Patterns of cell death in the embryonic antenna of the grasshopper Schistocerca gregaria.

We have investigated the pattern of apoptosis in the antennal epithelium during embryonic development of the grasshopper Schistocerca gregaria. The molecular labels lachesin and annulin reveal that the antennal epithelium becomes subdivided into segment-like meristal annuli within which sensory cell clusters later differentiate. To determine whether apoptosis is involved in the development of such sensory cell clusters, we examined the expression pattern of the cell death labels acridine orange and TUNEL in the epithelium. We found stereotypic, age-dependent, wave-like patterns of cell death in the antenna. Early in embryogenesis, apoptosis is restricted to the most basal meristal annuli but subsequently spreads to encompass almost the entire antenna. Cell death then declines in more basal annuli and is only found in the tip region later in embryogenesis. Apoptosis is restricted throughout to the midregion of a given annulus and away from its border with neighboring annuli, arguing against a causal role in annular formation. Double-labeling for cell death and sensory cell differentiation reveals apoptosis occurring within bands of differentiating sensory cell clusters, matching the meristal organization of the apical antenna. Examination of the individual epithelial lineages which generate sensory cells reveals that apoptosis begins peripherally within a lineage and with age expands to encompass the differentiated sensory cell at the base. We conclude that complete lineages can undergo apoptosis and that the youngest cells in these lineages appear to die first, with the sensory neuron dying last. Lineage-based death in combination with cell death patterns in different regions of the antenna may contribute to odor-mediated behaviors in the grasshopper.

Ontogeny and development of the tritocerebral commissure giant (TCG): an identified neuron in the brain of the grasshopper Schistocerca gregaria.

The tritocerebral commissure giant (TCG) of the grasshopper Schistocerca gregaria is one of the best anatomically and physiologically described arthropod brain neurons. A member of the so-called Ventral Giant cluster of cells, it integrates sensory information from visual, antennal and hair receptors, and synapses with thoracic motor neurons in order to initiate and regulate flight behavior. Its ontogeny, however, remains unclear. In this study, we use bromodeoxyuridine incorporation and cyclin labeling to reveal proliferative neuroblasts in the region of the embryonic brain where the ventral giant cluster is located. Engrailed labeling confirms the deutocerebral identity of this cluster. Comparison of soma locations and initial neurite projections into tracts of the striate deutocerebrum help identify the cells of the ventral cluster in both the embryonic and adult brain. Reconstructions of embryonic cell lineages suggest deutocerebral NB1 as being the putative neuroblast of origin. Intracellular dye injection coupled with immunolabeling against neuron-specific horseradish peroxidase is used to identify the VG1 (TCG) and VG3 neurons from the ventral cluster in embryonic brain slices. Dye injection and backfilling are used to document axogenesis and the progressive expansion of the dendritic arbor of the TCG from mid-embryogenesis up to hatching. Comparative maps of embryonic neuroblasts from several orthopteroid insects suggest equivalent deutocerebral neuroblasts from which the homologous TCG neurons already identified in the adult brain could originate. Our data offer the prospect of identifying further lineage-related neurons from the cluster and so understand a brain connectome from both a developmental and evolutionary perspective.

Octopaminergic innervation and a neurohaemal release site in the antennal heart of the locust Schistocerca gregaria.

A detailed account is given by the octopaminergic innervation of the antennal heart in Schistocerca gregaria using various immunohistochemical methods. Anterograde axonal filling illustrates the unilateral innervation on the medial ventral surface of the pumping muscle of the antennal heart via the paired corpora cardiaca nerve III. In addition, antibody staining revealed that ascending axons of this nerve terminate at the ampullae of the antennal heart forming synaptoid structures and extensive neurohaemal release sites. Due to the innervation by two dorsal unpaired median neurons, the presence of the biogenic amines octopamine and tyramine could be visualized by immunocytochemistry in an insect antennal heart for the first time. The data suggest that tyramine acts as a precursor and not purely as an independent transmitter. While the octopaminergic fibers innervating the pumping muscle of the antennal heart indicate a cardioregulatory role, we conclude that octopamine released from the neurohaemal area is pumped into the antennae and an involvement in the modulation of the antennal sensory sensitivity is discussed.

Hyperthermia enhances methyl methanesulfonate-induced adaptive response in meiotic cells of grasshopper Poecilocerus pictus.

To understand the role of hyperthermia (HT) in adaptive response, methyl methanesulfonate (MMS) adapted meiotic cells of Poecilocerus pictus were used. Poecilocerus pictus were treated with conditioning (L) or challenging (H) dose of MMS and 2-h time lag (TL) between these doses (L-2h-H) (combined) was employed. Different treatment schedules were used to analyse the influence of HT on MMS-induced adaptive response namely pre; inter; post-treatment and cross-adaptation. After each treatment schedules, chromosomal anomalies were analysed. The frequencies of chromosomal anomalies induced by conditioning and challenging doses of MMS were significantly higher (P < 0.0001) compared to that of the control or HT groups. The combined treatments resulted in significant reduction of chromosomal anomalies compared to additive effect of MMS (P < 0.0001). The pre, inter, post and cross-adaptation treatments with HT reduced the frequencies of chromosomal anomalies compared to the challenge and combined treatments with MMS. There is a protection against MMS-induced chromosomal anomalies by HT in in vivo P.pictus. This is the first report to demonstrate that HT enhances the MMS-induced adaptive response in in vivo meiotic cells.

A conserved plan for wiring up the fan-shaped body in the grasshopper and Drosophila.

The central complex comprises an elaborate system of modular neuropils which mediate spatial orientation and sensory-motor integration in insects such as the grasshopper and Drosophila. The neuroarchitecture of the largest of these modules, the fan-shaped body, is characterized by its stereotypic set of decussating fiber bundles. These are generated during development by axons from four homologous protocerebral lineages which enter the commissural system and subsequently decussate at stereotypic locations across the brain midline. Since the commissural organization prior to fan-shaped body formation has not been previously analyzed in either species, it was not clear how the decussating bundles relate to individual lineages, or if the projection pattern is conserved across species. In this study, we trace the axonal projections from the homologous central complex lineages into the commissural system of the embryonic and larval brains of both the grasshopper and Drosophila. Projections into the primordial commissures of both species are found to be lineage-specific and allow putatively equivalent fascicles to be identified. Comparison of the projection pattern before and after the commencement of axon decussation in both species reveals that equivalent commissural fascicles are involved in generating the columnar neuroarchitecture of the fan-shaped body. Further, the tract-specific columns in both the grasshopper and Drosophila can be shown to contain axons from identical combinations of central complex lineages, suggesting that this columnar neuroarchitecture is also conserved.

Ontogeny of pioneer neurons in the antennal nervous system of the grasshopper Schistocerca gregaria.

The nervous system of the antenna of the grasshopper Schistocerca gregaria consists of two nerve tracts in which sensory cells project their axons to the brain. Each tract is pioneered early in embryogenesis by a pair of identified cells located apically in the antennal lumen. The pioneers are thought to originate in the epithelium of the antenna and then delaminate into the lumen where they commence axogenesis. However, unambiguous molecular identification of these cells in the epithelium, of an identifiable precursor, and of their mode of generation has been lacking. In this study, we have used immunolabeling against neuron-specific horseradish peroxidase and against Lachesin, a marker for differentiating epithelial cells, in combination with the nuclear stain DAPI, to identify the pioneers within the epithelium of the early embryonic antenna. We then track their delamination into the lumen as differentiated neurons. The pioneers are not labeled by the mesodermal/mesectodermal marker Mes3, consistent with an epithelial (ectodermal) origin. Intracellular dye injection, as well as labeling against the mitosis marker phospho-histone 3, identifies precursor cells in the epithelium, each associated with a column of cells. Culturing with the S-phase label 5-ethynyl-2'-deoxyuridine (EdU) shows that both a precursor and its column have incorporated the label, confirming a lineage relationship. Each set of pioneers can be shown to belong to a separate lineage of such epithelial cells, and the precursors remain and are proliferative after generating the pioneers. Analyses of mitotic spindle orientation then enable us to propose a model in which a precursor generates its pioneers asymmetrically via self-renewal.

Neurons in the brain of the desert locust Schistocerca gregaria sensitive to polarized light at low stimulus elevations.

Desert locusts (Schistocerca gregaria) sense the plane of dorsally presented polarized light through specialized dorsal eye regions that are likely adapted to exploit the polarization pattern of the blue sky for spatial orientation. Receptive fields of these dorsal rim photoreceptors and polarization-sensitive interneurons are directed toward the upper sky but may extend to elevations below 30°. Behavioral data, however, suggests that S. gregaria is even able to detect polarized light from ventral directions but physiological evidence for this is still lacking. In this study we characterized neurons in the locust brain showing polarization sensitivity at low elevations down to the horizon. In most neurons polarization sensitivity was absent or weak when stimulating from the zenith. All neurons, including projection and commissural neurons of the optic lobe and local interneurons of the central brain, are novel cell types, distinct from polarization-sensitive neurons studied so far. Painting dorsal rim areas in both eyes black to block visual input had no effect on the polarization sensitivity of these neurons, suggesting that they receive polarized light input from the main eye. A possible role of these neurons in flight stabilization or the perception of polarized light reflected from bodies of water or vegetation is discussed.

Axogenesis in the antennal nervous system of the grasshopper Schistocerca gregaria revisited: the base pioneers.

The antennal nervous system of the grasshopper Schistocerca gregaria comprises two parallel pathways projecting to the brain, each pioneered early in embryogenesis by a pair of sibling cells located at the antennal tip. En route, the growth cones of pioneers from one pathway have been shown to contact a guidepost-like cell called the base pioneer. Its role in axon guidance remains unclear as do the cellular guidance cues regulating axogenesis in the other pathway supposedly without a base pioneer. Further, while the tip pioneers are known to delaminate from the antennal epithelium into the lumen, the origin of this base pioneer is unknown. Here, we use immunolabeling and immunoblocking methods to clarify these issues. Co-labeling against the neuron-specific marker horseradish peroxidase and the pioneer-specific cell surface glycoprotein Lazarillo identifies not only the tip pioneers but also a base pioneer associated with each of the developing antennal pathways. Both base pioneers co-express the mesodermal label Mes3, consistent with a lumenal origin, whereas the tip pioneers proved Mes3-negative confirming their affiliation with the ectodermal epithelium. Lazarillo antigen expression in the antennal pioneers followed a different temporal dynamic: continuous in the tip pioneers, but in the base pioneers, only at the time their filopodia and those of the tip pioneers first recognize one another. Immunoblocking of Lazarillo expression in cultured embryos disrupts this recognition resulting in misguided axogenesis in both antennal pathways.

Pioneer neurons of the antennal nervous system project to protocerebral pioneers in the grasshopper Schistocerca gregaria.

The twin nerve tracts of the antenna of the grasshopper Schistocerca gregaria are established early in embryogenesis by sibling pairs of pioneers which delaminate from the epithelium into the lumen at the antennal tip. These cells can be uniquely identified via their co-expression of the neuronal labels horseradish peroxidase and the lipocalin Lazarillo. The apical pioneers direct axons toward the antennal base where they encounter guidepost-like cells called base pioneers which transiently express the same molecular labels as the apical pioneers. To what extent the pioneer growth cones then progress into the brain neuropil proper, and what their targets there might be, has remained unclear. In this study, we show that the apical antennal pioneers project centrally beyond the antennal base first into the deutocerebral, and then into the protocerebral brain neuropils. In the protocerebrum, we identify their target circuitry as being identified Lazarillo-positive cells which themselves pioneer the primary axon scaffold of the brain. The apical and base antennal pioneers therefore form part of a molecularly contiguous pathway from the periphery to an identified central circuit of the embryonic grasshopper brain.

Dynamics of cohesin subunits in grasshopper meiotic divisions.

The cohesin complex plays a key role for the maintenance of sister chromatid cohesion and faithful chromosome segregation in both mitosis and meiosis. This complex is formed by two structural maintenance of chromosomes protein family (SMC) subunits and two non-SMC subunits: an α-kleisin subunit SCC1/RAD21/REC8 and an SCC3-like protein. Several studies carried out in different species have revealed that the distribution of the cohesin subunits along the chromosomes during meiotic prophase I is not regular and that some subunits are distinctly incorporated at different cell stages. However, the accurate distribution of the different cohesin subunits in condensed meiotic chromosomes is still controversial. Here, we describe the dynamics of the cohesin subunits SMC1α, SMC3, RAD21 and SA1 during both meiotic divisions in grasshoppers. Although these subunits show a similar patched labelling at the interchromatid domain of metaphase I bivalents, SMCs and non-SMCs subunits do not always colocalise. Indeed, SA1 is the only cohesin subunit accumulated at the centromeric region of all metaphase I chromosomes. Additionally, non-SMC subunits do not appear at the interchromatid domain in either single X or B chromosomes. These data suggest the existence of several cohesin complexes during metaphase I. The cohesin subunits analysed are released from chromosomes at the beginning of anaphase I, with the exception of SA1 which can be detected at the centromeres until telophase II. These observations indicate that the cohesin components may be differentially loaded and released from meiotic chromosomes during the first and second meiotic divisions. The roles of these cohesin complexes for the maintenance of chromosome structure and their involvement in homologous segregation at first meiotic division are proposed and discussed.

Timelines in the insect brain: fates of identified neural stem cells generating the central complex in the grasshopper Schistocerca gregaria.

This study employs labels for cell proliferation and cell death, as well as classical histology to examine the fates of all eight neural stem cells (neuroblasts) whose progeny generate the central complex of the grasshopper brain during embryogenesis. These neuroblasts delaminate from the neuroectoderm between 25 and 30 % of embryogenesis and form a linear array running from ventral (neuroblasts Z, Y, X, and W) to dorsal (neuroblasts 1-2, 1-3, 1-4, and 1-5) along the medial border of each protocerebral hemisphere. Their stereotypic location within the array, characteristic size, and nuclear morphologies, identify these neuroblasts up to about 70 % of embryogenesis after which cell shrinkage and shape changes render progressively more cells histologically unrecognizable. Molecular labels show all neuroblasts in the array are proliferative up to 70 % of embryogenesis, but subsequently first the more ventral cells (72-75 %), and then the dorsal ones (77-80 %), cease proliferation. By contrast, neuroblasts elsewhere in the brain and optic lobe remain proliferative. Apoptosis markers label the more ventral neuroblasts first (70-72 %), then the dorsal cells (77 %), and the absence of any labeling thereafter confirms that central complex neuroblasts have exited the cell cycle via programmed cell death. Our data reveal appearance, proliferation, and cell death proceeding as successive waves from ventral to dorsal along the array of neuroblasts. The resulting timelines offer a temporal blueprint for building the neuroarchitecture of the various modules of the central complex.

Physical mapping of the Period gene on meiotic chromosomes of South American grasshoppers (Acridomorpha, Orthoptera).

The single-copy gene Period was located in five grasshopper species belonging to the Acridomorpha group through permanent in situ hybridization (PISH). The mapping revealed one copy of this gene in the L1 chromosome pair in Ommexecha virens, Xyleus discoideus angulatus, Tropidacris collaris, Schistocerca pallens, and Stiphra robusta. A possible second copy was mapped on the L2 chromosome pair in S. robusta, which should be confirmed by further studies. Except for the latter case, the chromosomal position of the Period gene was highly conserved among the four families studied. The S. robusta karyotype also differs from the others both in chromosome number and morphology. The position conservation of the single-copy gene Period contrasts with the location diversification of multigene families in these species. The localization of single-copy genes by PISH can provide new insights about the genomic content and chromosomal evolution of grasshoppers and others insects.

Wolbachia induced cytogenetical effects as evidenced in Chorthippus parallelus (Orthoptera).

The cytoplasmic incompatibility induced by the bacterial endosymbiont Wolbachia is attributed to chromatin modification in the sperm of infected individuals and is only 'rescued' by infected females after fertilization. Chorthippus parallelus is a grasshopper with 2 subspecies that form a hybrid zone in the Pyrenees in which this Wolbachia-generated cytoplasmic incompatibility has recently been described. The analysis of certain cytogenetic traits (sex chromosome-linked heterochromatic bands, nucleolar organizing region expression, spermatid size and morphology, and number of chiasmata formed) in pure and hybrid Chorthippus parallelus that are infected and not infected by this bacterium indicates that the infection affects some of these traits and, in the case of the spermatids, reveals a synergism between the infection and the hybrid condition. These results are interpreted as being secondary effects of the chromatin modification induced by Wolbachia which thereby support this model of modification/rescue. The possible effects of these cytogenetic variations on affected individuals are also considered.

Gliogenesis in the embryonic brain of the grasshopper Schistocerca gregaria with particular focus on the protocerebrum prior to mid-embryogenesis.

I investigate the pattern of gliogenesis in the brain of the grasshopper Schistocerca gregaria prior to mid-embryogenesis, with particular focus on the protocerebrum. Using the glia-specific marker Repo and the neuron-specific marker HRP, I identify three types of glia with respect to their respective positions in the brain: surface glia form the outmost cell layer ensheathing the brain; cortex glia are intermingled with neuronal somata forming the brain cortex; and neuropil glia are associated with brain neuropils. The ontogeny of each glial type has also been studied. At 24% of embryogenesis, a few glia are observed in each hemisphere of the proto-, deuto- and tritocerebrum. In each protocerebral hemisphere, such glia form a cluster that expands rapidly during later development. Closer examination reveals proliferative glia in such clusters at ages spanning from 24 to 36% of embryogenesis, indicating that glial proliferation may account for the expansion of the clusters. Data derived from 33-39% of embryogenesis suggest that, in the protocerebrum, each type of glia is likely to be generated by its respective progenitor-forming clusters. Moreover, the glial cluster located at the anterior end of the brain can give rise to both surface glia and cortex glia that populate the protocerebrum via subsequent migration. Proliferation is observed for all three glial types, indicating a possible source for the glia.

Postembryonic development of astrocyte-like glia of the central complex in the grasshopper Schistocerca gregaria.

Central complex modules in the postembryonic brain of the grasshopper Schistocerca gregaria are enveloped by Repo-positive/glutamine-synthetase-positive astrocyte-like glia. Such cells constitute Rind-Neuropil Interface glia. We have investigated the postembryonic development of these glia and their anatomical relationship to axons originating from the w, x, y, z tract system of the pars intercerebralis. Based on glutamine synthetase immunolabeling, we have identified four morphological types of cells: bipolar type 1 glia delimit the central body but only innervate its neuropil superficially; monopolar type 2 glia have a more columnar morphology and direct numerous gliopodia into the neuropil where they arborize extensively; monopolar type 3 glia are found predominantly in the region between the noduli and the central body and have a dendritic morphology and their gliopodia project deeply into the central body neuropil where they arborize extensively; multipolar type 4 glia link the central body neuropil with neighboring neuropils of the protocerebrum. These glia occupy type-specific distributions around the central body. Their gliopodia develop late in embryogenesis, elongate and generally become denser during subsequent postembryonic development. Gliopodia from putatively type 3 glia within the central body have been shown to lie closely apposed to individual axons of identified columnar fiber bundles from the w, x, y, z tract system of the central complex. This anatomical association might offer a substrate for neuron/glia interactions mediating postembryonic maturation of the central complex.

Hebbian STDP in mushroom bodies facilitates the synchronous flow of olfactory information in locusts.

Odour representations in insects undergo progressive transformations and decorrelation from the receptor array to the presumed site of odour learning, the mushroom body. There, odours are represented by sparse assemblies of Kenyon cells in a large population. Using intracellular recordings in vivo, we examined transmission and plasticity at the synapse made by Kenyon cells onto downstream targets in locusts. We find that these individual synapses are excitatory and undergo hebbian spike-timing dependent plasticity (STDP) on a +/-25 ms timescale. When placed in the context of odour-evoked Kenyon cell activity (a 20-Hz oscillatory population discharge), this form of STDP enhances the synchronization of the Kenyon cells' targets and thus helps preserve the propagation of the odour-specific codes through the olfactory system.

A cellular network of dye-coupled glia associated with the embryonic central complex in the grasshopper Schistocerca gregaria.

The central complex of the grasshopper (Schistocerca gregaria) brain comprises a modular set of neuropils, which develops after mid-embryogenesis and is functional on hatching. Early in embryogenesis, Repo-positive glia cells are found intermingled among the commissures of the midbrain, but then redistribute as central complex modules become established and, by the end of embryogenesis, envelop all midbrain neuropils. The predominant glia associated with the central body during embryogenesis are glutamine synthetase-/Repo-positive astrocyte-like glia, which direct extensive processes (gliopodia) into and around midbrain neuropils. We used intracellular dye injection in brain slices to ascertain whether such glia are dye-coupled into a communicating cellular network during embryogenesis. Intracellular staining of individual cells located at any one of four sites around the central body revealed a population of dye-coupled cells whose number and spatial distribution were stereotypic for each site and comparable at both 70 and 100% of embryogenesis. Subsequent immunolabeling confirmed these dye-coupled cells to be astrocyte-like glia. The addition of n-heptanol to the bathing saline prevented all dye coupling, consistent with gap junctions linking the glia surrounding the central body. Since dye coupling also occurred in the absence of direct intersomal contacts, it might additionally involve the extensive array of gliopodia, which develop after glia are arrayed around the central body. Collating the data from all injection sites suggests that the developing central body is surrounded by a network of dye-coupled glia, which we speculate may function as a positioning system for the developing neuropils of the central complex.

[Identification of the meiotic events in grasshopper spermatogenesis].

The grasshoppers are ideal materials to study various meiotic stages of spermatogenesis due to their easy availability, fairly large chromosomes, and fewer numbers of chromosomes. It is easy to make temporary squash preparation of grasshopper testes; however, it is usually difficult for the beginners to differentiate between stages of meiosis. In view of this, we demonstrated the method of identification of meiotic stages by chromosome number and chromosome conformation, taking spermatogonial meiosis of Locusta migratoria manilensis as an example. We described briefly the mitosis of spermatogonia and the spermatogenesis of this species as well.

Patterns of dye coupling involving serotonergic neurons provide insights into the cellular organization of a central complex lineage of the embryonic grasshopper Schistocerca gregaria.

All eight neuroblasts from the pars intercerebralis of one protocerebral hemisphere whose progeny contribute fibers to the central complex in the embryonic brain of the grasshopper Schistocerca gregaria generate serotonergic cells at stereotypic locations in their lineages. The pattern of dye coupling involving these neuroblasts and their progeny was investigated during embryogenesis by injecting fluorescent dye intracellularly into the neuroblast and/or its progeny in brain slices. The tissue was then processed for anti-serotonin immunohistochemistry. A representative lineage, that of neuroblast 1-3, was selected for detailed study. Stereotypic patterns of dye coupling were observed between progeny of the lineage throughout embryogenesis. Dye injected into the soma of a serotonergic cell consistently spread to a cluster of between five and eight neighboring non-serotonergic cells, but never to other serotonergic cells. Dye injected into a non-serotonergic cell from such a cluster spread to other non-serotonergic cells of the cluster, and to the immediate serotonergic cell, but never to further serotonergic cells. Serotonergic cells tested from different locations within the lineage repeat this pattern of dye coupling. All dye coupling was blocked on addition of an established gap junctional blocker (n-heptanol) to the bathing medium. The lack of coupling among serotonergic cells in the lineage suggests that each, along with its associated cluster of dye-coupled non-serotonergic cells, represents an independent communicating pathway (labeled line) to the developing central complex neuropil. The serotonergic cell may function as the coordinating element in such a projection system.

Cell fate decisions in the grasshopper central nervous system.

The central nervous system (CNS) of grasshopper embryos is similar in organization to the embryonic Drosophila CNS, but its neurons are much larger. The recent development of a culture system in which extensive CNS development occurs has allowed new types of experiments to be performed, including perturbation of gene expression within single neuroblast lineages.