From bristle to brain: embryonic development of topographic projections from basiconic sensilla in the antennal nervous system of the locust Schistocerca gregaria.

The antennal flagellum of the locust S. gregaria is an articulated structure bearing a spectrum of sensilla that responds to sensory stimuli. In this study, we focus on the basiconic-type bristles as a model for sensory system development in the antenna. At the end of embryogenesis, these bristles are found at fixed locations and then on only the most distal six articulations of the antenna. They are innervated by a dendrite from a sensory cell cluster in the underlying epithelium, with each cluster directing fused axons topographically to an antennal tract running to the brain. We employ confocal imaging and immunolabeling to (a) identify mitotically active sense organ precursors for sensory cell clusters in the most distal annuli of the early embryonic antenna; (b) observe the subsequent spatial appearance of their neuronal progeny; and (c) map the spatial and temporal organization of axon projections from such clusters into the antennal tracts. We show that early in embryogenesis, proliferative precursors are localized circumferentially within discrete epithelial domains of the flagellum. Progeny first appear distally at the antennal tip and then sequentially in a proximal direction so that sensory neuron populations are distributed in an age-dependent manner along the antenna. Autotracing reveals that axon fasciculation with a tract is also sequential and reflects the location and age of the cell cluster along the most distal annuli. Cell cluster location and bristle location are therefore represented topographically and temporally within the axon profile of the tract and its projection to the brain.

A locust embryo as predictive developmental neurotoxicity testing system for pioneer axon pathway formation.

Exposure to environmental chemicals during in utero and early postnatal development can cause a wide range of neurological defects. Since current guidelines for identifying developmental neurotoxic chemicals depend on the use of large numbers of rodents in animal experiments, it has been proposed to design rapid and cost-efficient in vitro screening test batteries that are mainly based on mixed neuronal/glial cultures. However, cell culture tests do not assay correct wiring of neuronal circuits. The establishment of precise anatomical connectivity is a key event in the development of a functional brain. Here, we expose intact embryos of the locust (Locusta migratoria) in serum-free culture to test chemicals and visualize correct navigation of identified pioneer axons by fluorescence microscopy. We define separate toxicological endpoints for axonal elongation and navigation along a stereotyped pathway. To distinguish developmental neurotoxicity (DNT) from general toxicity, we quantify defects in axonal elongation and navigation in concentration-response curves and compare it to the biochemically determined viability of the embryo. The investigation of a panel of recognized DNT-positive and -negative test compounds supports a rather high predictability of this invertebrate embryo assay. Similar to the semaphorin-mediated guidance of neurites in mammalian cortex, correct axonal navigation of the locust pioneer axons relies on steering cues from members of this family of cell recognition molecules. Due to the evolutionary conserved mechanisms of neurite guidance, we suggest that our pioneer axon paradigm might provide mechanistically relevant information on the DNT potential of chemical agents on the processes of axon elongation, navigation, and fasciculation.

RNAi-Mediated Knockdown of Transcription Factor E93 in Nymphs of the Desert Locust (Schistocerca gregaria) Inhibits Adult Morphogenesis and Results in Supernumerary Juvenile Stages.

Postembryonic development of insects is coordinated by juvenile hormone (JH) together with ecdysteroids. Whereas the JH early response gene krüppel-homolog 1 (Kr-h1) plays a crucial role in the maintenance of juvenile characteristics during consecutive larval stages, the ecdysteroid-inducible early gene E93 appears to be a key factor promoting metamorphosis and adult morphogenesis. Here, we report on the developmental and molecular consequences of an RNAi-mediated knockdown of SgE93 in the desert locust, Schistocerca gregaria, a hemimetabolan species. Our experimental data show that injection of gregarious locust nymphs with a double-stranded RNA construct targeting the SgE93 transcript inhibited the process of metamorphosis and instead led to supernumerary nymphal stages. These supernumerary nymphal instars still displayed juvenile morphological features, such as a nymphal color scheme and body shape, while they reached the physical body size of the adult locusts, or even surpassed it after the next supernumerary molt. Interestingly, when compared to control locusts, the total duration of the fifth and normally final nymphal (N5) stage was shorter than normal. This appeared to correspond with temporal and quantitative changes in hemolymph ecdysteroid levels, as well as with altered expression of the rate-limiting Halloween gene, Spook (SgSpo). In addition, the levels of the ecdysone receptor (SgEcR) and retinoïd X receptor (SgRXR) transcripts were altered, indicating that silencing SgE93 affects both ecdysteroid synthesis and signaling. Upon knockdown of SgE93, a very potent upregulation of the SgKr-h1 transcript levels was observed in both head and fat body, while no significant changes were detected in the transcript levels of SgJHAMT and SgCYP15A1, the enzymes that catalyze the two final steps in JH biosynthesis. Moreover, the process of molting was disturbed in these supernumerary nymphs. While attempting ecdysis to the next stage, 50% of the N6 and all N7 nymphal instars eventually died. S. gregaria is a very harmful, swarm-forming pest species that destroys crops and threatens food security in many of the world's poorest countries. We believe that a better knowledge of the mechanisms of postembryonic development may contribute to the discovery of novel, more selective and sustainable strategies for controlling gregarious locust populations. In this context, identification of molecular target candidates that are capable of significantly reducing the fitness of this devastating swarming pest will be of crucial importance.

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.

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.

Surviving a flood: effects of inundation period, temperature and embryonic development stage in locust eggs.

The Australian plague locust, Chortoicetes terminifera (Walker), is an important agricultural pest and oviposits into compacted soil across vast semi-arid and arid regions prone to irregular heavy summer rainfall. This study aimed to quantify the effects of flooding (control, 7, 14, 21, 28 and 35 days) at different temperatures (15, 20 and 25°C) and embryonic development stages (25 and 75%) on egg viability, hatchling nymph body mass and survival to second-instar. Egg viability after flooding was dependent on temperature and flood duration. Eggs inundated at 15°C showed ≥53.5% survival regardless of flood duration and development stage compared with ≤29.6% for eggs at 25°C for ≥21 days early in development and ≥14 days late in development. Hatchling nymphs did not differ in body mass relative to temperature or flood duration, but weighed more from eggs inundated early in development rather than late. Survival to second-instar was ≤55.1% at 15 and 20°C when eggs were flooded for ≥28 days late in development, ≤35.6% at 25°C when flooded for ≥28 days early in development, and zero when flooded for ≥21 days late in development. These results suggest that prolonged flooding in summer and early autumn may cause very high egg mortality and first-instar nymph mortality of any survivors, but is likely to only ever affect a small proportion of the metapopulation. More common flash flooding for ≤14 days is unlikely to cause high mortality and have any direct effect on distribution and abundance.

On the origin of grasshopper oviposition behavior: structural homology in pregenital and genital motor systems.

In female grasshoppers, oviposition is a highly specialized behavior involving a rhythm-generating neural circuit, the oviposition central pattern generator, unusual abdominal appendages, and dedicated muscles. This study of Schistocerca americana (Drury) grasshoppers was undertaken to determine whether the simpler pregenital abdominal segments, which do not contain ovipositor appendages, share common features with the genital segment, suggesting a roadmap for the genesis of oviposition behavior. Our study revealed that although 5 of the standard pregenital body wall muscles were missing in the female genital segment, homologous lateral nerves were, indeed, present and served 4 ovipositor muscles. Retrograde labeling of the corresponding pregenital nerve branches in male and female grasshoppers revealed motor neurons, dorsal unpaired median neurons, and common inhibitor neurons which appear to be structural homologues of those filled from ovipositor muscles. Some pregenital motor neurons displayed pronounced contralateral neurites; in contrast, some ovipositor motor neurons were exclusively ipsilateral. Strong evidence of structural homology was also obtained for pregenital and ovipositor skeletal muscles supplied by the identified neurons and of the pregenital and ovipositor skeletons. For example, transient embryonic segmental appendages were maintained in the female genital segments, giving rise to ovipositor valves, but were lost in pregenital abdominal segments. Significant proportional differences in sternal apodemes and plates were observed, which partially obscure the similarities between the pregenital and genital skeletons. Other changes in reorganization included genital muscles that displayed adult hypertrophy, 1 genital muscle that appeared to represent 2 fused pregenital muscles, and the insertion points of 2 ovipositor muscles that appeared to have been relocated. Together, the comparisons support the idea that the oviposition behavior of genital segments is built upon a homologous, segmentally iterated motor infrastructure located in the pregenital abdomen of male and female grasshoppers.

Glia associated with central complex lineages in the embryonic brain of the grasshopper Schistocerca gregaria.

We have investigated the pattern of glia associated with central complex lineages in the embryonic brain of the grasshopper Schistocerca gregaria. Using the glia-specific marker Repo, we identified glia associated externally with such lineages, termed lineage-extrinsic glia, and glia located internally within the lineages, termed lineage-intrinsic glia. Populations of both glial types increase up to 60 % of embryogenesis, and thereafter decrease. Extrinsic glia change their locations over time, while intrinsic ones are consistently found in the more apical part of a lineage. Apoptosis is not observed for either glial type, suggesting migration is a likely mechanism accounting for changes in glial number. Proliferative glia are present both within and without individual lineages and two glial clusters associated with the lineages, one apically and the other basally, may represent sources of glia.

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.

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.

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.

Astrocyte-like glia associated with the embryonic development of the central complex in the grasshopper Schistocerca gregaria.

In this study we employed the expression of the astrocyte-specific enzyme glutamine synthetase, in addition to the glia-specific marker Repo, to characterize glia cell types associated with the embryonic development of the central complex in the grasshopper Schistocerca gregaria. Double labeling experiments reveal that all glutamine synthetase-positive cells associated with the central complex are also Repo-positive and horseradish peroxidase-negative, confirming they are glia. Early in embryogenesis, prior to development of the central complex, glia form a continuous population extending from the pars intercerebralis into the region of the commissural fascicles. Subsequently, these glia redisperse to envelop each of the modules of the central complex. No glial somata are found within the central complex neuropils themselves. Since glutamine synthetase is expressed cortically in glia, it allows their processes as well as their soma locations to be visualized. Single cell reconstructions reveal one population of glia as directing extensive ensheathing processes around central complex neuropils such as the central body, while another population projects columnar-like arborizations within the central body. Such arborizations are only seen in central complex modules after their neuroarchitecture has been established suggesting that the glial arborizations project onto a prior scaffold of neurons or tracheae.

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.

Proliferative cell types in embryonic lineages of the central complex of the grasshopper Schistocerca gregaria.

The central complex of the grasshopper Schistocerca gregaria develops to completion during embryogenesis. A major cellular contribution to the central complex is from the w, x, y, z lineages of the pars intercerebralis, each of which comprises over 100 cells, making them by far the largest in the embryonic protocerebrum. Our focus has been to find a cellular mechanism that allows such a large number of cell progeny to be generated within a restricted period of time. Immunohistochemical visualization of the chromosomes of mitotically active cells has revealed an almost identical linear array of proliferative cells present simultaneously in each w, x, y, z lineage at 50% of embryogenesis. This array is maintained relatively unchanged until almost 70% of embryogenesis, after which mitotic activity declines and then ceases. The array is absent from smaller lineages of the protocerebrum not associated with the central complex. The proliferative cells are located apically to the zone of ganglion mother cells and amongst the progeny of the neuroblast. Comparisons of cell morphology, immunoreactivity (horseradish peroxidase, repo, Prospero), location in lineages and spindle orientation have allowed us to distinguish the proliferative cells in an array from neuroblasts, ganglion mother cells, neuronal progeny and glia. Our data are consistent with the proliferative cells being secondary (amplifying) progenitors and originating from a specific subtype of ganglion mother cell. We propose a model of the way that neuroblasts, ganglion mother cells and secondary progenitors together produce the large cell numbers found in central complex lineages.

Multipotent neuroblasts generate a biochemical neuroarchitecture in the central complex of the grasshopper Schistocerca gregaria.

We have examined the developmental expression of the neuromodulators locustatachykinin, leucokinin-1, allatostatin and serotonin in a subset of lineages (Y, Z) of the central complex in the brain of the grasshopper Schistocerca gregaria. First, we show that all these neuromodulators are expressed in the same lineages during embryogenesis. The neuroblasts generating these lineages are therefore biochemically multipotent. Second, the neurons expressing the different neuromodulators are found clustered at stereotypic locations in their respective lineages. Locustatachykinin and leucokinin-1 map to the apical region of the lineage, allatostatin medially and serotonin to the base of the lineage. Since the location in these lineages translates into their birth order, we have been able ontogenetically to analyse their biochemical expression patterns. The age-profile within a lineage reveals that locustatachykinin- and leucokinin-1-expressing neurons are born first, then allatostatin neurons and finally serotoninergic neurons. Co-expression has been tested for serotonin with locustatachykin, leucokinin-1 or allatostatin and is negative but is positive for locustatachykinin and leucokinin-1, consistent with the stereotypic location of cells in the lineages. The delay between the birth of a neuron and the expression of its neuromodulator is stereotypic for each substance. Combined with a known birth date, this delay translates into a developmental expression pattern for the central complex itself.

A developmental study of enteric neuron migration in the grasshopper using immunological probes.

Motility of enteric plexus neurons in the grasshopper Locusta migratoria depends critically on the NO/cGMP signaling cascade. This is reflected in a strong NO-dependent cGMP staining in migrating enteric midgut neurons. In contrast, first cGMP immunoreactivity (cGMP-IR) in the foregut enteric ganglia was detected clearly after the main migratory processes have taken place. Thus, expression of cGMP-IR in migrating neurons is a distinct phenomenon restricted to neurons forming midgut and foregut plexus, that does not occur during convergence of neurons forming the enteric ganglia. Analysis of time lapse video microscopy of migrating midgut neurons together with an immunofluorescence study of midgut cellular structures suggests a contribution of the midgut musculature to enteric neuron guidance. Additionally, during midgut plexus formation a fasciculating signal for enteric neuron neurites appears to be provided by the cell adhesion molecule Fasciclin I.

Epithelial domains and the primordial antennal nervous system of the embryonic grasshopper Schistocerca gregaria.

The antenna is a key sensory organ in insects. Factors which pattern its epithelium and the spacing of sensillae will play an important role in shaping its contribution to adaptive behavior. The antenna of the grasshopper S. gregaria has three major articulations: scape, pedicel, and flagellum. During postembryonic development, the flagellum lengthens as segments (so-called meristal annuli) are added at each molt. However, the five most apical annuli do not subdivide; thus, their epithelial domains must already be defined during embryogenesis. We investigated epithelial compartmentalization and its relationship to the developing primordial nervous system of the antenna by simultaneous immunolabeling against the epithelial cell surface molecule Lachesin, against neuron-specific horseradish peroxidase, and against the mitosis marker phospho-histone 3. We found that Lachesin is initially expressed in a highly ordered pattern of "rings" and a "sock" in the apical antennal epithelium of the early embryo. These expression domains appear in a stereotypic order and prefigure later articulations. Proliferative cells segregate into these developing domains and pioneer- and sensory-cell precursors were molecularly identified. Our study allows pioneer neurons, guidepost cells, and the earliest sensory cell clusters of the primordial nervous system to be allocated to their respective epithelial domain. As the apical-most five domains remain stable through subsequent development, lengthening of the flagellum must originate from more basal regions and is likely to be under the control of factors homologous to those which regulate boundary and joint formation in the antenna of Drosophila.

Scanning laser optical tomography resolves developmental neurotoxic effects on pioneer neurons.

Developmental neurotoxic compounds impair the developing human nervous system at lower doses than those affecting adults. Standardized test methods for assessing developmental neurotoxicity (DNT) require the use of high numbers of laboratory animals. Here, we use a novel assay that is based on the development of an intact insect embryo in serum-free culture. Neural pathways in the leg of embryonic locusts are established by a pair of afferent pioneer neurons, extending axons along a well-defined pathway to the central nervous system. After exposure to test chemicals, we analyze pioneer neuron shape with conventional fluorescence microscopy and compare it to 3D images, obtained by scanning laser optical tomography (SLOT) and processed by a segmentation algorithm. The segmented SLOT images resolve the 3D structure of the pioneers, recognize pathfinding defects and are thus advantageous for detecting DNT-positive compounds. The defects in axon elongation and pathfinding of pioneer axons caused by two DNT-positive reference compounds (methylmercury chloride; sodium(meta)arsenite) are compared to the biochemically measured general viability of the embryo. Using conventional fluorescence microscopy to establish concentration-response curves of axon elongation, we show that this assay identifies methylmercury chloride and the pro-apoptotic compound staurosporine as developmental neurotoxicants.

Microdissection and chromosome painting of X and B chromosomes in the grasshopper Eyprepocnemis plorans.

The relative location of 2 repetitive DNAs, i.e. ribosomal (rDNA) and a tandemly repeated satellite DNA (satDNA), with respect to the centromere, suggested that B chromosomes in the grasshopper Eyprepocnemis plorans derived intraspecifically from the X chromosome. To test this hypothesis, we microdissected X and B chromosomes and amplified the obtained DNA by 2 different procedures, the conventional DOP-PCR method and the single-cell whole-genome amplification GenomePlex method. We then generated DNA probes to perform chromosome painting. Our results have confirmed that X and B chromosomes share many DNA sequences between them and with most of the autosomes, especially at locations where the satDNA and rDNA reside, in consistency with previous information. This supports the hypothesis of an intraspecific origin of B chromosomes in E. plorans. Nevertheless, the present results did not help to clarify whether Bs were derived from the X chromosome or else from 1 or more autosomes.