Structure of the thoracic spiracular valves and their contribution to unidirectional gas exchange in flying blowflies Calliphora vicina.

The operation of the thoracic spiracular valves was analysed using anatomical and physiological techniques. Dense spiracular filter trichomes impede a diffusive gas exchange. However, the hinged posterior filter flap of the metathoracic spiracle (Sp2) opens passively during upstroke of the wings and closes by the suction of the sub-atmospheric tracheal pressure during the downstroke, which supports a unidirectional respiratory airflow. The action of the interior spiracular valve lids was recorded by photocell sensors oriented above the enlarged spiracles and projected onto the screen of a video camera. The thoracic spiracles opened much quicker (approximately 0.1 s) than they closed (1 s), suggesting that the spiracular muscles are openers, as confirmed by experimental induction of muscle contraction. Simultaneous photocell measurement revealed that the first and second thoracic spiracles act concordantly. At rest, the spiracles were mostly closed or only slightly open (<1%). During intermittent short flights, the valves opened wide at the start of the flight for a short time, and in many cases opened again after the flight ended. Often, the opening was wider after the flight ended than during the flight itself. During long spontaneous continuous flight phases (up to 2 h), the valves were only slightly open (<5%), widening shortly after transient increases of wing stroke intensity. It is an amazing paradox that the spiracles were only slightly open when the requirement for O2 was high during sustained flight. The advantage of generating sub-atmospheric pressure, supporting a unidirectional airflow with a PO2  increase above the resting level, is discussed.

Flight-motor-driven respiratory airflow increases tracheal oxygen to nearly atmospheric level in blowflies (Calliphora vicina).

It is widely accepted that an efficient oxygen supply and removal of CO2 in small flying insects are sufficiently performed by diffusion with open spiracles. This paper shows that in the tethered flying blowfly, gas exchange occurs by autoventilation and unidirectional airflow. The air is inspired through the mesothoracic spiracles (Sp1) during the downstroke of the wings and is expired through the metathoracic spiracles (Sp2) during the upstroke. This directed airflow through the thoracic tracheal system was documented by pre-atrial pressure measurements at the Sp1 and Sp2, revealing a sub-atmospheric mean pressure at the Sp1 and an over-atmospheric mean pressure at the Sp2. In the mesothoracic air sacs, the mean pressure is sub-atmospheric, conditioned by the only slightly open spiracles. In a split flow-through chamber experiment, the CO2 released through the Sp2 confirmed this unidirectional respiratory gas flow, implicating an inner tracheal valve. In the thoracic tracheal system, the PO2  during flight exceeds the high resting PO2  by 1-2 kPa, reaching nearly atmospheric values. In the abdominal large air sacs, the PO2  drops during flight, probably due to the accumulation of CO2. Periodic heartbeat reversals continue during flight, with a higher period frequency than at rest, supporting the transport of CO2 via the haemolymph towards the metathoracic tracheae and abdominal air sacs.

Periodic heartbeat reversals cause cardiogenic inspiration and expiration with coupled spiracle leakage in resting blowflies, Calliphora vicina.

Respiration in insects is thought to be independent of the circulatory system because insects typically lack respiratory pigments and because oxygen transport occurs in the gaseous phase through a ramified tracheal system by diffusion and convection directly to the tissues. In the blowfly, as in other insects with periodic heartbeat reversal, the haemolymph is periodically shifted between the anterior body and abdomen, exerting alternating pressure changes on the compliant tracheae in the thorax and in the abdomen. Simultaneous pressure and O2 optode measurements show that, during negative pressure periods, the tracheal partial pressure of oxygen (PO2) increases by 0.5 kPa. In the quiescent fly, tracheal PO2 is rather high (17.5-18.9 kPa), although the thoracic spiracles remain constricted. Microscopic video recordings and reflectance measurements revealed that the dorsal soft edges of the valve lips of the second spiracle leave a very small leak, which is passively widened during backward pulses of the heart. Thus, negative pressure, combined with increased leakage of the spiracle Sp2 valve enable inspiration in the thorax. The positive pressure periods are correlated with a new type of convective CO2 micro-bursts as shown in flow-through measurements. The bulk of the CO2 is, however, released after longer interbursts in macro-bursts with actively opening valves reminiscent of the open phase in a cyclic gas exchange. When the valves open, the PO2 in the thoracic air sacs unexpectedly drops by a mean of 2.75±1.09 kPa, suggesting a displacement of O2 by the transient accumulation of CO2 in the tracheal system before its release.

Drosophila flies combine periodic heartbeat reversal with a circulation in the anterior body mediated by a newly discovered anterior pair of ostial valves and 'venous' channels.

Heartbeat activity in tethered adult drosophilids was recorded using a linear optosensor chip and an IR-light beam. Recording from two to five sensor elements within 250 mum along the anterior heart, it was possible to analyze periodic reversals. In intact Drosophila melanogaster and D. hydei, longer anterograde pulse periods with lower pulse rates generally alternated with shorter retrograde pulse periods having higher pulse rates. These differences are dependent on heart anatomy: a newly discovered first pair of ostia is connected to bilateral thoraco-abdominal hemolymph channels. These channels are part of a venous space separated from the abdominal hemocoel by a septum, consisting of a metanotal ridge and the pericardial diaphragm lined by a special form of fat body. The channels are sealed, and their lumen is possibly controlled by the metathoracic tergo-pleural muscle. During retrograde pulses, the heart chamber works like a suction pump, aspiring hemolymph through the first ostia from the venous channels and discharging it through a newly described caudal opening. During forward beating, the anterior chamber receives hemolymph via all inflow ostia from the entire heart and drives it like a pressure pump through the narrow aorta. Also, during forward pulses, a lateral circulation occurs in the thorax as a result of the venous supply. Inhibition of abdominal mobility leads to an irregular heart rate, with pulse-wise alternating heartbeat reversals. The possible involvement of slow abdominal movements in heartbeat periodicity is discussed. The heartbeat periods are superimposed with intermittent bouts of abdominal pumping movements.

Expression of UV-, blue-, long-wavelength-sensitive opsins and melatonin in extraretinal photoreceptors of the optic lobes of hawk moths.

Lepidopterans display biological rhythms associated with egg laying, eclosion and flight activity but the photoreceptors that mediate these behavioural patterns are largely unknown. To further our progress in identifying candidate light-input channels for the lepidopteran circadian system, we have developed polyclonal antibodies against ultraviolet (UV)-, blue- and extraretinal long-wavelength (LW)-sensitive opsins and examined opsin immunoreactivity in the adult optic lobes of four hawk moths, Manduca sexta, Acherontia atropos, Agrius convolvuli and Hippotion celerio. Outside the retina, UV and blue opsin protein expression is restricted to the adult stemmata, with no apparent expression elsewhere in the brain. Melatonin, which is known to have a seasonal influence on reproduction and behaviour, is expressed with opsins in adult stemmata together with visual arrestin and chaoptin. By contrast, the LW opsin protein is not expressed in the retina or stemmata but rather exhibits a distinct and widespread distribution in dorsal and ventral neurons of the optic lobes. The lamina, medulla, lobula and lobula plate, accessory medulla and adjacent neurons innervating this structure also exhibit strong LW opsin immunoreactivity. Together with the adult stemmata, these neurons appear to be functional photoreceptors, as visual arrestin, chaoptin and melatonin are also co-expressed with LW opsin. These findings are the first to suggest a role for three spectrally distinct classes of opsin in the extraretinal detection of changes in ambient light and to show melatonin-mediated neuroendocrine output in the entrainment of sphingid moth circadian and/or photoperiodic rhythms.

Contribution of the maxillary muscles to proboscis movement in hawkmoths (Lepidoptera: Sphingidae)--an electrophysiological study.

The role of the maxillary muscles in the uncoiling and coiling movements of hawkmoths (Sphingidae) has been examined by electromyogram recordings, combined with video analysis. The maxillary muscles of adult Lepidoptera can be divided into two groups, galeal and stipital muscles. The galea contains two basal muscles and two series of oblique longitudinal muscles, which run through the entire length of the galea. Three muscles insert on the stipes, taking their origin on the tentorium and on parts of the cranium and gena, respectively. Proboscis extension is initiated by an elevation of the galea base caused by the basal galeal muscles. The actual uncoiling of the proboscis spiral is accompanied by rapid compressions of the stipites which are caused by two of the stipital muscles. The study provides strong support for the hypothesis that uncoiling is brought about by an increase of hemolymph pressure by the stipites forcing hemolymph into the galeae. Recoiling is caused by the contraction of both sets of oblique longitudinal galeal muscles supported by elasticity of the galea cuticle. Finally, the remaining stipital muscle pulls down the galea base which brings the coiled proboscis back to its resting position where it is held in the U-shaped groove of the labium without further muscle activity.

Tympanal and atympanal 'mouth-ears' in hawkmoths (Sphingidae).

The labral pilifers and the labial palps form ultrasound-sensitive hearing organs in species of two distantly related hawkmoth subtribes, the Choerocampina and the Acherontiina. Biomechanical examination now reveals that their ears represent different types of hearing organs. In hearing species of both subtribes, the labral pilifer picks up vibrations from specialized sound-receiving structures of the labial palp that are absent in non-hearing species. In Choerocampina, a thin area of cuticle serves as an auditory tympanum, whereas overlapping scales functionally replace a tympanum in Acherontiina that can hear. The tympanum of Choerocampina and the scale-plate of Acherontiina both vibrate maximally in response to ultrasonic, behaviourally relevant sounds, with the vibrations of the tympanum exceeding those of the scale plate by ca. 15 dB. This amplitude difference, however, is not reflected in the vibrations of the pilifers and the neural auditory sensitivity is similar in hearing species of both subtribes. Accordingly, morphologically different - tympanal and atympanal - but functionally equivalent hearing organs evolved independently and in parallel within a single family of moths.