Discontinuous respiration in diapausing pupae of Cecropia silkworms was monitored by means of several electronic methods, including recording changes in haemocoelic pressure, monitoring respiratory movements by strain-gauge sensors and nanorespirographic recording of O2 consumption and CO2 output. It appears that, in contrast to previous concepts of stereotypic discontinuous respiration cycles (DGC) driven by accumulation of gaseous CO2 in the body, the new results indicate that CO2 remains dissolved in liquid carbonate buffers during interburst periods. In other words, there is no accumulation of gaseous CO2 within the air filled tracheal space between the bursts. The bursts of CO2 are caused by homeostatically regulated enzymatic hydration by carbonic anhydrase of metabolically produced carbonic acid. The chemically produced gaseous CO2 was exhaled mainly by a bulk outflow through selectively opened or pulsating spiracles. The output of CO2 was enhanced by actively regulated, unidirectional ventilation. The deep depressions in haemocoelic pressure, caused by permanent closure of all spiracular valves for long periods, appeared to be a specific feature of diapausing saturniid pupae. Physiologically, it has circulatory, not respiratory functions. The original definition of spiracular "fluttering" resulted from a misinterpretation of previously unknown extracardiac pulsations in haemocoelic pressure. The coordinated pulsation of the spiracular valves with extracardiac pulsations produce a very efficient, unidirectional ventilation of the whole tracheal system. According to the new results, the discontinuous respiration cycles of diapausing Cecropia pupae can be briefly described as follows: (1) Spiracular valves are kept permanently closed during the periods of deep depressions, they remain closed for some 99% of the time with occasional snap opening (passive inspirations) during prolonged interburst periods and more than 50% closed during the bursts; (2) During the long interburst periods, CO2 is retained in liquid carbonate buffers, while the relatively high (after the burst) or low (toward the next burst) rate of O2 consumption creates an internal vacuum, which is homeostatically compensated for by the snap-opening of one or just a few spiracular valves (passive suction inspirations); (3) The CO2 gas, produced enzymatically by carbonic anhydrase, enters the air filled tracheal system and leaves the body by diffusion, a bulk outflow, or actively regulated unidirectional ventilation ("fluttering" spiracles). The selective advantage of this actively regulated respiratory system for water retention in pupae is discussed.
Genetic characteristics of the first three mutants found in P. apterus L.; white (w/w) 1965, yellow (y/y) 1966 and melanotic (m/m) 1973 have been described in detail. Exact Mendelian proportions of 1 : 1 and 3 : 1 in all standard test crosses and absence of sexual linkage revealed that each of these mutations was inherited as a single autosomal recessive gene. The dihybrid and trihybrid crosses showed that the w gene is epistatic over y. The absence of linkage shows that each of the described mutant genes is situated on a different chromosome. During 30 years of sustained rearings of P. apterus, the white (w/w) and yellow (y/y) mutants never originated de novo, whereas the melanotic (m/m) mutants originated independently from the macropterous strain three times. Triple recessive (w y m) white melanotic strain has been maintained and used for some genetic investigations for over 20 years.
Respiratory metabolism of developing eggs of Schistocerca gregaria has been individually monitored by means of scanning microrespirography. The freshly oviposited eggs consumed 7 nl of O2 /min./egg (50 µl O2/g/h) while the pharate 1st instar larvae shortly before hatching consumed 141 nl of O2/min./egg (550 µl O2/g/h), which shows 20-fold metabolic increase during the egg stage. The output of CO2 was also regular, without discontinuous bursts throughout the whole embryonic development. The amounts of CO2 produced were constantly close to R.Q. ratio of 0.7, suggesting that lipid was the main energetic source. The vermiform, pharate 1st instar larvae emerging from the eggs exhibited very high respiratory rates (up to 3,000 µl O2/g/h). During initial phases of the egg stage, O2 consumption steadily increased until day 6, which was associated with katatrepsis or blastokinesis stage of the embryo (61 nl of O2/egg/min. = 240 µl O2/g/h). Since blastokinesis, respiratory metabolism of the egg remained constant or decreased steadily until day 10, when it rose sharply again towards hatching. The temporary metabolic depression was closely correlated with endogenous peak in ecdysteroid concentration within the embryo. These results corroborate validity of the reciprocal, high ecdysteroid - low metabolism rule previously known from insect metamorphosis. They extend its application into the period of embryogenesis. Practical implications of certain physiological, morphological and evolutionary consequences of these findings are discussed with special emphasis on the connecting links between embryogenesis and metamorphosis.
Reversal of heartbeat was monitored in vivo by noninvasive, multisensor, thermo-cardiographic and pulse-light, opto-cardiographic techniques. The dorsal vessel was sectioned at the beginning, in the middle and near the end of the abdomen. Changes in the heartbeat were simultaneously monitored in both the disconnected anterior and posterior sections of the heart. The results revealed the existence of a caudal regulatory cardiac centre located in the fused A7-A10 abdominal segments. Posterior sections, containing this terminal ampulla of the heart always exhibited a more or less normal heartbeat reversal, including both anterograde and retrograde pulsations. This shows that the forward-oriented as well as the reciprocal, backward-oriented peristaltic waves of the heart are both regulated from the posterior regulatory center, without involvement of the cephalic region. The cardiac pulsations in the anterior sections of the heart were paralysed and seriously impaired by the lesions. During the acute phase after the lesions, anterior sections showed only some convulsion-like, unidirectional, backward-oriented peristaltic pulsations of low frequency. Within one or two days after the lesions, isolated anterior sections of the heart developed a subsidiary heartbeat regulation associated with the oscillating, bi-directional peristaltic waves running alternatively, forward and backward in opposite directions.
After a few days, the previously paralysed anterior sections of the heart were able to develop perfectly coordinated patterns of heartbeat reversal. At this time, the two asynchronous heartbeat patterns ran separately in each of the divided sections of the heart. One or two weeks later, reversal of the heartbeat occasionally occurred synchronously along the entire length of the dorsal vessel. Sectioning of the ventral nerve cord, removal of the cephalic nervous system (brain, frontal ganglion, suboesophageal ganglion and the associated nerves) or removal of the fused terminal abdominal ganglionic mass and adjacent caudal nerves, had no effect on the pattern of heartbeat reversal. These facts indicate that the pupal heart of M. sexta operates purely myogenically, like the human heart. The myogenic pacemakers of the caudal regulatory cardiac centre (analogous to the atrio-ventricular nodes of the human heart) are autonomous, generating inherent rhythmicity without intervention from the nervous system. Development of subsidiary pacemakers regulating rhythmicity in the lesioned myocardium and restitution of the synchronized contracting integrity between the two disconnected sections of the heart are new cardiological features, which merit further investigation.
Heartbeat patterns were monitored in the living bodies of decapitated adult flies using several electrocardiographic methods (pulse-light optocardiography, thermocardiography, strain-gauge cardiography). Unlike other insect species, in which there is a peristaltic segmental propagation of cardiac contractions, Drosophila uses extremely efficient synchronic cardiac contractions. The rate of synchronic cardiac pulsation, which is characterized by simultaneous propagation of anterograde systolic contractions along all the segments of the heart, is relatively fast (~ 4 Hz at room temperature). This pulsation is used mainly for the vigorous pumping of haemolymph into the head and thorax through a narrow elastic tube, the aorta (anterograde I heartbeat). In addition, this synchronic pulsation is also used to enhance the circulation of haemolymph throughout the abdominal body cavity (anterograde II heartbeat). The switch between thoracic (anterograde I) and abdominal (anterograde II) haemolymph circulation is regulated by periodically alternating, tetanic contractions and relaxations of the conical heart chamber (ventricle). In the latter there is a pair of slit-like apertures, which are closed or opened by contraction or relaxation of the organ, respectively. During contraction of the conical chamber, the apertures are tightly constricted for several seconds and haemolymph is pumped forwards into the aorta (anterograde I heartbeat). Conversely, during relaxation of the conical chamber, the apertures are wide open for a few seconds, haemolymph leaves the heart and leaks out through open apertures and circulates from the tail to the base of the abdomen. The backward oriented, retrograde heartbeat recorded in other insects, has a lower frequency (1 to 2 Hz), occurs in Drosophila only sporadically and usually in the form of individual or twinned systolic peaks of large amplitude. Unlike the synchronic nature of the anterograde I and II cardiac contractions, propagation of the relatively slow retrograde heartbeat is by peristalsis. The newly discovered, compact ventricle with atrium and synchronic functioning of the insect heart shows structural and functional analogies with the functioning of the human heart.
Pulsations of dorsal vessel were monitored by the noninvasive techniques of contact thermography on the dorsal cuticle and by strain gauge detection of abdominal elongation movements. Diapausing pupae exhibited periods of forward-oriented, or anterograde pulsations (average duration of each pulsation 5-8 min, frequency of individual systolic strokes 10-15 per min) alternating with somewhat slower, backward-oriented or retrograde cardiac pulsations (average duration of each pulsation 6-10 min, frequency of systolic strokes 7-12 per min). The highest rate of hemolymph flow was associated with the anterograde pulsations. We studied cardiac functions in diapausing pupae because of the almost complete absence of extracardiac hemocoelic pulsations, which are much stronger and could interfere with the recordings of heartbeat in all other developing stages. The movement of abdomen associated with the heartbeat was extremely small, only some 0.14 to 0.9 µm (i.e. from one 428000th to one 66000th of the body length) and thus was not practical for routine recordings of heartbeat.
Simultaneous recordings from multiple thermographic sensors revealed the complete absence of retrograde cardiac pulsations in the head region. There are some indications that the retrograde pulsations were also lacking in the thoracic region of the aorta. The retrograde peristalsis appeared to be used for circulatory functions in the abdomen alone. By contrast, the anterograde cardiac pulsations underwent a profound amplification in the anterior part of the abdomen, entering thoracic aorta with considerable strength before reaching the final destination in the head region. The amplification of anterograde peristalsis was manifested by enhanced hemolymph flow towards the head associated with a two-fold increase in frequency of anterograde heartbeat before reaching the head region. The sensors distributed along the dorsal vessel revealed that the rate of the backward-oriented, retrograde cardiac flow of the hemolymph was also location specific. The rate of flow was lowest at the front of the abdomen, medium in the middle and highest close to the end of the abdomen. The finding of lowest hemolymph circulation at the beginning of the cardiac peristaltic waves suggested that the physiological "raison d' être" for heartbeat reversal was a need for differential enhancement of hemolymph flow towards the extremities of the immobile pupal body. The switchovers from the retrograde to anterograde cardiac pulsations were usually immediate, while the reciprocal, antero- to retro-switchovers were mostly associated with a brief cardiac arrest. Increasing temperature gradients (in 5°C steps) progressively diminished duration of both reciprocal heartbeat periods. The amplitudes of the cardiac systolic strokes also decreased with increasing temperature while the frequencies were substantially elevated.