Cockroach Embryos Research Articles

Juvenile hormone and methyl farnesoate production in cockroach embryos in relation to dorsal closure and the reproductive modes of different species of cockroaches

Juvenile hormone (JH), produced by the corpora allata (CA), is first detectable after dorsal closure, a conspicuous event in embryogenesis. The present research found that the timing of dorsal closure was consistently at about 45% of the total embryonic development time across most of the oviparous and ovoviviparous cockroach species examined. These included the ovoviviparous cockroaches Blaberus discoidalis, Byrsotria fumigata, Rhyparobia maderae, Nauphoeta cinerea, Phoetalia pallida, Schultesia lampyridiformis, and Panchlora nivea, as well as the oviparous cockroaches Blatta orientalis, Periplaneta americana, Eurycotis floridana, and Supella longipalpa. However, the only known viviparous cockroach Diploptera punctata completed dorsal closure at 20.8% of embryo development time. Methyl farnesoate (MF), the immediate precursor of JH III, is considered a functional molecule in crustaceans; however, in insects its function is still unclear. To understand the role of JH and MF in cockroach embryos, I compared JH and MF biosynthesis and release in several cockroach species of known phylogenetic relationships. Using a radiochemical assay, the present research showed that cockroach embryos representing all three reproductive modes produced and released both JH and MF, as previously shown for B. germanica, N. cinerea, and D. punctata. Members of a pair of embryonic CA from B. discoidalis, B. fumigata, R. maderae, and D. punctata were incubated with and without farnesol. MF accumulated in large amounts only in CA of R. maderae in the presence of farnesol, which indicates that control of the last step of biosynthesis of JH, conversion of MF into JH by MF epoxidase, is probably a rate-limiting step in this species.

The Role of Allatostatins in Juvenile Hormone Synthesis in Insects and Crustaceans

Allatostatins are pleiotropic neuropeptides for which one function in insects is the inhibition of juvenile hormone synthesis. Juvenile hormone, an important regulator of development and reproduction in insects, is produced by the corpora allata. Mandibular organs, the crustacean homologs of insect corpora allata, produce precursors of juvenile hormone with putatively similar functions. Three types of allatostatins in insects have been isolated: FGLamides, W(X)(6)Wamides, and PISCFs. All act rapidly and reversibly; however, although these types occur in all groups of insects studied, they act as inhibitors of juvenile hormone production in only some groups. Only the FGLamide-type peptides have been isolated in crustaceans, in which they may function to stimulate production of hormone by the mandibular glands, as occurs in early cockroach embryos. Much remains to be learned in order to understand the role of allatostatins in the modulation of hormone production.

Neuropeptides in perisympathetic organs of Manduca sexta: specific composition and changes during the development

We used a combination of matrix-assisted laser desorption-ionization time-of-flight mass spectrometry and immunocytochemistry to investigate the peptides from abdominal perisympathetic organs of Manduca sexta. Altogether three mass peaks, detected in mass spectra from single abdominal perisympathetic organs were identical with already known neuropeptides, namely CAP 2b, CCAP, and Manduca-allatotropin. Only CAP 2b was found throughout the postembryonic development. In larvae, perisympathetic organs of the abdominal ganglia 1 and 7 do not accumulate neuropeptides. During the metamorphosis, the number of putative hormones stored in the abdominal perisympathetic organs, increases dramatically. Not a single substance, however, obtained in mass spectra of larval perisympathetic organs disappeared in the respective adult neurohemal organs. Peptides from abdominal perisympathetic organs are different from those of thoracic perisympathetic organs and the retrocerebral complex. Manduca-FLRFa-2 and -3 are enriched in thoracic perisympathetic organs; FLRFa-1, corazonin and adipokinetic hormone are abundant peptides of the retrocerebral complex. The majority of ion signals, however, represent unknown substances. An antiserum which recognized CAP 2b allowed the morphological characterization of a median neurosecretory system in the abdominal ventral nerve cord of M. sexta, which resembles that of cockroach embryos. Double stainings confirmed that crustacean cardioactive peptide (CCAP) becomes colocalized with CAP 2b in median neurosecretory cells during the last larval instar. This colocalization continues in adult insects.

A Paradoxical Gradient of a Basal Lamina-Associated Repellent Is Essential for Pathfinding by the Ti1 Pioneer Axons in Cockroach Embryos

Perturbations of in situ axon growth with proteolytic enzymes and monoclonal antibodies were used to determine the role of gradient guidance cues in the formation of the Ti1 pioneer axon trajectory in cultured cockroach embryos. Treatment with enzymes that degrade the basal lamina indicated that this substrate contains both an elastase-sensitive proximal directing cue and a collagenase-sensitive distal directing cue. The latter is shown to be a repellent of axon growth and is identical to the PROD-2 antigen that is distributed in a gradient along the proximal-distal axis of the leg with high levels in proximal regions. This means that throughout the course of their growth the axons extend in the direction of increasing levels of repellent. At a critical decision point in the trajectory the axons change both the direction of growth and the substrate to which their growth cones adhere. PROD-2 plays an essential role in both of these processes.

Thein VivoRegulation of Pioneer Axon Growth by FGF-2 and Heparan Sulfate Proteoglycans in Cultured Embryos of the Cockroach

Antibody perturbation experiments on cultured cockroach embryos demonstrated that a localized source of an FGF-2-like immunoreactive molecule in the head is required for the proper growth of pioneer axons in the leg. The study of axon growth in various fragments of cultured embryos and in the presence of various conditioned media showed that FGF-2 is needed to counteract the effects of an inhibitor of axon growth produced in the body trunk of the embryo. Endogenous heparan sulfate proteoglycans mediate these effects of FGF-2 on axon growth. The results of experiments with FGF-2 and/or body trunk axon growth inhibitor added to the culture medium indicate that more globally and uniformly distributed molecules may play as important a role in axon guidance as the more spatially restricted guidance cues. The results are interpreted in terms of a model that is consistent with a role for the FGF-2 receptor in axon growth.

Error correction during guidance of pioneer axons in the leg of the cockroach embryo.

Axons of the Til and Fe2 pioneer neurons in the legs of insect embryos possess separate and highly stereotyped proximal projections towards the CNS. However, quantitative analyses of deviations from the standard paths during the period of axon growth indicate that transient errors occur unexpectedly often. The distribution of legs with axons following deviant paths among the embryos analyzed is used to determine whether these errors are caused by random developmental noise or by non-random genetic or environmental factors. During the formation of the Til pathway all the errors are characterized by defasciculation of the 2 axons, occur with an average incidence of 7% and are statistically shown to be randomly caused. In comparison, during the formation of the Fe2 pathway the errors are characterized by both defasciculation and elongation in an inappropriate distal direction, occur with an incidence of 16%, and as revealed by statistical analyses, are caused by a non-random factor. Therefore, during pathfinding by these 2 pairs of axons there is a need for error-correcting mechanisms to insure the stereotypy of the final projections. These error-correcting mechanisms are suggested to have properties similar to those producing canalization as proposed by Waddington.

Genetics and Expression of Mottled Body: Mosaic Mutant of the German Cockroach (Dictyoptera: Blattellidae) with Possible Cytoplasmic Inheritance

Mottled body (Mt) is a mutant of the German cockroach, Blattella germanica (L.), which is characterized by extensive color variegation on the surface of the body. Irregular light patches appear in the integument of wild-type black color. Mottling is more conspicuous on abdominal segments than on thoracic segments. The character is clearly observed in the nymphal stage but also can be seen on the small dorsal tergites of adults. Expression of mottling is variable. The trait is mostly maternally inherited, and cytoplasmic factor(s) may be involved. A method for judging the degree of mottling was established. Degree of mottling was scored on the surfaces from 1st to 3rd thoracic and 1st to 7th abdominal segments. Mottling patterns did not change during late nymphal development. Mottling expression did not decrease from the Mt stock to the F1 (Mt female × wild-type male), and F2 (F1Mt female × wild-type male). Mottling patterns were often different between left and right sides. Patterns were often discontinuous across the median line of the body and sometimes one side was mottled and the other side almost wild-type color. Separate scoring between left and right surfaces and also between dorsal and ventral surfaces revealed that the degree of mottling was more correlated dorsoventrally than laterally. This suggests that mottling patterns were determined before dorsal closure during embryogenesis and are influenced by the shape peculiar to cockroach embryos packed in the ootheca, which is broad dorsoventrally and compressed laterally.

A multifunctional cell surface developmental stage-specific antigen in the cockroach embryo: involvement in pathfinding by CNS pioneer axons.

mAb DSS-8 binds to a 164-kD developmental stage-specific cell surface antigen in the nervous system of the cockroach, Periplaneta americana. The antigen is localized to different subsets of cells at various stages of development. The spatial and temporal distributions of DSS-8 binding were determined and are consistent with this antigen playing multiple roles in the development of the nervous system. Direct identification of some of these functions was made by perturbation experiments in which pioneer axon growth occurs in embryos that are cultured in vitro in the presence of mAb DSS-8 or its Fab fragment. Under these conditions the pioneer axons of the median fiber tract grow but follow altered pathways. In a smaller percentage of the ganglia, the immunoreagents additionally produce defasciculation of a subset of DSS-8 labeled axons. Therefore, direct roles for the DSS-8 antigen in both the guidance of pioneer axons and selective fasciculation have been demonstrated.

A role for proteoglycans in the guidance of a subset of pioneer axons in cultured embryos of the cockroach

Several molecules involved in the development of the nervous system have specific binding sites for the glycosaminoglycan (GAG) side chains of proteoglycans. Exogenous GAGs should bind to these sites, competitively inhibit interactions with proteoglycans, and perturb development. GAGS added to the culture medium perturb the in situ growth of pioneer axons in cultured cockroach embryos by producing axon defasciculation and growth in incorrect directions. The specificity of this phenomenon is evident from the following observations: Of all the GAGS tested only heparin and heparan sulfate produced perturbation; of the six axon tracts being pioneered during the culture period only two of them are perturbed by the GAGs; and similar perturbations are produced when embryos are cultured in the presence of heparinase II and heparitinase.

The reappearance of a developmental stage-specific antigen in adult regenerating neurons of the cockroach

A monoclonal antibody has previously been described that binds to all neurons in the 15 d (50% development) cockroach embryo but to only a small subset of neurons in the adult (Denburg et al., 1989). Experiments were performed in order to determine whether the developmental stage-specific antigen recognized by this antibody would reappear in adult neurons that were induced to undergo axonal regeneration by axotomy. It is demonstrated here that after nerve crush motor, sensory and interneurons undergo axonal regeneration and regain their ability to bind this antibody. This indicates that the developing and regenerating states of these neurons selectively use the same molecules to perform apparently similar cellular functions. The increase and subsequent decrease of antibody binding as a function of time after nerve crush was determined for each of these adult neurons. Correlations between the temporal distribution of the antigen and cellular events occurring during axonal regeneration are consistent with a role for this molecule in axon growth and the elimination of inappropriate synaptic connections. The antigen was localized to the external surface of the plasma membrane, and preliminary biochemical characterization has led to the tentative identification of the antigen as a glycolipid. These characteristics distinguish this growth-associated antigen from other previously described molecules whose temporal distribution has implicated a role for them in axon growth.

An axon growth associated antigen is also an early marker of neuronal determination

We have previously described the generation of a monoclonal antibody (DSS-3) that binds to all neurons in cockroach embryos at 50% development and to only a small subset of interneurons in the adult nervous system. This developmental stage-specific antigen was observed to reappear in all axotomized adult neurons that were undergoing axonal regeneration. In the present study the time course of the appearance of this growth-associated antigen during embryonic development was determined. Unexpectedly, the antigen was observed to be present in embryonic neurons long before axon growth. In addition, all cells in the CNS neuronal lineage (neuroblasts, ganglion mother cells, and neurons) bind the antibody as soon as they can be morphologically identified. However, the antigen is also transiently present in all neuroepithelial cells at a stage prior to the morphological differentiation of some of them to neuroblasts. Analogous patterns of DSS-3 binding to cells involved in the development of sensory neurons and leg pioneer neurons are observed. The DSS-3 antigen is therefore a very early marker for the capacity of ectodermal epithelial cells to develop along a neuronal lineage.

Developmental stage-specific antigens in the nervous system of the cockroach

A specific effort was made to obtain monoclonal antibodies that bind to macromolecules that play a role in the development of the nervous system. It was considered that good candidates for such molecules were those that were only transiently present in the embryonic nervous system. Hybridomas were prepared from spleen cells taken from mice that had been immunized with nerve cords from cockroach embryos at the 43–50% stage of development. The hybridoma supernatants were screened for antibody binding to frozen sections of both embryonic and adult thoracic ganglia. Cell lines that produced monoclonal antibodies that transiently bound to the embryonic nervous system were saved and cloned. These developmental stage-specific monoclonal antibodies either did not bind to the adult nervous system or bound to it with a pattern very different from that in the embryonic nervous system. The developmental stage-specific antigens detected by these monoclonal antibodies were organized into four categories based on the part of the embryonic nervous system in which they were transiently localized. These include binding to the cell bodies of all neurons, cell bodies of subsets of neurons or neuroblasts, subsets of axons, and the neuropile. Preliminary biochemical characterization of the antigens showed that many of these antibodies were recognizing carbohydrate epitopes. Functions for these antigens, most of which are components of the cell surface, are tentatively proposed.

Multiplicity of unitary potassium channels recorded in the somata of cultured neurones from cockroach embryos.

Multiplicity of unitary potassium channels recorded in the somata of cultured neurones from cockroach embryos.

Unusual structural features and assembly of gap and pleated septate junctions in embryonic cockroach CNS.

Junctional assembly in the developing CNS in cockroach embryos has been studied during the last half of neurogenesis. Atypical linear tracts of gap junctions are found to develop between attenuated cytoplasmic glial cell processes and their overlying perineurial cells during the last third of development. During both perineurial and glial gap-junctional formation, 13 nm E face (EF) intramembrane particles (IMPs), such as are characteristic of arthropod gap junctions, are seen initially as free IMPs; these then become arranged in loose irregular clusters or alignments and finally are aggregated in plaques. P face ridges (or EF grooves), typical of tight junctions, are found on the same perineurial membrane face as assembling gap-junctional PF pits (or EF particles). Pleated separate junctions also develop between adjacent perineurial processes during the last third of embryogenesis; these form by the apparent migration of individual 8 nm PF IMPs into meandering rows, which then become aligned in numerous orderly parallel stacks. Although all these junctions occur on the same perineurial membrane face, the IMPs that form the different junctional types never appear to be confused during junctional assembly. The cues to signal the advent of these precise patterns, however, are unknown.

Development of chemosensitivity of an identified insect interneurone

Characteristic features of cockroach embryos (Periplaneta americana) are determined for different stages in development. Morphological changes in giant interneurone 2 (GI 2) in the terminal abdominal ganglion during embryogenesis are described based on cobalt injections. A sequential proliferation of branches preceded by filopodial extension is observed between 45 and 80% embryogenesis, by which time the characteristic arborization of the first instar cell is established. The cholinergic agonist carbamylcholine was ionophoretically applied to the cell body and dendritic region of GI 2 at various stages in development, and the responses were recorded intracellularly. Chemosensitivity of GI 2 appears at 40 to 45% embryogenesis, to a similar degree in both cell body and dendrites. The sensitivity of these areas subsequently diverges, and after the 60 to 65% stage, the dendrites are approximately 1000 times more sensitive than the cell body. At 80 to 85% embryogenesis, there is a temporary peak in dendritic sensitivity, and cell body sensitivity increases during postembryonic development. The changes in sensitivity take place before synaptogenesis, and the decline in dendritic sensitivity after the 80% stage may be related to synaptic maturation.

Cuticulogenesis correlated with ultrastructural changes in oenocytes and epidermal cells in the late cockroach embryo

During late embryogenesis in a cockroach, the epidermal cells secrete two cuticles: the embryonic cuticle and the pharate first larval cuticle. Late embryogenesis begins with the deposition of the cuticulin layer of the embryonic cuticle. The embryonic cuticle is an atypical one. It remains relatively thin and a well lamellated endocuticle is usually lacking. After general apolysis of the embryonic cuticle the epidermis secretes the epicuticle of the first larval cuticle and, subsequently, a typical lamellate procuticle. During the penultimate phase of late embryogenesis (i.e. before general apolysis) the epidermis becomes larvally committed. Some epidermal cells start to differentiate into specialized structures of the dermal glands, whereas the differentiated oenocytes appear to have acquired some stability. Nevertheless, shortly before general apolysis some oenocytes display signs of an increased alteration of the SER. When general apolysis occurs, the oenocytes contain a well-developed SER. The whole of the oenocyte population is programmed to regress after epicuticle deposition of the first larval cuticle. The correlation of oenocyte regression with available data on cuticulogenesis, ecdysteroid titres and cuticular lipid synthesis is discussed.

DNA synthesis and epidermal differentiation in the cockroach embryo and pharate first instar larva: Moulting hormone and mitomycin

The second period of the life occurring in ovo, which corresponds to the end of the embryonic development and to the pharate first instar larva stage, may be divided into 4 phases with respect to DNA synthesis: the first one lasting about one week may correspond to proliferative divisions ensuring appendage growth; the second (maximal DNA synthesis) and the third correspond to the synthesis involved in spine and hair formation: the fourth one, during which the epidermis differentiates. does not show DNA synthesis. Fifty μg/ml of inokosterone added to the culture medium stimulates DNA synthesis and then blocks it (no more synthesis by day 4). A detailed analysis of the stimulating effect of the hormone on DNA synthesis shows that it could trigger the synthesis involved in spine and hair formation. Further experiments using mitomycin C confirmed this hypothesis: 100 μg/ml of mitomycin added to the cultured medium at the time of DNA synthesis triggered by exogenous hormone, provokes the death of the cells passing through the S phase and thus prevents spine and hair formation. Mitomycin does not suppress cuticle secretion. It can be concluded that epidermal cell differentiation which occurs in response to moulting hormone is not a single phenomenon, but consists of at least 2 independent processes. In our experimental conditions inokosterone seems to trigger only the DNA synthesis linked to differentiation.

Effects of moulting hormone on RNA and cuticle synthesis in the epidermis of cockroach embryos cultured in vitro

In epidermal cells of embryos of Blabera cultured in a medium without exogenous moulting hormone, 2 peaks in RNA synthesis occur respectively on days 7 and 11, followed by two peaks in cuticular products synthesis: a peak in protein synthesis on day 16 and a peak in chitin synthesis on day 17. Addition of exogenous moulting hormone to the culture medium hastens the two peaks in RNA synthesis and brings them closer together on days 1 and 4. The peaks in protein and chitin synthesis are similarly brought forward, and both appear on day 5. Localisation of the synthesised cuticular products is a function of the stage of differentiation reached in culture by the epidermal cells. Initially, there are little of the synthesised products and they are distributed uniformly throughout the cells. Progressively, the rate of synthesis increases and the products appear concentrated at the tips of the cells and then pass into the developing cuticle. At the beginning, the whole thickness of the deposited cuticle is labelled, then only the basal regions. Finally, the intensity of the label decreases. The results of incorporation rates of uridine, proline and glucosamine, the observation of the localisation of the label, and the secretion of the cuticle show that RNA synthesis occur before protein and chitin syntheses which are tied to the secretion of the procuticle. The hormone acts directly on the epidermal cells by triggering, in a general manner, the events which usually occur under the endogenous moulting hormone control.

Formation of cockroach interganglionic connectives: an in vitro analysis.

Individual ganglia of the cockroach embryo (Periplaneta americana) were explanted on clean glass coverslips immersed in a chemically defined liquid medium and incubated for periods up to eight weeks. Substantial, straight interganglionic connections were formed between: (1) rows of ganglia arranged in the normal in vivo configuration; (2) rows of ganglia placed in abnormal orders; (3) rows of ganglia which never form connections in vivo because they occur singly in the embryo; and (4) rows of ganglia in natural sequences but which have had their rostro-caudal axes rotated 90 degrees in relation to the line of the row. Therefore fascicles and interganglionic connectives were formed without regard to normal in vivo relationships. Daily observations with a Nomarski microscope indicated that several processes are involved in connective formation. (1) Initial outgrowth is in a random, radial pattern. (2) Intersecting fibers from adjacent ganglia are deflected toward each others' perikarya. (3) Initially bowed fiber connectives are straightened, perhaps by increases in fiber tension or by fiber shortening which may be brought about by neuronal or extraneuronal (glial) processes. (4) Outgrowing fibers follow already established fiber pathways. The present results indicate that fiber-fiber and fiber-target interactions play a significant role in the formation of interganglionic connectives. In this system, the spatial relationships between ganglia determine the patterns and varieties of permissible neuronal connections. Thus, major, straight nerve trunks may be formed between adjacent ganglia which are growing out fibers on a glass surface submerged in a liquid medium which offers minimal orientation cues and provides a growth substrate vastly different and simpler than that encountered by outgrowing fibers in vivo.

Cycles embryonnaires et sécrétion de la cuticule chez l'embryon de blatte, Blabera craniifer

Cuticle deposition has been studied with the electron microscope in cockroach embryos ( Blabera craniifer) during normal incubation in situ and in culture in vitro, in the absence or presence of inokosterone (a phytoecdysone). Two cuticles are deposited successively during embryonic life, respectively between stages 11 and 17, and stage 21 and 24 hr after hatching. The occurrence of two embryonic cycles is thus demonstrated, the first ending at stage 17 without exuviation since there is no old cuticle to be shed, the second one at hatching. In embryos explanted at stage 17 and cultured in vitro, the formation of cuticle 2 occurred at the same rate as in situ. The addition of inokosterone (50 μg/ml) to the medium resulted in the early onset of cuticle deposition (in 3 days as compared with 15 days in situ) in legs previously cut at the base of the tarsus. Cuticle 2 was completed within 9 days after explantation (as compared with about 20 days elapsing in the normal embryo between stage 17 and the completion of cuticle 2). Unsectioned appendages were insensitive to the hormone. Regeneration of sectioned legs, which occurred normally n vitro in non-treated embryos, was completely inhibited in the presence of inokosterone, presumably because the hormone caused early immobilization of cells through accelerated cuticle formation. Results suggest that embryonic cycles are controlled by the same hormonal mechanism as larval cycles.