Innervation and control of tension in abdominal muscles of Blaberus discoidalis and Gromphadorhina portentosa

In Blaberus discoidalis and Gromphadorhina portentosa, the distribution of motor axons to the muscles which control movements of the spiracular valves at both respiratory and non-respiratory spiracles is identical. Both fast and slowly contracting heads of the opener muscles are innervated by an excitatory motor axon. Physiological properties of the opener excitor axon correlate with valve function. The slowly contracting head of the opener muscle is, in addition, innervated by a common inhibitor which also occasionally innervates closer muscle fibers. Activation of the common inhibitor terminates contraction of slowly contracting opener muscle fibres and initiates a rapid relaxation of these fibres.     

Tradeoffs between metabolic rate and spiracular conductance in discontinuous gas exchange of Samia cynthia (Lepidoptera, Saturniidae)

The insect tracheal system is a unique respiratory system, designed for maximum oxygen delivery at high metabolic demands, e.g. during activity and at high ambient temperatures. Therefore, large safety margins are required for tracheal and spiracular conductance. Spiracles are the entry to the tracheal system and play an important role in controlling discontinuous gas exchange (DGC) between tracheal system and atmosphere in moth pupae. We investigated the effect of modulated metabolic rate (by changing ambient temperature) and modulated spiracular conductance (by blocking all except one spiracles) on gas exchange patterns in Samia pupae. Both, spiracle blocking and metabolic rates, affected respiratory behavior in Samia cynthia pupae. While animals showed discontinuous gas exchange cycles at lower temperatures with unblocked spiracles, the respiratory patterns were cyclic at higher temperatures, with partly blocked spiracles or a combination of these two factors. The threshold for the transition from a discontinuous (DGC) to a cyclic gas exchange (^cycGE) was significantly higher in animals with unblocked spiracles (18.7 nmol g⁻¹ min⁻¹ vs. 7.9 nmol g⁻¹ min⁻¹). These findings indicate an important influence of spiracle conductance on the DGC, which may occur mostly in insects showing high spiracular conductances and low metabolic rates.     

Co-ordinating interneurones of the locust which convey two patterns of motor commands: their connexions with ventilatory motoneurones.

1. The interneurones which make widespread connexions with flight motoneurones also synapse upon ventilatory motoneurones so that in all 50 motoneurones receive synapses. They influence three aspects of ventilation; (a) the closing and opening movements of the thoracic spiracles, (b) some aspects of abdominal pumping movements and (c) the recruitment of some motoneurones controlling head pumping. 2. The two closer motoneurones of a particular thoracic spiracle receive the same excitatory synaptic inputs (EPSPs) during expiration. The EPSPs match those in appropriate flight motoneurones. 3. The closer motoneurones of each thoracic spiracle whose somata are in the pro-, meso- or metathoracic ganglia all receive the same excitatory synaptic inputs. These inputs are an adequate explanation of the pattern of spikes in the closer motoneurones. Both the slow ventilatory and fast rhythms of synaptic potentials are expressed as spikes; the slow as the overall expiratory burst of spikes and the fast as the groups of spikes within that burst. This establishes a ventilatory function for the interneurones. All thoracic closer motoneurones therefore receive the same excitatory commands which will tend to synchronize the movements of each spiracle. 4. Spiracular opener motoneurones are inhibited during expiration, their IPSPs matching the EPSPs in flight or closer motoneurones. Therefore the interneurones have reciprocal effects on the antagonistic motoneurones of the spiracles. 5. The interneurones synapse upon some motoneurones which control the pumping movements of the abdomen and which have their somata in the metathoracic or first unfused abdominal ganglion. Motoneurones in four separate ganglia therefore receive inputs from these interneurones. 6. The interneurones also synapse upon motoneurones which control an auxiliary form of ventilation, head pumping.     

Spiracular closing mechanisms in the firebrat, Thermobia domestica (packard) (Thysanura)

Mechanisms for regulating the degree of opening of its spiracles are present in Thermobia. That of the mesothoracic spiracle is of the external type with a flap-like hood guarding the spiracular aperture. Contraction of muscles open the spiracle by raising the hood. Closure is brought about by muscular relaxation and elastic cuticular recoil. Opening is either partial, with small-scale oscillatory movements ('fluttering'), or complete ('wide-opening'). Wide-opening follows bouts of muscular activity. Carbon dioxide anaesthesia relaxes the opener muscles causing the spiracles to close by elastic recoil. This explains continued low tracheal water loss during anaesthesia, and also in death. The control mechanisms of the metathoracic and 8 pairs of abdominal spiracles are of the internal type, with a crypt-like atrium leading into the slit-like neck region of the spiracular pit, one side of which has an elastic cuticular rod running along it. Muscles inserted on the opposite side widen the aperture. As with the mesothoracic spiracle, closure is brought about by muscular relaxation and elastic cuticular recoil.     

The respiratory basis of locomotion in Drosophila

The respiratory system of insects has evolved to satisfy the oxygen supply during rest and energetically demanding processes such as locomotion. Flapping flight in particular is considered a key trait in insect evolution and requires an increase in metabolic activity of 10-15-fold the resting metabolism. Two major trade-offs are associated with the extensive development of the tracheal system and the function of spiracles in insects: the risk of desiccation because body water may leave the tracheal system when spiracles open for gas exchange and the risk of toxic tracheal oxygen levels at low metabolic activity. In resting animals there is an ongoing debate on the function and evolution of spiracle opening behavior, focusing mainly on discontinuous gas exchange patterns. During locomotion, large insects typically satisfy the increased respiratory requirements by various forms of ventilation, whereas in small insects such as Drosophila diffusive processes are thought to be sufficient. Recent data, however, have shown that during flight even small insects employ ventilatory mechanisms, potentially helping to balance respiratory currents inside the tracheal system. This review broadly summarizes our current knowledge on breathing strategies and spiracle function in the genus Drosophila, highlighting the gas exchange strategies in resting, running and flying animals.     

Transitions in insect respiratory patterns are controlled by changes in metabolic rate

We examined the respiratory patterns of Rhodnius prolixus and Gromphadorhina portentosa as metabolic rates varied with temperature to determine whether insects transition from discontinuous (DGC), cyclical and continuous respiration as a response to increasing aerobic demand. Using flow through respirometry we: (1) determined the effects of temperature on metabolic rate; (2) objectively defined periods of spiracular closure; (3) observed whether there was a correlation between metabolic rate and length of spiracular closure. At low temperatures both species exhibit lengthy periods of spiracular closure reflecting a discontinuous respiratory pattern. As metabolic rate increased, periods of spiracular closure decreased and insects displayed a more cyclical pattern of respiration. As metabolic rates increased even further under the highest experimental temperatures, periods of spiracular closure decreased even more and a continuous respiratory pattern was employed by both species. Our results suggest that the three described respiratory patterns in insects are not distinct but are instead a continuum of respiratory responses driven by the metabolic demand experienced by the insect.     

Btk-dependent epithelial cell rearrangements contribute to the invagination of nearby tubular structures in the posterior spiracles of Drosophila

The Drosophila respiratory system consists of two connected organs, the tracheae and the spiracles. Together they ensure the efficient delivery of air-borne oxygen to all tissues. The posterior spiracles consist internally of the spiracular chamber, an invaginated tube with filtering properties that connects the main tracheal branch to the environment, and externally of the stigmatophore, an extensible epidermal structure that covers the spiracular chamber. The primordia of both components are first specified in the plane of the epidermis and subsequently the spiracular chamber is internalized through the process of invagination accompanied by apical cell constriction. It has become clear that invagination processes do not always or only rely on apical constriction. We show here that in mutants for the src-like kinase Btk29A spiracle cells constrict apically but do not complete invagination, giving rise to shorter spiracular chambers. This defect can be rescued by using different GAL4 drivers to express Btk29A throughout the ectoderm, in cells of posterior segments only, or in the stigmatophore pointing to a non cell-autonomous role for Btk29A. Our analysis suggests that complete invagination of the spiracular chamber requires Btk29A-dependent planar cell rearrangements of adjacent non-invaginating cells of the stigmatophore. These results highlight the complex physical interactions that take place among organ components during morphogenesis, which contribute to their final form and function.     

Spiracle activity in moth pupae—The role of oxygen and carbon dioxide revisited

After decades of intensive research, the actual mechanism behind discontinuous gas exchange in insects has not been fully understood. One open question concerns the actual way (closed, flutter, and open) of how spiracles respond to tracheal gas concentrations. As the results of a classic paper [Burkett, B.N., Schneiderman, H.A., 1974. Roles of oxygen and carbon dioxide in the control of spiracular function in cecropia pupae. Biological Bulletin 147, 274-293] allow ambiguous interpretation, we thus reexamined the behavior of the spiracles in response to fixed, controlled endotracheal gas concentrations.The tracheal system of diapausing pupae of Attacus atlas (Saturniidae, Lepidoptera) was flushed with gas mixtures varying in PO₂ and PCO₂ while the behavior of the spiracles was monitored using changes in the pressure signal. This novel pressure based technique proved to be superior to classic visual observation of single spiracles. A two-dimensional map of the spiracle behavior in response to endotracheal PO₂ and PCO₂ was established. Typically, it contained two distinct regions only, corresponding to “closed” and “open” spiracles. A separate “flutter” region was missing. Because fluttering is commonly observed in moth pupae, we suggest that the intermittent spiracle opening during a flutter phase is an effect of non-steady-state conditions within the tracheal system. For low PCO₂ the minimum PO₂ resulting in open spiracles was linearly dependent upon PCO₂. Above a threshold of 1-1.5 kPa CO₂ the spiracles were open irrespective of PO₂. We propose a hypothetical spiracular control model, which is simple and explains the time course of endotracheal partial pressures during all phases of discontinuous gas exchange.     

The role of the spiracles in gas exchange during development of Samia cynthia (Lepidoptera, Saturniidae)

Spiracles and the tracheal system of insects allow effective delivery of respiratory gases. During development, holometabolous insects encounter large changes in the functional morphology of gas exchange structures. To investigate changes in respiratory patterns during development, CO₂-release was measured in larvae, pre-pupae and pupae of Samia cynthia (Lepidoptera, Saturniidae). Gas exchange patterns showed great variability. Caterpillars had high metabolic rates and released carbon dioxide continuously. Pre-pupae and pupae showed typical discontinuous gas exchange cycles (DGC) at reduced metabolic rates. Changes in gas exchange patterns can partly be explained with low metabolic rates during pupation. Sequential blocking of spiracles in pre-pupae and pupae reduced spiracle conductance with tracheal conductance remaining unaffected. Analysis of gas exchange patterns indicates that caterpillars and pre-pupae use more than 14 spiracles simultaneously while pupae only use 8 to 10 spiracles. Total conductance is not a simple multiple of single spiracles, but may be gradually adaptable to gas exchange demands. Surprisingly, moth pupae showed a DGC if all except one spiracle were blocked. The huge conductance of single spiracles is discussed as a pre-adaptation to high metabolic demands at the beginning and the end of the pupal as well as in the adult stage.     

Tracheal compression in pupae of the beetle Zophobas morio

Insects that are small or exhibit low metabolic rates are considered to not require active ventilation to augment diffusive gas exchange. Some pupae with low metabolic rates exhibit abdominal pumping, a behaviour that is known to drive tracheal ventilation in the adults of many species. However, previous work on pupae suggests that abdominal pumping may serve a non-respiratory role. To study the role of abdominal pumping in pupa of the beetle Zophobas morio, we visualized tracheal dynamics with X-rays while simultaneously measuring haemolymph pressure, abdominal movement, and CO₂ emission. Pupae exhibited frequent tracheal compressions that were coincident with both abdominal pumping and pulsation of pressure in the haemolymph. However, more than 63% of abdominal pumping events occurred without any tracheal collapse and hence ventilation, suggesting that the major function of the abdominal pump is not respiratory. In addition, this study shows that the kinematics of abdominal pumping can be used to infer the status of the spiracles and internal behaviour of the tracheal system.     

The role of the spiracles in gas exchange during development of Samia cynthia (Lepidoptera, Saturniidae).

Spiracles and the tracheal system of insects allow effective delivery of respiratory gases. During development, holometabolous insects encounter large changes in the functional morphology of gas exchange structures. To investigate changes in respiratory patterns during development, CO2-release was measured in larvae, pre-pupae and pupae of Samia cynthia (Lepidoptera, Saturniidae). Gas exchange patterns showed great variability. Caterpillars had high metabolic rates and released carbon dioxide continuously. Pre-pupae and pupae showed typical discontinuous gas exchange cycles (DGC) at reduced metabolic rates. Changes in gas exchange patterns can partly be explained with low metabolic rates during pupation. Sequential blocking of spiracles in pre-pupae and pupae reduced spiracle conductance with tracheal conductance remaining unaffected. Analysis of gas exchange patterns indicates that caterpillars and pre-pupae use more than 14 spiracles simultaneously while pupae only use 8 to 10 spiracles. Total conductance is not a simple multiple of single spiracles, but may be gradually adaptable to gas exchange demands. Surprisingly, moth pupae showed a DGC if all except one spiracle were blocked. The huge conductance of single spiracles is discussed as a pre-adaptation to high metabolic demands at the beginning and the end of the pupal as well as in the adult stage.     

Comparative morphology of the spiracles of the Papilionidae,Sphingidae, and Saturniidae (Insecta: Lepidoptera)

The morphology, ultrastructure, and innervation of the spiracles of the instars and adults of representatives of three lepidopteran families were examined: Ornithoptera priamus poseidon and Pachliopta aristolochiae (Papilionidae), Attacus atlas (Saturniidae), and Acherontia atropos (Sphingidae).Peritreme and atrium show stage- and family-specific structures for protecting the internal valve apparatus. The gross morphology of the cuticular valve mechanism is uniform within the three families, consisting of a rigid bow and a movable bar with a lever. In adult Papilionidae, all cuticular parts (bow, bar and lever) of the valve are innervated by multipolar dendrites. Internal or external cuticular chemo- or hygroreceptors, which could participate in the regulation of respiration, could not be detected in any stage. The closing muscle inserts between the tip of the lever and the base of the bar, and is innervated only by motor neurons. The elasticity of the cuticular system and an opener are the antagonists to the closing muscle. The spiracular opener of the adult Papilionidae and of all instars of the moths is an elastic ligament. The opener of the larval and pupal spiracles of the Papilionidae, however, is a single thickened muscle fiber surrounded by an elastic sheath of connective tissue. As it contains motor and multipolar sensory neurons, we assume that it may function as a stretch receptor for controlling the spiracular opening state.     

Hold your breath beetle—Mites!

Respiratory gas exchange in insects occurs via a branching tracheal system. The entrances to the air‐filled tracheae are the spiracles, which are gate‐like structures in the exoskeleton. The open or closed state of spiracles defines the three possible gas exchange patterns of insects. In resting insects, spiracles may open and close over time in a repeatable fashion that results in a discontinuous gas exchange (DGE) pattern characterized by periods of zero organism‐to‐environment gas exchange. Several adaptive hypotheses have been proposed to explain why insects engage in DGE, but none have attracted overwhelming support. We provide support for a previously untested hypothesis that posits that DGE minimizes the risk of infestation of the tracheal system by mites and other agents. Here, we analyze the respiratory patterns of 15 species of ground beetle (Carabidae), of which more than 40% of individuals harbored external mites. Compared with mite‐free individuals, infested one's engaged significantly more often in DGE. Mite‐free individuals predominantly employed a cyclic or continuous gas exchange pattern, which did not include complete spiracle closure. Complete spiracle closure may prevent parasites from invading, clogging, or transferring pathogens to the tracheal system or from foraging on tissue not protected by thick chitinous layers.     

Oxygen reperfusion damage in an insect

The deleterious effects of anoxia followed by reperfusion with oxygen in higher animals including mammals are well known. A convenient and genetically well characterized small-animal model that exhibits reproducible, quantifiable oxygen reperfusion damage is currently lacking. Here we describe the dynamics of whole-organism metabolic recovery from anoxia in an insect, Drosophila melanogaster, and report that damage caused by oxygen reperfusion can be quantified in a novel but straightforward way. We monitored CO(2) emission (an index of mitochondrial activity) and water vapor output (an index of neuromuscular control of the spiracles, which are valves between the outside air and the insect's tracheal system) during entry into, and recovery from, rapid-onset anoxia exposure with durations ranging from 7.5 to 120 minutes. Anoxia caused a brief peak of CO(2) output followed by knock-out. Mitochondrial respiration ceased and the spiracle constrictor muscles relaxed, but then re-contracted, presumably powered by anaerobic processes. Reperfusion to sustained normoxia caused a bimodal re-activation of mitochondrial respiration, and in the case of the spiracle constrictor muscles, slow inactivation followed by re-activation. After long anoxia durations, both the bimodality of mitochondrial reactivation and the recovery of spiracular control were impaired. Repeated reperfusion followed by episodes of anoxia depressed mitochondrial respiratory flux rates and damaged the integrity of the spiracular control system in a dose-dependent fashion. This is the first time that physiological evidence of oxygen reperfusion damage has been described in an insect or any invertebrate. We suggest that some of the traditional approaches of insect respiratory biology, such as quantifying respiratory water loss, may facilitate using D. melanogaster as a convenient, well-characterized experimental model for studying the underlying biology and mechanisms of ischemia and reperfusion damage and its possible mitigation.     

Complex innervation of three neck muscles by motor and dorsal unpaired median neurons in crickets

Summary In the crickets, Gryllus campestris and Gryllus bimaculatus, the innervation of the dorso-ventral neck muscles M62, M57, and M59 was examined using cobalt staining via peripheral nerves and electrophysiological methods. M62 and M57 are each innervated by two motoneurons in the suboesophageal ganglion. The four motoneurons project into the median nerve to bifurcate into the transverse nerves of both sides. M62 and M57 are the only neck muscles innervated via this route. These bifurcating axon-projections are identical to those of the spiracular motoneurons in the prothoracic ganglion innervating the opener and closer muscle of the first thoracic spiracle in the cricket. The morphology of their branching pattern is described. The neck muscle M57 and the opener muscle of the first thoracic spiracle are additionally innervated by one mesothoracic motoneuron each, with similar morphology. These results suggest, that in crickets, the neck muscles M57 and M62 are homologous to spiracular muscles in the thoracic segments. The two neck muscles M62 and M59 (the posterior neighbour of M57) receive projections from a prothoracic dorsal unpaired median (DUM) neuron that also innervates dorsal-longitudinal neck muscles but not M57. In addition, one or two mesothoracic DUM neurons send axon collaterals intersegmentally to M59. This is the first demonstration of the innervation of neck muscles by DUM neurons.     

Respiratory significance of the external morphology of adults and fifth instar nymphs of Notonecta undulata Say (Aquatic Heteroptera; Notonectidae)

The bodies of adult and fifth instar Notonecta possess external air stores which are periodically renewed at the surface of the water. Both nymphs and adults have large ventral air stores on the thorax and abdomen and obtain atmospheric air at the posterior end of the latter; the adult also has dorsal subalar and supra-alar air stores on both these regions. Ten pairs of spiracles open onto the air stores. Although the seven small, ventrally placed abdominal spiracles are probably both exhalant and inhalant in nymphs and adults, the three large anterior spiracles (mesothoracic, metathoracic, and first abdominal), which play a more important respiratory role, appear to function differently in mature and immature Notonecta. In the nymph they are probably both inhalant and exhalant, and communicate broadly with each other and with the ventral air stores. In the adult, however, they open onto separate, air-filled chambers, each of which communicates differently with various parts of the air stores. Although all three probably function in exhalation, only the first abdominal spiracle, whose spiracular chamber is widely continuous with the dorsal and ventral air stores, appears to be well suited for inhalation. Several morphological features, most notably the development of long prothoracic lobes, separate spiracular chambers, and long, movable forewings, allow the adult a greater variety of respiratory modes than are available to the nymph. Some of the respiratory advantages of the adult are: (1) a larger amount of stored air; (2) a longer subalar air store, which can serve as an alternate pathway between the air stores and the atmosphere; (3) a greater capacity to utilize dissolved as well as atmospheric oxygen; (4) greater separation and functional specialization of the three anterior spiracles, thus allowing more separation of exhaled air from oxygen-rich air on the external surface of the thorax; (5) the probable ability to regulate the continuity between various parts of the air stores, thus utilizing alternate pathways of air circulation and/or changing the functions of the three anterior spiracles; and (6) better protection of the latter against the entry of water during prolonged submergence.     

A combinatorial enhancer recognized by Mad, TCF and Brinker first activates then represses dpp expression in the posterior spiracles of Drosophila

A previous genetic analysis of a reporter gene carrying a 375-bp region from a dpp intron (dppMX-lacZ) revealed that the Wingless and Dpp pathways are required to activate dpp expression in posterior spiracle formation. Here we report that within the dppMX region there is an enhancer with binding sites for TCF and Mad that are essential for activating dppMX expression in posterior spiracles. There is also a binding site for Brinker likely employed to repress dppMX expression. This combinatorial enhancer may be the first identified with the ability to integrate temporally distinct positive (TCF and Mad) and negative (Brinker) inputs in the same cells. Cuticle studies on a unique dpp mutant lacking this enhancer showed that it is required for viability and that the Filzkorper are U-shaped rather than straight. Together with gene expression data from these mutants and from brk mutants, our results suggest that there are two rounds of Dpp signaling in posterior spiracle development. The first round is associated with dorsal-ventral patterning and is necessary for designating the posterior spiracle field. The second is governed by the combinatorial enhancer and begins during germ band retraction. The second round appears necessary for proper spiracle internal morphology and fusion with the remainder of the tracheal system. Intriguingly, several aspects of dpp posterior spiracle expression and function are similar to demonstrated roles for Wnt and BMP signaling in proximal-distal outgrowth of the mammalian embryonic lung.     

Tracheal ultrastructure of protura (Insecta : Apterygota)

The tracheal systems of Sinentomon and Eosentomon (Apterygota : Protura) were examined in thin sections and compared with the tracheae of collembolan, Allacma. The tracheal system of Protura consists of spiracles and tracheae. The spiracle is a simple, concave cuticular cavity known as an atrium. A globular chamber is present between the atrium and trachea. The atrium of Eosentomon is decorated with ridges and has 2 small openings to tracheal recesses beside the central tracheal opening. The tracheae of Protura are characterized by a high frequency of taenidia and the absence of intima folds and intertaenidial spaces. The taenidia of Sinentomon have a rectangular section and those of Eosentomon are gable-shaped. The results also suggest that the tracheal recess of Eosentomon is a kind of stigmatic gland. The tracheal structure of Protura was compared with that of collembolans, insects, and other arthropods, and discussed in terms of phylogeny.     

Communication in the courtship of a madagascan hissing cockroach. I. Normal courtship

The hissing Madagascar cockroach, Gromphadorhina portentosa, has a prolonged and complex courtship involving signals in several sensory modalities. Courtship was described for 13 pairs of cockroaches and the frequencies and sequencing of 16 behavioural units were analysed. Particular attention was paid to the function and interactions of acoustic, chemical and tactile components. The results indicate that posturing and sound production by males, and antennation by both males and females, are important in courtship. They also suggest that courtship in G. portentosa, rather than depending on a rigid sequence of behaviour determined by a series of discrete releasers, is quite flexible, using ‘behavioural monologues’ by both sexes as a means of achieving transitions from one stage of courtship to the next.