J OURNAL OF C RUSTACEAN B IOLOGY, 33(4), 586-589, 2013

ANATOMICAL EVALUATION OF THE ORGANS IN THE RED SWAMP CRAYFISH,

PROCAMBARUS CLARKII, BY DIAGNOSTIC ULTRASOUND EXAMINATION

Francesco Macrì 1,∗ , Simona Di Pietro 1 , Rossella Bonfiglio 2 , Carmelo De Stefano 2 ,

Pietro Giorgianni 2 , and Teresa Bottari 3
1 Department of Veterinary Sciences, University of Messina, Polo Universitario dell’Annunziata,
98168 Messina, Italy
2 Freelance practitioners
3 Institute for Coastal Marine Environment (IAMC), National Research Council (CNR),

Spianata S. Raineri, 86-98122 Messina, Italy

ABSTRACT
We describe the ultrasound examination of the cephalothorax and pleon in red swamp crayfish. Real-time B-mode, Color and Power
Doppler examinations were performed on 10 crayfish, Procambarus clarkii (Girard, 1852). We envisage that use of this technique will
provide baseline information for further imaging studies to investigate the cardiovascular and digestive physiology and can be applied to
identify disorders in crayfish, to say nothing of use in other crustaceans.
K EY W ORDS: anatomy, crayfish, ultrasound
DOI: 10.1163/1937240X-00002156

I NTRODUCTION Before the ultrasound examination, the subjects were maintained in

Recently, EU member states have moved to apply Directive
2010/63/EU that for the first time includes an entire class
of invertebrate species, i.e., cephalopods, on the list of
organisms to be regulated for experimental use in research
and testing. In consideration of this, our effort herein, despite
being focused to a non-included taxon, viz, crustaceans,
has a particular relevance towards, and can be of interest
for augmenting consideration of, the application of the
principles of the 3Rs in experimental approaches (Russell
and Burch, 1959). These entail: “replacement,” or use of
non-animal methods; “reduction,” or methods to reduce
the numbers of animals used; “refinement,” or methods to
improve animal welfare. The present study, in the spirit of
the EU directive, aims to illustrate the characterization of the
organs of the cephalothorax and pleon of the Procambarus
clarkii (Girard, 1852) by using ultrasound examination.
M ATERIALS AND M ETHODS
A total of 10 individual P. clarkii, with a minimum straight carapace length
from 11.5-13 cm and weights from 65-70 g, were used in this study. The
crayfish were collected in the Trasimeno Lake District in Umbria (Central
Italy) and housed for about two months at the Center for Experimental Fish
Pathology of Sicily (C.I.S.S.).
Ultrasound examinations were performed with a real-time, B-mode
scanner (Mylab Vet 40, Esaote™) using linear array electronic transducers
at a frequency of 12.0 MHz. The two-dimensional (2-D) imaging mode and
Color and Power Doppler provided both a real-time snapshot of the heart in
motion, and scrutiny of the large blood vessels. Ultrasound examination
was performed by placing the transducer on the dorsal body surface of
the animals to collect the sagittal (cranio-caudal) and short axis (cross-
sectional) images for the morphological study.
600-l freshwater aquaria at a standard controlled-temperature of 22°C
with continuous aeration. At 48 hours before the time of ultrasound
examination, feeding was stopped to enhance scanning results. During
the actual investigation, the crayfish were placed in a tank containing
fresh water (Fig. 1A). The animals were manually restrained in ventral
recumbancy without sedation, the claws were bound with rubber bands.
R ESULTS
We provide a schematic rendition of normal internal anatomy
to facilitate the interpretation of the ultrasound images
(Fig. 1B).
The ultrasound examination of the circulatory system
of the crayfish showed the inside of the pericardial sinus
(anechoic cavity) and the echoic wall of the heart (ventricle)
in diastole and systole. The ventricle had a homogenously
echoic appearance, with distinction of a thick wall with a
fine texture. The heart as a muscular structure resembling a
rhombus (Fig. 2).
When the beam struck an individual perpendicularly,
the pericardial sinus exhibited a hyperechoic wall (Fig. 2).
The oxygenated blood flow from pericardial sinus enters
the heart through three pairs of valved slits called ostia
(Fig. 2C2). The heart pumps the blood into the body cavities
through seven large arteries, which were seen as anechoic
tubular structures with presence of the normal echoic walls,
either in transverse or longitudinal sections. The ophthalmic
artery carries blood anteriorly, just below the dorsal body
wall and supplies the eyestalks, the brain, and the antennules
(Fig. 2B). The paired antennary arteries extend from the an-
terior portion of the heart, travel deep around the heart, and

∗ Corresponding author; e-mail: framac@alice.it

© The Crustacean Society, 2013. Published by Brill NV, Leiden DOI:10.1163/1937240X-00002156

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 MACRÌ ET AL.: ULTRASOUND EXAMINATION OF CRAYFISH ORGANS 587

Fig. 1. A, positioning for ultrasound examination in a Procambarus clarkii; ventral recumbency, transducer position for scanning the cephalothoracic and
of pleonal organs. B, illustration of the relationship of the major organs in the crayfish: a = heart; b = stomach (anterior cardiac chamber); c = stomach
(smaller piloric chamber); d = intestine; e = hepatopancreas; f = gonad; g = green gland; h = brain; i = dorsal pleonal artery; l = sternal artery; m =
ophthalmic artery; n = antennary artery; o = artery to hindgut; p = hepatic artery; q = ventral pleonal artery; r = ventral thoracic artery.

go to the antennae, pericardium, cardiac stomach, eyes and
renal organs (Fig. 2D). Two hepatic arteries arise from the
heart on the ventro-lateral sides and plunges downwards into
the hepatopancreas (Fig. 2D). The large dorsal artery into
the pleon originates from the heart and extends posteriorly
along the dorsal surface of the intestine (Fig. 2D). Pairs of
segmental arteries branch off, supplying the intestine and the
pleonal muscles (Fig. 2C4). The sternal artery carries blood
obliquely downwards, and supplies blood to the ventral re-
gion of the pleon and thorax (Fig. 2A). Images obtained by

Fig. 2. Sagittal scan. A, 1 = cardiac muscle; 2 = pericardial sinus; 3 = dorsal pleonal artery; 4 = sternal artery; 5 = ventriculus. B, 1 = ophthalmic artery;
2 = hepatopancreas. C, images illustrate cardiac cycle of Procambarus clarkii (Color Doppler); C2, arrows indicate the ostii; C4, arrow indicates the artery
to hindgut. D, doppler waveforms of ventriculus heart. 1 = hepatic artery; 2 = antennary artery.

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 588 JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 33, NO. 4, 2013

two-dimensional (2-D) echocardiography provides real-time
imaging of heart structures throughout the cardiac cycle, in-
cluding changes in the diameter of the ventricular cavity and
can be used to infer cardiac systolic function (Fig. 2C).
Pulsed-wave Doppler was used to study flow direction and
blood velocity, the spectrum of flow velocities is represented
graphically on the y-axis and time on the x-axis. The mean
heart rate was 103 ± 2 beats/min. (Fig. 2D).
The gonads, testes or ovaries, could not be identified
because they were examined after spawning. The gut of
the crayfish is divided into five parts: esophagus, large
cardiac stomach (Fig. 3A and 3C), smaller pyloric stomach

Fig. 3. A, sagittal scan: 1 = stomach (anterior cardiac chamber); 2 = hepatopancreas. B, sagittal scan: stomach (smaller piloric chamber) and heart. C,
short axis of the cervical region: 1 = stomach (anterior cardiac chamber); 2 = green glands. D, sagittal scan: 1 = intestine; 2 = dorsal pleoal artery. E,
sagittal scan: 1 = green gland; 2 = ostium. F, short axis of the cervical region; the arrows indicate the gills (Power Doppler).

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 MACRÌ ET AL.: ULTRASOUND EXAMINATION OF CRAYFISH ORGANS
(Fig. 3B), midgut (intestine) and hindgut (rectum). The
esophagus was not identified in this study. The stomach
occupies most of the cephalothoracic cavity. The intestine
runs from the stomach through the pleon, ending at the
anus (Fig. 3D). Transverse and longitudinal views of the
gastrointestinal tract segments are necessary to complete the
study of the wall thickness and the echogenicity of segments.
In the stomach one can see the folding invaginations of
the wall into the lumen (Fig. 3B). Luminal patterns of the
gastrointestinal tract can be described on the basis of the
contents. If it is fluid-filled, the luminal content is anechoic;
if it contains sonolucent ingesta, it is echoic.
The large digestive gland, or hepatopancreas, was visual-
ized as an echoic structure with granular parenchyma placed
laterally, ventrally, and posteriorly to the stomach and placed
antero-ventrally to the heart (Figs. 2B and 3A). The green
glands, located at the base of each antenna, had a circular
dense appearance and looked echoic, similar to the ultra-
sound pattern of the digestive gland (Fig. 3C and 3E). The
gills are featherlike structures found underneath the carapace
and attached to the chelipeds and to the walking legs. Each
gill has blood vessels running through it; a constant flow of
blood, observed with the Doppler (Fig. 3F).
D ISCUSSION
The use of ultrasound examination offers new opportunities
to evaluate parts of the internal organ systems of the
crayfish, and by extension other macrocrustaceans as well.
We provide here the first description of an ultrasound
examination of the cephalothorax and pleon in the P.
clarkii. The ultrasound examinations lasted a maximum of
5 minutes. When necessary in order to avoid stress on the
crayfish employed for longer studies, the use of anesthetics
must be considered. The carapace did not limit the extent
of the ultrasound examination of the internal organs. Lastly,
Doppler echocardiography can be used to study blood
flow patterns and to determine heart rates in crustaceans.
Although several studies on heart rate variation under stress
in crayfish are available in the literature, there is no unique
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589
reference on the resting heart rate (McMahon et al., 1974;
Reiber et al., 1997).
Several invasive (electrocardiography) and non-invasive
techniques (fibre-optic) are used to evaluate the cardiocircu-
latory system of crayfish (Kozák et al., 2011). Ultrasound ex-
amination, compared to electrocardiography and fibre-optic
methods, is a practical tool to assess cardiac morphology
and function in P. clarkii. We envisage that this work can
be utilized to provide baseline information for further imag-
ing studies in investigations of cardiovascular and digestive
physiology, and can serve to identify disorders in crayfish.
ACKNOWLEDGEMENTS
The authors thank Professor Eugenio Cianflone for reading the English draft
and for his revision of this article. Collaboration is the place where the tree
of knowledge grows.
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R ECEIVED: 6 November 2012.
ACCEPTED: 25 February 2013.
AVAILABLE ONLINE: 29 March 2013.