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First published online October 19, 2007
Journal of Experimental Biology 210, 3707-3719 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.007864
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
Institute of Biology, University of Erlangen-Nuernberg, Staudtstrasse 5, D-91058 Erlangen, Germany
e-mail: ltwthal{at}biologie.uni-erlangen.de
Accepted 21 August 2007
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Key words: anatomy, cardiogenesis, dorsal vessel, Drosophila melanogaster, Drosophila hydei, fruitfly, heart development, inflow tract, insect heart, linear optosensor array, ostium, optocardiography, svp-lacZ
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). However,
even after about 100 years of Drosophila research, the regular function of the
adult heart in this insect and its role in circulation are not understood
because the morphological and physiological data are incomplete.
One characteristic of heart activity in many insects is periodic heartbeat
reversal. It is present even in prepupae and pupae of Lepidoptera and Diptera
and was first noted by Malpighi (Malpighi,
1669) (reviewed by Jones,
1977; Miller,
1997; Wasserthal,
1996; Wasserthal,
2003b). It has been shown to be a regular event in resting adults
from several insect orders, including flies
(Gerould, 1929;
Queinnec and Campan, 1975;
Thon and Queinnec, 1976;
Thon, 1982;
Jones, 1977;
Wasserthal, 1975;
Wasserthal, 1976;
Wasserthal, 1982a;
Wasserthal, 1982b;
Wasserthal, 1996;
Wasserthal, 2003a;
Wasserthal, 2003b;
Angioy, 1988;
Angioy and Pietra, 1995). In
prepupae and pupae the function of periodic heartbeat reversal is unclear. It
is suggested that in pupae of Manduca sexta heartbeat reversals
selectively enhance the hemolymph flow towards both body extremities
(Slama and Miller, 2001;
Slama, 2003). In adult moths
and butterflies, scarabaeid beetles and the blowfly, a periodic hemolymph
shift between anterior and posterior body compartments occurs causing an
alternating tracheal ventilation
(Wasserthal, 1998;
Wasserthal, 2003a). In D.
melanogaster, however, physiological effects on heart rate have been
investigated without taking heartbeat reversal into account
(Zornik et al., 1999;
Paternostro et al., 2001).
Periodic changes in heartbeat frequency have recently been recorded by
optoelectric registration (Slama and
Farkás, 2005) and heartbeat reversals documented by
indirect microscopic video techniques, and the regulating effect of
neurotransmitters on anterior and posterior pacemakers has been demonstrated
(Dulcis and Levine, 2005;
Dulcis et al., 2005). There
is, however, a lack of physiological measurements in intact adult Drosophila
over longer periods of time and no comparison of the somewhat controversial
data with results from other flies. It remains an open question whether
heartbeat reversals cause a periodic hemolymph shift between the anterior and
posterior compartments in drosophilids, which could assist in tracheal
ventilation.
A correct physiological analysis in drosophilids is difficult because of their small size and the relatively high pulse rate of the heart. Finding a method of recording the heartbeat from at least two measuring points, while avoiding damage and minimizing the stress caused by the need to restrain the insects, remains a challenge.
The anatomy of the Drosophila heart is generally thought to be well known,
following the anatomical work of Miller
(Miller, 1950;
Rizki, 1978;
Curtis et al., 1999;
Dulcis and Levine, 2005). The
focus of interest has now shifted to embryology of the heart at the level of
gene activation and molecular signalling during morphogenesis
(Bodmer et al., 1997; Cripps et
al., 1999; Gajewski et al.,
2000; Molina and Cripps,
2001; Lovato et al.,
2002).
The ostial precursor cells of the heart tube are determined very early in
development (Gajewski et al.,
2000; Molina and Cripps,
2001). The number of inflow tracts is greater (seven pairs) than
the three pairs of expressed ostia in the larva
(Rizki, 1978), the three pairs
in the pupa (Curtis et al.,
1999) and the four pairs in the adult
(Miller, 1950;
Rizki, 1978). Comparison of
the drosophilid heart with the heart in adult calliphorid flies, which have
five pairs of inflow ostia and one pair of caudal outflow openings
(Angioy et al., 1999;
Wasserthal, 1999), suggests
that the anterior enlarged heart chamber in the drosophilid heart should also
have two pairs of valves, instead of only one pair as hitherto described
(Miller, 1950;
Rizki, 1978;
Curtis et al., 1999).
Furthermore it is postulated that there is an excurrent opening at the caudal
end of the heart, based on the assumption that the heart transports hemolymph
effectively during retrograde pulses. In order to elucidate the fly's late
morphogenetic development with respect to these findings it is essential to
have accurate knowledge of the adult anatomy. Therefore, preparations of the
complete heart and the caudal end of Drosophila were analyzed using the light
microscope and scanning microscope. The focus lies on the anterior heart
chamber, which was analyzed in serial semithin sections and scanning electron
microscope (SEM) preparations.
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Recording of heartbeat by a modified linear optosensor chip
Insects are partly transparent for IR-radiation, and IR light or shorter
wavelength light in the visible spectrum have been used repeatedly to record
heart pulses in other insects or developmental instars (see Discussion). The
pulse signal obtained by IR-transmittance measurements using two sensor diodes
has been validated in detail in butterfly pupae, combining this technique with
contact-thermography and ultrasonic doppler velocimetry
(Hetz et al., 1999). The shape
of the IR-transmittance curves of a high-resolution single pulse showed that
during systole of the heartbeat the condensed tissues are less transparent,
reducing light transmission, and that the extension of the heart during
diastole and the presystolic wave increases the transmission as the amounts of
more transparent hemolymph increase. At least two measuring points are
necessary to determine the direction of peristaltic contractions. However, the
dimensions of the IR-sensitive diodes are much too large to be able to use
more than one diode near the abdomen of the small Drosophila. To solve the
problem, a linear multichannel sensor chip was chosen
(Fig. 1A). A light beam from an
IR-emitting diode (TSHA 6203, 940 nm: Conrad Electronics,
www.conrad.biz)
was projected by a glass fibre (230 µm diameter) onto the side of the
anterior heart region (Fig. 2).
On the opposite side of this heart region a modified optosensor chip (TAOS:
TSL 1402: 256x1 sensor array, maximum sensivity at 800 nm: Plano, TX 75074,
USA) was positioned along the first to fourth tergite by a micromanipulator.
The fly was fixed to a piece of aluminium foil by clamping the wings under
glass slides after CO2-narcosis
(Fig. 1B). Attachment of the
fly with its wings in the upstroke position was the best precondition for
avoiding damage. To prevent struggling and to obtain consistent results, flies
were given a substrate to hold, a minute ball of soft plastic impregnated with
the smell of the breeding box (see Movie 1, supplementary material). The flies
were 3–7 days old. They were supplied with water and fruit juice. The
measurements were performed in a temperature-controlled darkroom at
20±0.7°C and 55% relative humidity (RH). It was essential for the
investigation that the flies felt as unrestrained as possible. Therefore, only
flies that accepted the running ball and remained alive and active for at
least one overnight period were considered for data evaluation. A
micromanipulator allowed precise orientation of the mounted fly between the IR
emitter and sensor array until the response intensity was optimized. The
signals of up to five neighbouring sensor elements, covering a surface of
50x300 µm, were processed (Fig.
2, Fig. 5). The
distance between sensor elements and insect was reduced by milling and
polishing a central furrow into the polymer surface above the sensor chip. A
measured delay of 1 ms between consecutive elements of the linear sensor array
was considered in the calculations and recording traces. The sampling rate of
the 8-channel-computer interface (Powerlab: Chart 5: CB Sciences, Milford, MA
01757, USA) was 1000–40 000 Hz, depending on the number of recording
channels (5, 3 or 2), respectively.
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Anatomy
Serial sectioning was done in preparations after removal of the prothorax
and abdominal tip only to avoid deformation of cuticular parts in the waist,
keeping the heart and surrounding tissues in a natural position. The flies
were fixed in glutaraldehyde–paraformaldehyde with an initial 20 min
evacuation and post-fixed in OsO4, both in phosphate buffer.
Details of fixation and embedding were as previously described
(Wasserthal, 1999). Semithin
sections were cut on a Reichert Ultracut 3 (Vienna, Austria) with diamond
knives, stained with Toluidine Blue, analyzed in a light microscope with
interference contrast (Leitz) and photographed using a Nikon D2X camera. For
in situ preparations, the abdomen was dissected and rinsed in saline
(Ephrussi and Beadle, 1936),
fixed in formaldehyde and photographed in the fluid
(Fig. 8A) or after embedding in
purified camphene at 45°C, cutting with broken razor blades and
sublimation of the embedding medium. The photographs were taken with a Nikon
DX2 on a Leica macroscope (Fig.
11C) and a scanning EM (LEO Gemini 1530, Oberkochen, Germany).
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0.5 Hz for suppression of the periodic up- and
down-changes in mean transmittance (Fig.
4).
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There are no fundamental differences in the optical recordings, frequencies
and period lengths between the two Drosophila species: the heart alternates
between pumping hemolymph in a forward (=anterograde) and a reverse
(backward=retrograde) direction with a very regular periodicity. The
anterograde heartbeat periods of D. melanogaster are generally longer
(14.1±2.9 s) than the retrograde periods (4.9±1.4 s)
(18.1±11.1 s and 6.9±3.2 s, respectively, in D. hydei)
(Table 1 and
Fig. 3B). In younger, fully
hydrated flies, the retrograde pulse periods are almost as long as the
anterograde periods (Fig. 6B).
The anterograde pulses of D. melanogaster generally have a lower
frequency (4.2±0.93 Hz) than the retrograde pulses (5.3±0.8 Hz);
N=4 flies, with 24 periods calculated per fly (D. hydei:
3.4±0.7 Hz and 4.9±0.3 Hz, respectively, N=9)
(Table 1). The velocity of the
peristaltic wave is extremely variable and ranges from 1.5 mm to 100
mm s–1 in both directions. This means that a peristaltic wave
needs about 5–240 ms to traverse the 1 mm long heart of D.
melanogaster. The anterograde pulses are less regular and single pulses
often look more complex by the addition of one or more peaks
(Fig. 4B,
Fig. 5A). These additional
peaks are generally not visible at the beginning of the forward pulse periods
(Fig. 5B) but become obvious in
the course of the period (Fig.
5A). It is assumed that the anterior heart chamber during diastole
is filled by hemolymph during the presystolic wave, which in the course of the
forward pulse period flows against an increasing back-pressure and therefore
becomes inhomogenous (graduated) and reduces the pulse speed (compare
Fig. 5A,B). The retrograde
pulses are more regular and are characterized by a single peak
(Fig. 4C,
Fig. 5B). The original
unfiltered recordings with the slow periodic changes in mean level of
transmittance provide additional information. At the second and third segment
the mean level of transmittance during retrograde pulses is generally higher
than during anterograde pulse periods (Fig.
3, Fig. 6B). The
transmittance increases during the first retrograde pulses and achieves a
nearly constant level, slightly undulating. At the beginning of the
anterograde pulse periods the transmittance decreases rapidly during the first
few pulses and remains on a lower but variable level. It is noteworthy that
with mobility of the abdomen restricted by gluing the caudal end, length
changes are prevented, so that the mean level of IR-transmittance remains
rather constant during anterograde and retrograde pulses. Under these
conditions, the heart reverses its beating direction irregularly or alternates
its pulse direction repetitively with each pulse
(Fig. 7). The heart seems to be
unable to perform the normal sequences of periodic reversals. It is concluded
that apart from the above-described intermittent bouts of abdominal movements,
which are independent from the heartbeat periodicity, the abdomen changes its
length and volume in parallel with the heartbeat reversals. The increase of
mean transmittance during retrograde pulses is a consequence of the augmenting
hemolymph volume combined with abdominal expansion. The rapid decrease of mean
transmittance during anterograde pulses is caused by abdominal contraction
under reduction of hemolymph volume in the abdomen and the denser arrangement
of the tissues.
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Here in the waist, the abdominal movements and volume changes have only little effect due to the dense arrangement of organs and fat body. However, another type of pulse with greater amplitude and low frequency of 0.3–0.4 Hz interferes with the heart pulses (Fig. 6A, arrows). This interference is obvious during the anterograde pulse periods, but almost hidden during the retrograde pulses. In the waist, the vertical part of the anterior heart and the aorta directly adjoin the intestine. It is inevitable that in this anterior, narrow part of the abdomen the peristalsis of the intestine influences the IR light beam. These peristaltic movements occur continuously and should not be confused with the intermittent abdominal pumping movements.
Anatomy of heart and associated structures
As the postulated ostia were suspected to be in the anterior part of the
heart, which is curved ventrally, the waist region was sectioned horizontally
to obtain cross sections of this part at its site of contact with the adjacent
mesophragma. A pair of ostial valves was found in the anterior part of the
first heart chamber immediately behind the mesophragma, just above the
transition to the aorta (Fig.
8C, Fig. 9A,
Fig. 10B,
Fig. 11B). Here the frontal
surface of the first heart chamber is directly attached to the mesophragma by
threads of connective tissue (Fig.
10E). The ostial valve lips are oriented into the heart lumen
(Fig. 10B). The anterior lips
arise directly from the attached frontal heart margin. This position is the
reason why, in most preparations, the heart is rupturing along the anterior
ostial slits and why the first ostia were overlooked in all previous analyses.
SEM micrographs of the longitudinally split anterior heart chamber show the
first ostial lips, which are tightly closed and deeply protrude into the heart
lumen in such a way that they allow an inflow only during diastole and are
closed by the action of being pressed against the anterior wall of the
mesophragma during systole (Fig.
10B). The second pair of ostia, which until now was thought to be
the first one, is smaller and about 100–300 µm behind the `new' first
pair, depending on the overall size of the fly
(Fig. 8C). In contrast to the
first ostia, all posterior ostial pairs (numbers 2–5) are visible as
deep inflections of the inwardly projecting lips in the liquid preparations
(Fig. 8A).
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At the ventral face, the heart is connected to and covered by the dorsal diaphragm along its whole length (Fig. 8B). In the first abdominal segment, the dorsal diaphragm fully surrounds the anterior part of the conical heart chamber and forms a dense muscular pericardial diaphragm. It is the inner layer of the pericardial septum around the `venous' space (Fig. 9A,B, Fig. 10). Along the rest of the heart, the dorsal diaphragm is tightly attached to the ventral heart and leaves the lateral heart with the ostia uncovered and accessible for the abdominal hemolymph. Its muscles are oriented longitudinally and may shorten the heart tube when contracted. There are weak alary muscles in the third and fourth abdominal segments but two pairs of strong fans of alary muscles suspend the heart in the fifth abdominal segment. A pair of caudal suspending muscles arise directly from the heart tube.
The new first pair of ostia has a central function because it is
exclusively connected to the thoracic hemocoel via a pair of hitherto
overlooked lateral hemolymph channels. These channels are a part of the
greatly modified posterior metathoracic hemocoel. The dipteran thorax is
characterized by the displacement of the mesothorax into the metathorax due to
the mighty dorso-longitudinal flight muscles
(Ferris, 1950). The
mesophragma medially intrudes into the first abdominal segment and serves as
attachment site of the anterior heart wall and the ventral part of the
pericardial septum (Fig. 10,
Fig. 11B). The metanotum is
reduced to a cuticular dorsal ridge extending laterally as a cuticular septum,
which borders the lateral channel. Ridges and septum serve as attachment site
for the lateral and dorsal part of the pericardial septum
(Fig. 10D,E).
The pericardial septum is composed of the pericardial diaphragm and an outer layer of dense `constitutive' fat body (Fig. 9A,B, Fig. 10A,C). It shields the venous space against the remaining abdominal hemocoel. As all other ostia, including the second pair, lie outside the venous space, only the first pair of ostia receives hemolymph from the thorax. Thus, in Drosophila a `venous supply' to the anterior heart is established by the lateral channels.
The channels continue between mesophragma and lateral metathorax along the
base of the haltere (Fig. 9E)
and communicate with the hemocoel behind the metathoracic spiracles
(Fig. 9F). At the level of the
aorta, the lateral channels are no longer bordered by the pericardial septum
but by a tubular muscle (Fig.
9B–D, Fig.
10C,F). This muscle is the tergo-pleural muscle of the metathorax.
It is partly identical with the upper part of the tergo-sternal depressor, No.
86, described by Miller (Miller,
1950). It originates from the outer dorsal free edge of the
cuticular septum (Fig. 9B) and
descends anterior-ventrally. This muscle is stretched over the convex-curved
mesophragma before it enters the ventral foramen of the thorax
(Fig. 10C,F). The ventral end
is fixed at the stiff pleural apophysis arising from the suture between the
pre-episternum 3 and epimeron 3 (Fig.
9F, Fig. 10F). The
function of this muscle remains unclear. As it is attached to the channel
septum of the metanotum, which is elastically connected to the mesonotum, it
may either approach the septum towards the mesophragma and reduce the lumen of
the channel or remove it and dilate the channel lumen. This muscle could
change the flow volume of the venous channel in coordination with the
heartbeat reversals.
In flies no ventral hemocoelic connection between thorax and abdomen (e.g.
a perineural sinus) exists, due to the lack of an abdominal ventral nerve cord
and a ventral diaphragm (Richards,
1963). The ventral foramen in the waist between thorax and abdomen
(Fig. 10D,F) is filled and
probably tightened by the intestine with its appendages and sealed by muscles
and fat body (Fig. 9E,F,
Fig. 10C). Thus no other than
the lateral hemocoel connection between thorax and abdomen is known. The
separation of the thoracic hemocoel from the abdominal hemocoel divides the
body into two compartments as a precondition for a periodic hemolymph shift by
heartbeat reversal.
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;
Brazeau and Campan, 1970;
Queinnec and Campan, 1975;
Jones, 1977;
Wasserthal, 1982b;
Angioy and Pietra, 1995).
Attribution of pulse rate to direction of peristaltic wave is, however,
controversial. In earlier investigations on the heart of the adult Calliphora
and Protophormia, the pulses with the lower pulse rate were interpreted to be
retrograde (Brazeau and Campan,
1970; Queinnec and Campan,
1975; Thon and Queinnec,
1976; Angioy and Pietra,
1995). However, with contact thermographic studies using local
heat-marking in intact Calliphora, the pulses with higher frequency are shown
to run retrograde (Wasserthal,
1982b; Wasserthal,
1999; Wasserthal,
2003a). The methodological dilemma due to the small size in
Drosophila is now solved by use of the IR-radiation+linear optosensor array
method introduced in this paper, which allowed measurement of the heartbeat in
tethered, intact flies over a long period (up to several days) and
determination of the pulse speed and direction.
Photosensors have been used to record insect heartbeat [Bombyx, Xylotrupes
(Tachibana and Nagashima,
1957); Schistocerca (Phaneuf
et al., 1973); Sarcophaga
(Williams et al., 1968);
Schistocerca, Sarcophaga (Lhotsky et al.,
1975); Ornithoptera pupae
(Hetz et al., 1999); Manduca
pupae (Slama, 2003)]. In
Drosophila pupae and a few adults, Slama and Farkas used a single IR
photodiode for heartbeat recordings under transmitting and reflecting light
(Slama and Farkás,
2005). But the pulse direction could not be recorded using a
single sensor element and thus they attributed the higher frequency pulses to
the anterograde direction on the basis of visual observation. Dulcis and
Levine processed heart videos electronically and transformed light density
fluctuations into curves of heart pulses
(Dulcis and Levine, 2005). They
analyzed up to three points along the heart in order to detect the pulse
direction. They also attributed the higher frequency pulses to the anterograde
direction and correlated anterograde and retrograde beating with phasic
volumetric changes of the conical heart chamber. They called the retrograde
beating phase with higher volume `diastolic' and the anterograde beating phase
with smaller volume `systolic'. As the heart continues to pulsate during these
phases, the terms `systole and diastole' and `mini systole and diastole' are
somewhat misleading and the force or mechanism for the slow volume changes of
the conical chamber (or is it the pericardial diaphragm? see below) remains
unclear.
In the IR-multisensor recordings in intact Drosophila (wings and legs intact, abdomen free to move), the phases with higher pulse rate are generally retrograde and these pulses are more regular than the slower anterograde pulses, which are characterized by double or even more complex peaks during diastole.
The latter diagnosis is confirmed by x-ray videos from the heart of
Calliphora performed at the ESRF-synchrotron facility, Grenoble. These clearly
show that the higher frequent pulses coincide with retrograde pulsations
(Wasserthal et al., 2006). The
contradictory results may be explained by the different experimental
procedures. If the wings and legs are severed and the hemolymph and tracheal
spaces are opened, as in the experiments of Dulcis and Levine
(Dulcis and Levine, 2005), the
conditions of pressure and hemolymph supply from the lateral thorax are
possibly modified and may influence the diastolic volume.
The newly discovered first ostium and caudal opening of the heart in Drosophila have important functions in hemolymph transport
Nearly all recent papers on the Drosophila heart focus on regulation and
innervation, with no special attention given to the functional design and
proportions of the heart tube, which completely differs from other –
even holometabolous – insect orders such as Lepidoptera and Coleoptera
(Gerould, 1929;
Jones, 1977;
Wasserthal, 1996;
Wasserthal, 2003a). In order
to understand the mechanism of heartbeat reversal it is essential to know the
number and position of heart openings. In adult Drosophila heart, all authors
have described only four pairs of inflow ostia and a closed caudal end
(Miller, 1950;
Rizki, 1978;
Curtis et al., 1999). In fact
just the newly discovered first pair of ostia reported here and the caudal
opening are necessary for retrograde hemolymph transport to function. The
morphology of the enlarged anterior heart chamber, with two pairs of ostia, is
homologous to that of calliphorid flies
(Angioy et al., 1999;
Wasserthal, 1999). The caudal
excurrent opening in Drosophila is, however, unpaired, in contrast to the
calliphorid flies with paired openings.
The difference in pulse rates of retrograde pulses with the higher
frequency in flies but with lower frequency in Lepidoptera reflects their
different anatomy with different refilling and outflow conditions. The
lepidopteran heart also has ostia in the thoracic aorta and two-way ostia in
the abdomen with no caudal opening
(Gerould, 1929;
Jones, 1977;
Wasserthal, 2003b). Diastolic
filling during retrograde pulses of the heart occurs in a complex way by
action of the tergal pulsatile organs via these aortal ostia, and the
hemolymph must be pressed through the posterior ostia
(Wasserthal, 1996). In flies,
during retrograde pulses, the anterior heart chamber with the first large
ostia receives hemolymph directly from the lateral thorax and discharges it
through the caudal opening. Thus, the retrograde outflow in flies encounters
less resistance than in moths and butterflies and this may be the reason why
the backward pulses have a relatively higher frequency in flies than the
forward pulses, similar to scarabaeid beetles, which also have an open caudal
heart end (Wasserthal,
1996).
The mechanism of thoraco-abdominal hemolymph flow by `venous' channels
As a special trait in Drosophila, the first heart ostia are found to be
connected to thoraco-abdominal channels. They provide a flow passage from the
lateral thorax along the bases of the halteres and further to the dorsal
abdomen, directly to the pericardial space around the anterior heart chamber.
The pericardial septum shields the pericardial space and the hemocoel of the
lateral channels against the rest of the abdominal hemocoel and thus functions
in a similar way to the anterior wall of the large abdominal air sacs in
Calliphora (Wasserthal, 1999).
The venous channels are crucial for understanding hemolymph transport from
thorax to abdomen during backward beating. Retrograde hemolymph transport
via the aorta has been considered by some authors
(Angioy and Pietra, 1995;
Angioy et al., 1999). Dulcis
and Levine note that `the conical chamber lumen rapidly increased to reach
the maximum diastolic volume soon as the retrograde (!) beat started'
(Dulcis and Levine, 2005),
leaving the question of inflow undiscussed. Effective aortal retrograde pulses
are, however, ruled out by structural constraints: the aorta is
dorso-ventrally flattened when relaxed. It is thin-walled and contains no
striated muscles (Miller,
1950). Without a suspending muscle or connective tissue layer it
cannot actively aspire hemolymph but must collapse when the diastolic heart
sucks hemolymph during retrograde pulsations. Thus functional preconditions
for retrograde heart pulses in Drosophila differ fundamentally from those of
most other insect orders with heartbeat reversals, which possess inflow ostia
in the thoracic aorta (see above). Preconditions for diastolic filling of the
heart are essential for any heart functioning
(Vogel, 1992). With its
thoraco-abdominal channels connected to its anterior pair of ostia, the
Drosophila circulatory system evolved a venous pathway for effective
retrograde transport from thorax to abdomen. This compensates for a number of
fundamental reductions compared to a phylogenetically older plan, such as
aortal ostia and the ventral diaphragm for retrograde hemolymph transport.
The alternating function of the anterior heart chamber as a pressure pump and suction pump
My results lead to the following conclusions. The diastolic filling process
of the anterior chamber during forward and backward pulses is fundamentally
different. During anterograde pulsations, the heart chamber receives hemolymph
from all ostia and gets dilated by the presystolic wave (presystolic notch)
(Yeager, 1939), represented by
the graduated peak of the anterograde pulses. The complete volume of the
anterior chamber is then pressed through the narrow aorta. The need to
overcome the internal resistance of the aorta is possibly the reason for the
lower pulse rate and the graduated pulse peaks. During retrograde pulses, the
hemolymph is sucked by the anterior heart chamber directly through the first
pair of large ostia from the lateral thorax via the venous channels
and is discharged through the caudal opening. While the first ostia are
essential for hemolymph aspiration during backward beating, during forward
pulses some hemolymph can enter the diastolic heart through the first ostia
too, thus providing a lateral circulation in the thorax
(Fig. 12). This explains why
the forward pulse periods generally last longer than the backward pulse
periods, without the problem of an over-accumulation of hemolymph in the
anterior body. Even when the heartbeat changes from forward to backward
pulse-wise (Fig. 7), the
hemolymph `circulates' in the anterior body.
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The possible role of abdominal movements and a modified pericardial septum in regulation of heartbeat reversal
In Lepidoptera and Calliphora, the direction of heartbeat is influenced by
the tension of the abdomen (Wasserthal,
1996; Wasserthal,
1999). In the present investigation it is observed that, when the
abdomen is fixed, the Drosophila heart beats irregularly or changes its pulse
direction with each pulse. It is concluded that in Drosophila too the abdomen
plays a leading role in lowering the hemocoelic pressure by active expansion
during retrograde beating of the heart and augmenting the pressure by
contraction during anterograde beating, as in Lepidoptera and in Calliphora
(Wasserthal, 1981;
Wasserthal, 1982b;
Wasserthal, 1996;
Wasserthal, 1999;
Wasserthal, 2003a). During
anterograde pulses a positive hemocoelic pressure in the abdomen probably
prevents the hemolymph from leaving the caudal opening. In addition, two large
spongy cells on the rear of the second pair of ostia project deeply inward
into the heart lumen (Miller,
1950). They probably prevent a back-flow towards the caudal
opening during anterograde systole. By contrast, the heart can transport
efficiently backward into the abdomen only if the hemolymph pressure in the
abdomen is lower than the diastolic pressure inside the heart, otherwise
hemolymph would be sucked back into the heart immediately after having left
it. This mechanism may also influence the filling process of the anterior
heart chamber directly or by support of the pericardial septum. It forms a
muscular sheath with a cavity around the anterior heart chamber. If the
internal hemolymph pressure by abdominal expansion during retrograde pulses
decreases, the suction should dilate the pericardial septum and may thus
explain the `diastolic volume increase' observed by Dulcis and Levine
(Dulcis and Levine, 2005),
which does not occur in their bath preparation when retrograde pulses are
stimulated by application of glutamate. The arrangement and attachment of the
muscles of the pericardial septum at the metanotal cuticular ridge and at the
ventral surface of the mesophragma suggest that their contraction increases
the pericardial cavity and assists in distending the first heart segment. An
active and important role of the pericardial septum can be deduced from the
rich supply by the anterior transverse nerves. They form unique synaptic
structures on its ventral longitudinal muscles
(Dulcis and Levine, 2003). In
addition, only the conical chamber receives input from the thorax via
the median nerve. This suggests that the assumed pacemaker function of the
anterior heart chamber (Ritzki, 1978) is under neuronal control
(Dulcis and Levine, 2003).
Moreover, some coordination of abdominal movements with heartbeat reversals
may be mediated by these nerves.
Periodic hemolymph shift by heartbeat reversal and the physiological significance of having the hemolymph supply separated between thorax and abdomen in higher flies
Adult flies are lightweight constructions adapted for flight with a reduced
hemolymph content (Wigglesworth,
1963; Nicholson,
1976). The body in many flies is divided into an anterior and a
posterior hemocoelic compartment separated by a huge pair of air sacs in the
anterior abdomen (Faucheux,
1974). In Calliphora they function like a septum and facilitate an
alternating periodic increase and decrease in hemolymph volume in both
compartments by hemolymph shift with a compensatory ventilation effect upon
the tracheal system (Wasserthal,
1999; Wasserthal,
2003a). In Drosophila without these large air sacs
(Wigglesworth, 1950;
Miller, 1950;
Faucheux, 1974), the described
venous channels and septum enable a similar periodic hemolymph shift. The
hydraulic effects of the small hemolymph volume can be more effectively used
by separation of both compartments. Periodic reduction of the hemolymph volume
in the anterior body is compensated by an increase of the tracheal air sacs
with consequential inspiration. The periodic tracheal volume changes in the
head of Calliphora and Drosophila could be visualized by x-ray videographs at
the ESRF-synchrotron (Wasserthal et al.,
2006).
Gene activation pattern and number of ostia
The gene activation pattern in Drosophila heart morphogenesis has been
elucidated including the ostial valve determination
(Cripps and Olson, 2002;
Gajewski et al., 2000;
Popichenko and Paululat, 2004;
Reim and Frasch, 2005). The embryological status shows seven pairs of ostial
precursor cells. The anterior four pairs of ostia predetermined in the embryo
are not manifested in the later larva, which possesses only three pairs. Until
now, in adult Drosophila, only four pairs of heart ostia in total have been
described, with the first pair in the second abdominal segment
(Curtis et al., 1999).
Preparations with seven double pairs of svp-expressing cells with two
accessory terminal ones have been shown in wild-type Drosophila
(Gajewski et al., 2000;
Zaffran et al., 2002). With
this newly described pair of imaginal heart ostia in the conical heart chamber
there are in fact five pairs developed in the adult Drososphila heart
tube. This is homologous to the situation in Protophormia terraenovae
(Angioy et al., 1999) and in
Calliphora vicina (Wasserthal,
1999). In Calliphora, with its very similar heart and a similar
pattern of heartbeat reversals, additional terminal openings have been found
in REM preparations (Wasserthal,
1982b; Wasserthal,
1999), and these have been confirmed for Protophormia in semithin
sections (Angioy et al., 1999).
The above-mentioned two svp cells at the end of the embryonic heart may be
precursor cells for the excurrent openings at the caudal heart of adult flies,
which are paired in Calliphora and Protophormia and unpaired in Drosophila.
Documentation of a new anterior pair of ostia in the first abdominal segment
of the conical chamber and of a caudal heart opening will be essential for
reconstructing the ontogenetic fate of predetermined heart tissue during
metamorphic changes (Molina and Cripps,
2001).
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Acknowledgments |
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Footnotes |
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References |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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