Matrix metalloproteinases are involved in eclosion and wing expansion in the American cockroach, Periplaneta americana.

Matrix metalloproteinases (MMPs) are the major proteinases that process or degrade numerous extracellular matrix (ECM) components and are evolutionarily conserved from nematodes to humans. During molting in insects, the old cuticle is removed and replaced by a new counterpart. Although the regulatory mechanisms of hormones and nutrients in molting have been well studied, very little is known about the roles of ECM-modifying enzymes in this process. Here, we found that MMPs are necessary for imaginal molting of the American cockroach, Periplaneta americana. Inhibition of Mmp activity via inhibitor treatment led to the failure of eclosion and wing expansion. Five Mmps genes were identified from the P. americana genome, and PaMmp2 played the dominant roles during molting. Further microscopic investigations showed that newly formed adult cuticles were attenuated and that then chitin content was reduced upon Mmp inhibition. Transcriptomic analysis of the integument demonstrated that multiple signaling and metabolic pathways were changed. Microscopic investigation of the wings showed that epithelial cells were restrained together because they were incapable of degrading the ECM upon Mmp inhibition. Transcriptomic analysis of the wing identified dozens of possible genes functioned in wing expansion. This is the first study to show the essential roles of Mmps in the nymph-adult transition of hemimetabolous insects.

Establishment of an embryonic cell line from the American cockroach Periplaneta americana (Blattaria: Blattidae) and a preliminary study of telomerase activity changes during the culturing process.

Despite the pest status and medicinal value of the American cockroach Periplaneta americana, few attempts have been made to establish cell lines from this insect owing to the difficulty of culturing Blattarian cells. Here, we describe the establishment of the RIRI-PA1 line from P. americana embryo tissue following primary culture in modified Grace's medium containing 20% fetal bovine serum. RIRI-PA1 was found to primarily consist of attached spindle-shaped and giant cells, which attach themselves to their container. The population-doubling time of 40th-passage cells was approximately 84.8 h. The average chromosome number at the 30th passage was 42, with 40% of cells demonstrating substantial variations, with the highest number of variations of 78 and lowest of 24. The identity of RIRI-PA1 was confirmed by comparing the COI gene of these cells to that of P. americana embryo tissue. Telomerase activity decreased in primary cells after 7 d of culture and 5th-passage cells in comparison to embryo tissues; however, compared to the other cultured cells tested, the telomerase activity significantly increased at the 20th passage. We propose that the stagnation periods and cessation of proliferation observed relate to cellular telomerase activity, but the relationship between insect cell proliferation and telomerase as well as the regulatory mechanism involved remains to be elucidated.

The contribution of the melanin pathway to overall body pigmentation during ontogenesis of Periplaneta americana.

The most prominent colors observed in insects are black or brown, whose production is attributed to the melanin pathway. At present, though, the contribution of this pathway to overall body pigmentation throughout ontogenesis is still lacking. To address this question we examined the roles of 2 key melanin genes (TH and DDC), in embryonic and postembryonic development of the American cockroach, Periplaneta americana. Our results show that the melanin pathway does not contribute to the light brown coloration observed in the first nymphs. However, the dark brown coloration in mid nymphs and adults is produced solely from the melanin pathway. In addition, the DDC RNAi results reveal that it is dopamine melanin, not DOPA melanin, acts as the main contributor in this process. Overall, present study provides a new insight into insect pigmentation suggesting that genetic mechanisms of coloration can change during ontogenesis. Future studies of additional basal insect lineages will be required to assess in more details the generality of this phenomenon.

Functional analysis of Scr during embryonic and post-embryonic development in the cockroach, Periplaneta americana.

The cockroach, Periplaneta americana represents a basal insect lineage that undergoes the ancestral hemimetabolous mode of development. Here, we examine the embryonic and post-embryonic functions of the hox gene Scr in Periplaneta as a way of better understanding the roles of this gene in the evolution of insect body plans. During embryogenesis, Scr function is strictly limited to the head with no role in the prothorax. This indicates that the ancestral embryonic function of Scr was likely restricted to the head, and that the posterior expansion of expression in the T1 legs may have preceded any apparent gain of function during evolution. In addition, Scr plays a pivotal role in the formation of the dorsal ridge, a structure that separates the head and thorax in all insects. This is evidenced by the presence of a supernumerary segment that occurs between the labial and T1 segments of RNAiScr first nymphs and is attributed to an alteration in engrailed (en) expression. The fact that similar Scr phenotypes are observed in Tribolium but not in Drosophila or Oncopeltus reveals the presence of lineage-specific variation in the genetic architecture that controls the formation of the dorsal ridge. In direct contrast to the embryonic roles, Scr has no function in the head region during post-embryogenesis in Periplaneta, and instead, strictly acts to provide identity to the T1 segment. Furthermore, the strongest Periplaneta RNAiScr phenotypes develop ectopic wing-like tissue that originates from the posterior region of the prothoracic segment. This finding provides a novel insight into the current debate on the morphological origin of insect wings.

Polyphosphate polymers during early embryogenesis of Periplaneta americana.

Inorganic polyphosphates (PolyP) are linear polymers of phosphate (Pi) residues linked by high-energy phosphoanhydride bonds. Despite a wide distribution, their role during insect embryogenesis has not been examined so far. In this study, we show the mobilization of PolyP polymers during the embryogenesis of the cockroach Periplaneta americana. PolyP was detected by enzymatic and fluorimetric assays and found to accumulate in two main sizes by agarose gel electrophoresis. Confocal microscopy showed their presence in small vesicles. In addition, X-ray microanalysis of small vesicles showed considerable amounts of calcium, sodium and magnesium, suggesting an association of PolyP with these elements. Variations of the free Ca+2, Pi and PolyP levels were observed during the first days of embryogenesis. Our results are consistent with the hypothesis that phosphate ions modulate PolyP variation and that PolyP hydrolysis result in increasing free Ca+2 levels. This is the first investigation of PolyP metabolism during embryogenesis of an insect and might shed light on the mechanisms involving Pi storage and homeostasis during this period. We suggest that PolyP, mainly stored in small vesicles, might be involved in the functional control of Ca+2 and Pi homeostasis during early embryogenesis of P. Americana.

Interplay between acid phosphatase and cysteine proteases in mediating vitellin degradation during early embryogenesis of Periplaneta americana.

In this work, we characterized the activities of two classes of proteases and AcP during early embryogenesis of Periplaneta americana. AcP activity was first detected at day 6 and reached a maximum level at day 10 of development. Using phosphoamino acids, phosphatase activity was shown to be directed only against phosphotyrosine at day 6 while at day 10 it was also active against phosphoserine. In parallel, two classes of proteases were detected and located within yolk granules: a clan CA-cysteine protease, which was inhibited by E-64, insensitive to CA 074 and activated by acidic pH at day 3; and a neutral serine protease, which was inhibited by aprotinin at day 6. Assays of vitellin (Vt) degradation evidenced that incubations at neutral pH induced slight proteolysis, while the incubations at acidic pH did not result in Vt degradation. However, pre-incubations of Vt with AcP increased the levels of Vt acidic proteolysis and this could be inhibited by the addition of phosphatase inhibitors. On the other hand, the same pre-incubations showed no effects on the profile of degradation at neutral pH. We propose that AcP and cysteine protease cooperate to assure Vt breakdown during early embryogenesis of P. americana.

Calcium-regulated fusion of yolk granules during early embryogenesis of Periplaneta americana.

This work reported membrane fusion of yolk granules (YGs) during early embryogenesis of the insect Periplaneta americana (P. americana). We showed that eggs from Day 5 of embryogenesis possess a greater amount of enlarged YGs in comparison with Day 1. Day 5 is also the period when the largest amount of free calcium is found (approximately 17 mM) within the oothecae from early embryogenesis. Treatment of Day 1-YGs fraction with 17 mM Ca2+ resulted in a YG size pattern very similar to the one observed in Day 5 eggs, where enlarged YGs were formed. YG membrane fusion was observed by fluorescent membrane dye transfer from previously labeled small YGs to larger ones and was also visualized by electron microscopy. We also showed that the small "in fusion" YGs seemed to be acidic, suggesting that acidification is correlated with YG membrane fusion. Hence, it was shown that YGs are capable of membrane fusion in a calcium-dependent manner and this process probably occurs in vivo during early embryogenesis of P. americana.

Identical cellular distribution of all abundant neuropeptides in the major abdominal neurohemal system of an insect (Periplaneta americana).

The median neurosecretory cells in abdominal ganglia of insects synthesize a number of putative hormones, which are abundant in the abdominal perisympathetic organs (PSOs). The peptide inventory of these prominent neurohemal release sites is best investigated in the American cockroach and strongly differs from that of head/thoracic neurohemal organs. In this study, we found a complete colocalization of all abundant neuropeptides in this hormonal system, including periviscerokinin-1 and -2, pyrokinin-5, YLSamide, VEAacid, and SKNacid. The first immunoreactive cells were detected on day 18 of embryonic development and already contained the complete set of peptides. By using antisera against the above-mentioned peptides, the development of this neurohormonal system could be studied and is described in detail. Subsequent electron microscopic immunogold stainings in PSO preparations revealed the costorage of PSO peptides in a single vesicle species. Surprisingly, all these peptides were found in axons containing clear vesicles, whereas all axons with dense core vesicles were totally devoid of immunoreactivity. Unlike the axons with dense core vesicles, immunostained axons ramify in the center of the PSO but exhibit only rare morphological signs of exocytosis. Instead, putative release sites of the clear vesicle-containing axons were detected peripherally to the PSOs, namely, on the hyperneural muscle.

Properties and development of calcium currents in embryonic cockroach neurons.

In freshly dissociated neurons from embryonic cockroach (Periplaneta americana L.) brains, voltage-dependent calcium currents appear early in development (E14). Their intensity increases progressively during embryonic life until eclosion (E35). Their time course and voltage dependency are characteristic of high voltage activated (HVA) currents although a 10 mV shift of the I/V curve towards more negative potentials was observed between E18 and E23. Their sensitivity to omega-AgaTx-IVA and omega-CgTx-GVIA and insensitivity to both amiloride and isradipine indicate that the corresponding channels are of the P/Q and N types. These channels, as well as a small proportion of toxin-resistant (R) channels (about 20%), are blocked by mibefradil and verapamil. The physiological significance of these currents and their modifications during embryonic life is discussed.

Evidence of poly(ADP-ribosylation) in the cockroach Periplaneta americana.

Poly(ADP-ribosylation) is a post-translational modification of nuclear proteins typical of most eukaryotic cells. This process participates in DNA replication and repair and is mainly regulated by two enzymes, poly(ADP-ribose) polymerase, which is responsible for the synthesis of polymers of ADP-ribose, and poly(ADP-ribose) glycohydrolase, which performs polymer degradation. The aim of this work was to investigate in the cockroach Periplaneta americana L. (Blattaria: Blattidae) the behaviour of poly(ADP-ribosylation). In particular, we addressed: (i) the possible modulation of poly(ADP-ribosylation) during the embryonic development; (ii) the expression of poly(ADP-ribose) polymerase and glycohydrolase in different tissues; and (iii) the role of poly(ADP-ribosylation) during spermatogenesis. In this work we demonstrated that: (i) as revealed by specific biochemical assays, active poly(ADP-ribose) polymerase and glycohydrolase are present exclusively in P. americana embryos at early stages of development; (ii) an activity carrying out poly(ADP-ribose) synthesis was found in extracts from testes; and (iii) the synthesis of poly(ADP-ribose) occurs preferentially in differentiating spermatids/spermatozoa. Collectively, our results indicate that the poly(ADP-ribosylation) process in P. americana, which is a hemimetabolous insect, displays catalytical and structural features similar to those described in the holometabolous insects and in mammalian cells. Furthermore, this process appears to be modulated during embryonic development and spermatogenesis.

Double-stranded RNA interference shows that Engrailed controls the synaptic specificity of identified sensory neurons.

The transcription factor Engrailed (En) controls the topography of axonal projections by regulating the expression of cell-adhesion molecules [1] [2] [3] [4] but it is not known whether it also controls the choice of individual synaptic target cells. In the cercal sensory system of the larval cockroach (Periplaneta americana), small numbers of identified wind-sensitive sensory neurons form highly specific synaptic connections with 14 identified giant interneurons [5] [6], and target-cell choice is independent of the pattern of axonal projections [6]. En is a putative positional determinant in the array of cercal sensory neurons [7]. In the present study, double-stranded RNA (dsRNA) interference [8] was used to abolish En expression. This treatment changed the axonal arborisation and synaptic outputs of an identified En-positive sensory neuron so that it came to resemble a nearby En-negative cell, which was itself unaffected. We thus demonstrate directly that En controls synaptic choice, as well as axon projections.

Quantitative morphological analysis of embryonic cockroach (Periplaneta americana) brain neurons developing in vitro.

Neurons dissociated from the brain of embryonic cockroaches (Periplaneta americana) can be maintained in culture for several weeks. The survival as well as the progressive organization of the neurons into a complex network was studied during a 5-week period under different culture conditions. About 10% of the dissociated cells adhered to the culture dish. This figure remained constant throughout the culture. The cell diameter ranged from 10 to 20 microns and did not change significantly over time in culture. Whereas only a few cells exhibited neurites at the start of the culture, the number of cells exhibiting neurites increased to reach about 99% after 2 weeks. The different cells were then connected to each other, forming a network, which became more and more complex. The number of cells per cluster as well as the length and the diameter of the "connectives" that linked the different clusters were found to increase with time. The morphology of individual neurons within the network was visualized after intracellular injection of biocytin. Labeling with antibodies raised against serotonin or GABA indicated that neurons were able to differentiate and to acquire specific neurotransmitter fates. The serotonergic phenotype was found to appear progressively throughout the culture, in parallel with the formation of the network. Cell density, addition of fetal calf serum, and ecdysone were shown to influence the development of the network.

Specificity of identified central synapses in the embryonic cockroach: appropriate connections form before the onset of spontaneous afferent activity.

The mechanisms by which neurons recognize the appropriate postsynaptic cells remain largely unknown. A useful approach to this problem is to use a system with a few identifiable neurons that form highly specific synaptic connections. We studied the development of synapses between two identified cercal sensory afferents and two giant interneurons (GIs) in the embryonic cockroach Periplaneta americana. By 46% of embryonic development, the axons of the filiform hair sensory neurons have entered the terminal ganglionic neuropil and grow alongside the GI primary dendrites, although they do not form synapses. From 50% of development, the GI dendrites grow outward from the center of the neuropil to contact the presynaptic axons and their branches. The sensory neurons begin to spike at 52% of development, and, from 55% of development, these action potentials evoked excitatory postsynaptic potentials in the GIs. Synaptic contacts were first seen at this time. The pattern of synaptic connections was highly specific from the outset. G12 had strong input from the medial (M) afferent and had almost negligible input from the lateral (L) afferent, whereas G13 had input from both. This specificity was present before bursts of spontaneous activity began in the sensory neurons at 59% of development. G12 filopodia selectively formed synaptic contacts with the M axon rather than the L axon. The few contacts made by G12 with the L axon had a normal morphology but fewer presynaptic densities. Filopodial insertions were not involved in selective synapse formation. In this system, highly specific synaptic recognition appears to be activity independent.

Influence of receptor axons on the formation of olfactory glomeruli in a hemimetabolous insect, the cockroach Periplaneta americana.

The embryonic development of the hemimetabolous insect Periplaneta americana requires approximately 31 days. Deafferentation experiments were used to investigate the role of ingrowing receptor axons during embryogenesis, specifically their influence 1) on the subdivision of the antennal lobe neuropil into glomeruli, 2) on the morphology and number of glial cells, and 3) on the arborization pattern of central neurons. The flagellum of one antenna was removed from embryos at different developmental stages starting with day 10. Subsequently, they were raised in culture until a total age of 26 days. At day 10, the deutocerebrum has received only a very small number (ca. 0.4%) of antennal receptor axons; deafferentation at this stage allowed us to deprive the deutocerebrum of approximately 99% of its normal antennal input. Deafferentation has marked effects on the organization of the antennal lobe neuropil. The deafferented lobe is reduced in volume compared to the control side; the characteristic glomeruli are missing. During normal development glomeruli are formed between day 19 and 22, first in dorsal and then in ventral antennal lobe regions. By removing the antenna before day 20, their formation is disturbed in all parts of the antennal lobe. If deafferentation is performed after stage 20, glomeruli persist in dorsal regions, but are missing in ventral regions. On day 24 or later, glomeruli in both dorsal and ventral regions are unaffected by deafferentation. Glial cells continue to extend fine processes into the neuropil in the absence of ingrowing receptor axons. The number of glial cells is reduced compared to control lobes. Multiglomerular local interneurons and other gamma-amino butyric acid-immunoreactive neurons, as well as projection neurons, fail to develop glomerular arborization patterns in antennal lobes deprived of sensory axons.

[Parasitism in the oothecae of Periplaneta americana (Linnaeus)]. / Parasitismo en ootecas de Periplaneta americana (Linnaeus).

Parasitism present in others of Periplaneta americana in 3 municipalities of Havana City was observed, and it was detected that Tetrastichus hagenowii (Ratz.) attained significant levels of parasitism for an average value of 21.5% in the oothecae revised. Their presence in locations where different pesticides are used and their biological characteristics give them the probability of being an efficient bioregulator of cockroaches.

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.

Patch-clamp analysis of the effects of the insecticide deltamethrin on insect neurones.

1. The mode of action of the pyrethroid insecticide deltamethrin on inexcitable embryonic cultured cockroach neurones has been investigated using the patch-clamp technique. 2. Whole-cell recordings of the current induced by step depolarizations of the cell membrane showed that concentrations of deltamethrin ranging from 10(-8) to 5 x 10(-6) mol l-1 induced a small tetrodotoxin (TTX)-sensitive inward current that peaked at around +10 mV and reversed at around +60 mV. The activation and inactivation kinetics of this current were much slower than those of the axonal sodium current in this same species and were relatively insensitive to membrane potential. Steady-state inactivation was almost absent. 3. Single-channel activity associated with the action of the insecticide was analyzed using the cell-attached configuration. Three distinct patterns of activity were found: (1) discrete single-channel events of relatively short duration, (2) long events of comparatively small amplitude and (3) complex bursts made up of a succession of openings and closings to several levels. These three patterns were analyzed quantitatively using specially designed programs. 4. The first pattern of activity could be seen in most patches. It consisted of short (1-10 ms) rectangular events of comparatively small amplitude (1.5 pA at rest) and very low open time probability (around 0.001). The current-voltage relationship of these small events was linear over the voltage range studied and the (extrapolated) reversal potential approximated ENa. 5. The second pattern of activity was observed less frequently. The channels could stay open for very long periods (up to several seconds) and occasionally flickered between two or more levels. 6. The third pattern of activity was observed in many patches. During the burst, which could last from a few milliseconds to a few hundred milliseconds, the single-channel current jumped almost continuously between several levels (up to 7 or 8).

Micromolar concentrations of veratridine activate sodium channels in embryonic cockroach neurones in culture.

The mode of action of the alkaloid veratridine has been reinvestigated on cultured cockroach neurones, which are normally inexcitable and do not have a detectable fast sodium current. The whole-cell and cell-attached configurations of the patch-clamp technique were used to record the macroscopic and single channel currents, respectively. Concentrations of veratridine ranging from 10(-8) to 10(-5) M were found to induce a small tetrodotoxin (TTX)-sensitive inward current, which peaked around +10 mV and reversed around +55 mV. This current exhibited a pronounced plateau and was insensitive to changes in the holding potential. Bath application of veratridine induced typical TTX-sensitive inwardly-directed single-channel activity, falling into two (apparently coupled) categories of events: first, relatively large events (1 pA at a hyperpolarized potential of -125 mV relative to rest) of short duration and, second, small bursting events (0.4 pA under similar conditions) of slightly longer duration. Pipette application of similar concentrations of veratridine had similar effects in that two categories of events were observed: first, bursts of large events with multiple conductance states and, second, small events of very long duration. The current/voltage relationship of these events was linear for the voltage range studied and the (extrapolated) reversal potential approximated +110 mV. These results support the hypothesis that veratridine, in small concentrations, induces a slow voltage-dependent activation of TTX-sensitive sodium channels, independent of the fast activating and inactivating sodium channels involved in action potential generation.