bruchpilot) revealed neuropils of the brain and the partitions in the visual pathway. nc82 positive presynaptic marker could distinguish synaptic densities associated with optic lobes, lateral horn, mushroom bodies, central complex, lateral accessory lobes, anterior optic tubercles, posterior optic tubercles, and antennal lobes (Fig. 2). Immunohistochemistry with anti-GABA antibodies revealed GABA immunoreactive neuron cell clusters and regions of dense arborizations. Stronger GABA positivity in some subcompartments visualized via nc82 pointed to synaptic regions with strong GABAergic interactions. Central complex consisting of the protocerebral bridge (PB), central body upper (CBU) and lower (CBL) are visible in the nc82 immunoreactivity. CBL is more reactive to the anti-GABA than PB and CBU (Fig. 2b). Anterior optic tubercle contains upper (AOTu_UU) and lower units (AOTu_LU). The upper unit is further divided into the outer (AOTu_UU_OL) and inner lobe (AOTu_UU_IL), and both are highly reactive for nc82 antibodies (Fig S1d).

Optic lobe of the H. banian

H. banian optic lobe is compartmentalized into lamina, medulla and lobula from distal to proximal end.

Lamina: Lamina is a single-layered structure, containing multiple columnar structures called cartridges (Fig. 1b).

Medulla: An autofluorescence image of the optic lobe scanned using laser 488 (Fig. 3a) was used to estimate the number of layers in the medulla based on the pixel intensity of the scanned image. Six ROIs from the medulla were chosen at different locations along the dorsoventral axis (proximal end left and distal right in each ROI), which is approximately 300 µm wide and 45 pixels vertically. They were all scaled to the same number of pixels along the latero-medial axis. They are stacked along the dorsoventral axis and shown in (Fig. 3b top). The mean of each ROI, shown in gray (Fig. 3b bottom), was taken along the dorsoventral axis. The mean over the ROIs is shown in red. Both the crest and trough of the mean plot are considered as the presence of a layer. Based on this analysis, 11 layers are observed in the medulla.

The accessory medulla is an anterior structure at the proximal medulla, visible in the nc82 staining (Fig. 2e).

Lobula complex

Based on the nc82 and anti-GABA staining (Fig. 4), the lobula is compartmentalized into outer lobula (OLO), inner lobula (ILO), dorsal lobula (DLO) and anterior lobula (ALO) (Figs. 4 and 5). The outer lobula has four layers (Fig. 4). The first and second layers of the OLO are highly GABA-positive, the fourth layer is moderately stained and the third layer of the OLO is devoid of GABA staining (Fig. 4a). The fourth layer is a triangular structure (maximal extension of 250µm). The anterior lobula (ALO) (Fig S1 a2),and lower unit of the inner lobula (ILO) are prominently GABA positive compared to the upper unit of ILOand dorsal lobula (DLO). Compartmentalization and 3D reconstruction of the lobula complex of H. banian based on the nc82 immunoreactivity is shown in (Fig. 5).

The arborization pattern of individual neurons also supports this anatomical segmentation of the lobula complex. Here we present two neurons arborizing in distinct regions of the lobula to show segmentation of the lobula complex in H. banian (Fig. 6). Figure 6a-c shows a neuron morphologically similar to LGMD type 1 with branches A, B, and C that innervate into the second layer of OLO, the fourth layer of OLO, and DLO respectively. Figure 6d shows another neuron morphologically similar to LGMD type 2 with innervation into the second layer of OLO.

Tracts from the optic lobe

Targeted injection of dextran conjugated dye at several subcompartments of the optic lobe neuropils, identifiable by landmarks revealed tracts connecting the optic lobe to the other regions in the visual pathway.

The optic lobe and anterior optic tubercle

Anterior optic tubercle (AOTu) (Fig. 7d) is one of the neuropils located in the medial protocerebrum anteriorly, latero-ventral to the vertical lobe tip visible in the nc82 staining (Fig. 2a). AOTu is divided into upper (AOTu_UU) and lower units (AOTu_LU). AOTu_UU is further divided into outer (AOTu_UU_OL) and inner lobes (AOTu_UU_IL) (Fig. 8c). AOTu is a small structure (maximal extension of 200 µm). AOTu can be seen connected to the rest of the brain through four major tracts as visualized by dextran-based tract tracing. 1) Connected to the optic lobe through the anterior optic tract (AOT) (Fig. 7a). 2) Inter-tubercule tract (ITT) connects it to the contralateral AOTu (Fig. 8c). 3) The tubercle protocerebral tract connects it to the superior protocerebrum (Fig. 8a, white arrows). 4) The tubercle accessory lobe tract connects it to the lateral accessory lobe (TuLALT) (Fig. 8b). The anterior optic tract connecting AOTu and the optic lobe has three prominent bundles of fibers. Dextran injections at the anterior-dorsal medulla revealed that the trans-medullary neurons having cell bodies distal to the medulla enter the medulla and have side branches in the upper proximal medulla. These tracts then cross the medulla and enter the OLO followed by the stratum 2 of the anterior lobula (ALO2). Cells from the same region of the medulla also densely innervated the accessory medulla (Fig. 7c). The AOT continues beyond ALO2 and innervates both the outer and inner lobes of the upper unit of the ipsilateral anterior optic tubercle ( AOTu_UU) (Fig S1d).

Dextran injection at the contralateral lower unit of the anterior optic tubercle (AOTu_LU) filled stratum 1 of the contralateral anterior lobula (ALO1) through the anterior optic tract (Fig. 8a), and it also filled the ipsilateral AOTu through inter-tubercle tract (Fig. 8c). This innervation of the ipsilateral AOTu could be confirmed with the AOTu visualized by dextran injection in the ipsilateral medulla leading to the filling of the ipsilateral AOTu (Fig. 8c).

Dextran injection in the AOTu filled many fibers arborizing in the lateral bulb (LBU) and medial bulb (MBU) in the lateral accessory lobe (LAL) through the tubercle accessory lobe tract (Fig. 8a-b). Dextran injection at the anterior ventral lobula also filled the medial bulb (MBU) of the LAL as well as many cell bodies in the superior protocerebrum through tubercle protocerebral tracts (Fig. 8a). Dorsoventral partitioning of lobula to AOTu connection is observed upon injecting Alexa Fluor conjugated dextran dyes in the dorsal and ventral lobula. The ventral lobula is connected to the AOTu_UU & AOTu_LU on contrary to the dorsal lobula, which is connected only to the OL and IL of AOTu_UU (Fig S1 b-c)

Novel structures PS1 in the posterior protocerebrum innervated by fibers from the medulla

Very prominent tracts from the anterior-dorsal medulla also innervated two novel oval structures near the ipsilateral side of the central protocerebrum which we call posterior structure 1 (PS1), posterior to the central complex (Fig. 7e). These fibers travel as a thick bundle and terminate in a distribution shaped around the droplet (Fig. 9, structure marked with dashed lines) at the very posterior protocerebrum with dense fine branches. No feature was visible in our nc82 or GABA immunohistochemistry demarcating this region.

Posterior optic tubercle and a novel structure, PS2:

Posterior optic tubercle (POTu) is another neuropil located posterior to the junction of the peduncle, vertical lobe, and medial lobe. POTu is a smaller structure (maximal extension of 80 µm), which is almost half of the size of AOTu. Dextran injection into the accessory medulla (aMe) filled the POTu (Fig. 8d) through the posterior optic tract. It has also filled a novel structure 120 µm lateral to the POTu (Fig. 8d and 9, filled circular arrow), which we call posterior structure 2 (PS2), posterior to the CBU at the protocerebral bridge terminal. Dextran injection at the anterior dorsal lobula also filled this posterior structure 2 (PS2) (Fig S1b, blue arrow). Neither POTu nor PS2 were identifiable from the nc82 images.

It has been shown that the Hieroglyphus banian olfactory circuit is conserved through fourth order neurons and is analogous to Schistocerca americana which has ~ 57 million years of divergence (Singh and Joseph, 2019). Unlike locusts, H. banian species is univoltine, is found to inhabit somewhat lush vegetation, and is rarely seen in open grounds. In this work, we have studied optic lobes and other visual processing centers of Hieroglyphus banian. We counted the number of ommatidia and found them to be similar to that reported in L. migratoria (Shaw, 1978). Variation in the number of ommatidia among individuals from a wild population of the species is not surprising given that there is a large variation (approximately 20%) observed in the number of ommatidia in a single strain, Canton-S^BH of Drosophila (Ramaekers et al., 2019).

Comparison of visual processing centers of H. banian and Schistocerca gregaria:

In visual systems of the locusts, Schistocerca gregaria is well studied. Similar to H. banian, S.gregaria optic lobe has the following neuropils lamina, medulla, and lobula complex arranged distal to proximal. Anti-GABA staining showed 10 layers in the medulla of S. gregaria (Beetz et al., 2015; Rosner et al., 2017). H. banian optic lobe scanned under 488 laser, showed layered structure of the medulla due to autofluorescence. Upon analyzing these images for brightness changes in the medulla portion of the brain, it also showed a 10–11 layered structure. Here the method used to calculate the number of layers is similar to the method used for calculating medulla and lobula layers based on pixel intensity plots on confocal scans of anti-synapsin and anti-serotonin stained brains of dragonflies (Fabian et al., 2020). The lobula complex of S. gregaria has OLO, DLO, ALO and ILO similar to H. banian. S. gregaria OLO showed four layered structure for anti-GABA. Distal most layers are stained strongly for GABA, third layer is free of GABA and the fourth layer has more densely stained fibers than the third layer. In H. banian, a similar pattern of GABA staining is observed in OLO. In H. banian, nc82 staining showed all four layers of the OLO and the second layer is the most densely stained among the four layers. In S. gregraria and Drosophila neurons in lobula have been shown to receive inhibitory inputs via GABAergic neurons (Raghu et al., 2007; Rind et al., 2016).

In H. banian, nc82 (similar to anti-synapsin immunoreactivity) is clear in ALO, DLO and ILO, same as in S. gregeria. DLO stained brighter than ALO and ILO, and ILO showed substructures of upper and lower units. In S. gregaria, antibodies against Locust Tachykinin II (Lom-TK II) revealed two layers in ALO, (ALO1 & ALO2). ALO2 showed three sub layers (Homberg et al., 2003). Similarly, H. banian also has four layers, including the three sublayers of ALO2 and one layer of ALO1 (Fig. 5d).

In S. gregaria, the optic lobes are connected to the rest of the brain, predominantly through two tracts, anterior optic tract (AOT) and posterior optic tract (POT). Through AOT, optic lobes are connected to the anterior optic tubercle (AOTu) and to regions in the rest of the brain. AOTu has upper and lower units. Upper unit is further divided into the outer and inner lobe. AOTu is connected to the contralateral AOTu through the inter tubercle tract (ITT) and to LAL through TuLAL tracts (TuLALT). Innervation in the LAL region shows two bulbous structures called medial bulb (MBU) and lateral bulb (LBU). AOTu also connects to the superior protocerebrum through tubercle protocerebral tracts (TuPBT) (Homberg et al., 2003). Through POT, optic lobes are connected to the posterior optic tubercle (POTu) (Homberg and Würden, 1997). Both the POTu’s are connected through inter tubercle tracts (ITT), POTu is connected to the protocerebral bridge through tangential neurons (el Jundi and Homberg, 2010).

Similarly in H. banian, the AOTu is connected to the optic lobe through AOT, LAL through TuLALT, superior protocerebrum through TuPBT and contralateral AOTu through ITT. Dextran fills from anterior ventral lobula filled MBU, this might be through TuLAL1 neuron which was observed in S. gregaria (Homberg et al., 2003).Though POTu is filled through the accessory medulla (aMe), we couldn’t visualize the tracts leaving the POTu, as it was difficult to access and target specifically. From these results, we can conclude that the visual system of Hieroglyphus banian is anatomically very similar to that of the Schistocerca gregaria.

Novel structures PS1 and PS2:

In our dextran tracings in H. banian, two novel tracts that have not been described before in locust visual systems could be seen. Dextran injection at the anterior dorsal medulla filled two oval structures, PS1, in the central protocerebrum, posterior to the central complex via one of the tracts. This tract was prominent and consistently filled in our preparations. Dextran injection in the anterior dorsal lobula also filled this structure, possibly because the tracts going from the medulla to these structures were hit. Why the prominent tract from the medulla to the posterior protocerebrum was not visible in S. gregaria though it was consistently visible in all our H. banian fills is puzzling. Dextran injection at the anterior dorsal lobula and aMe filled another novel structure, PS2, dorsal to the POTu, posterior to the CBU at the protocerebral bridge terminal, and dextran injection at the accessory medulla also filled this structure. It is tempting to call this structure POTu2 because the originally reported close-by structure is called POTu1 and they get filled from similar regions in the optic lobe. It might be possible that both these novel structures are sending output to the descending motor neurons or interacting with connections from the ocelli. In our attempts, no overlaps could be seen of PS1 or PS2 with dextran fills from the ocelli (Figure S2) or the ventral nerve cord (data not shown). The connectivity and functions of these structures are yet to be elucidated.

Conflict of Interest:

The authors declare that there are no conflicts of interest pertaining to this submission.

Compliance with Ethical Standards:

Not Applicable

Author Contribution

C.S. and J.J. designed the study. C.S. carried out the experiments and prepared the figures. C.S. and J.J. wrote the manuscript. All authors reviewed the manuscript.

Acknowledgement:

Sivaraju C was supported by a scholarship from ICMR, India. The research was partly supported by DST India Purse grant to the University of Hyderabad.

Data Availability

All the data, (original confocal stacks and photographs) are available on request.

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Gross anatomy of the visual processing centers of Hieroglyphus banian

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Version 1

Abstract

Figures

Introduction

Materials and Methods

Animals

Animal preparation

Ommatidia counting

Bulk tracing

Immunohistochemistry

Confocal microscopy and image processing

Intracellular dye injection

Results

Number of Ommatidia

Neuropils visualized by immunostaining with nc82 and anti-GABA antibodies

Lobula complex

The optic lobe and anterior optic tubercle

Novel structures PS1 in the posterior protocerebrum innervated by fibers from the medulla

Posterior optic tubercle and a novel structure, PS2:

Discussion

Novel structures PS1 and PS2:

Declarations

Conflict of Interest:

Compliance with Ethical Standards:

Author Contribution

Acknowledgement:

Data Availability

References

Additional Declarations

Supplementary Files

Status:

Version 1