12
Research for this chapter has been published previously as:
Gardner, C., Rush, M. and Bevilacqua, T. 1998. Non-lethal imaging
techniques for crab spermathecae. Journal of Crustacean Biology, 18: 64-69.
Abstract
Techniques for collecting information on spermathecae without dissection were evaluated on the giant crab Pseudocarcinus gigas. Techniques tested were: biopsy, ultrasound,
conventional x-radiography, computerised tomography (CT) scans, and magnetic
resonance imaging (MRI). Attempts at biopsy and ultrasound imaging were unsuccessful. Spermathecae were imaged by x-radiography, although resolution was poor, suggesting that it can be applied only to gain general information, such as determining whether mating has occurred. High resolution images were produced with CT and MRI. Resolution by MRI is of such detail that internal structure of spermathecae is imaged. Nonlethal techniques allow animals to be used repeatedly, which permits monitoring of changes during mating, sperm storage, and extrusion.
Introduction
The more advanced brachyuran crabs possess paired spermathecae which store sperm between mating and fertilisation. The function of these organs is of biological interest as layered storage of separate ejaculates can affect paternity of offspring (Koga et al., 1993; Sévigny and Sainte-Marie, 1996). Spermathecae have also been the subject of fisheries- oriented research where they provide a means of measuring the occurrence of copulation, which may be affected when males are harvested. The ability of females to utilise stored sperm to fertilise separate broods may buffer the impact of male-only fisheries (Paul and Paul, 1992). Examination of spermathecae for this range of research has traditionally involved dissection and analysis (e.g. histology) of the spermathecae, or assessment of the
spermathecae from the same individuals. In this way, changes can be tracked from mating, through storage, to extrusion. Nonlethal techniques tested were: biopsy, ultrasound, conventional x-radiography, computerised tomography scan (CTS), and magnetic resonance imaging (MRI).
Materials and methods
Female giant crabs Pseudocarcinus gigas, greater than 3.0 kg, were collected by commercial fishers from depths in the range of 300–380 m off the east coast of Tasmania
(41°,15'S;148°40'E) in May 1994. Crabs used for trials with conventional x-radiography were alive and restrained by claw ties; in all other trials, crabs were killed in baths of clove oil in sea water (0.125 ml/l; Gardner, 1997). Following imaging, crabs were dissected to relate imaged structures with actual tissues. In the case of CT and MRI, crabs were frozen and sliced by band-saw (5 mm sections) to duplicate the orientation of the images which were collected as transverse slices.
Attempts were made to biopsy contents of spermathecae using Pipelle de Cornier™ human endometrial biopsy catheters on dissected females with the gonoduct exposed. This allowed the biopsy tube to be observed as it was passed through the gonopore and along the gonoduct. Conventional x-radiography images were taken with standard veterinary equipment (AtomScope™ 100P). Ultrasound imaging was attempted with an ATL-8™ ultrasound using 7, 5, and 3.5 Mhz transducers. Computerised tomography scanning was performed with a GE™ scanner and MRI was performed with a 1.5 Tesla, Picker™ magnetic resonance imager.
Results
The collection of stored sperm by biopsy was not possible without injuring the crab. Although the gonopore of giant crabs is large, it is calcified during intermoult and force was required to introduce a biopsy tube. Once within the lumen of the gonoduct, the tube tore through the delicate walls rather than bending with the curvature of the duct.
Ultrasound penetrated the exoskeleton, but there was insufficient definition to discern the spermathecae. Factors contributing to the poor results with ultrasound appeared to be the echoing from internal calcified plates and the lack of sufficient acoustic difference between the spermathecae and surrounding tissue.
Spermathecae were successfully imaged using conventional x-radiography, although images were very unclear (Fig. 1). Optimal exposure was relatively high in order to penetrate the carapace (3 LV, 0.1 SIC). Consequently, most tissue definition was lost and the boundary layer between the spermathecae and surrounding tissue became blurred.
Figure 1. Image of female Pseudocarcinus gigas produced by conventional x-radiography showing spermathecae (S) as a pale region.
High quality images of the spermathecae were made using both MRI and CT scans so that details of substructure could be detected, especially with MRI (Fig. 2). Spermathecae were dissected after imaging. The dark bands detected by imaging related to separate areas of seminal plasma which bounded areas of spermatophore deposit, possibly from separate ejaculates. The optimal MRI setting for giant crabs was considered to be scan protocol 14.
Figure 2. Dorsal (upper) and longitudinal (mid) images of a female Pseudocarcinus gigas
produced by magnetic resonance imaging (MRI) showing high resolution of
spermathecae (S). Schematic (lower) is of the longitudinal image produced by MRI. Separate regions (EJ1, EJ2, and EJ3) can be seen within the spermatheca which may relate to different ejaculates.
Figure 3. Dorsal (upper) and longitudinal (lower) images of a female Pseudocarcinus gigas produced by computerised tomography (CT) showing high resolution of
spermathecae (S). Unlike MRI, CT is sensitive to calcified structures and the carapace is seen as a white band.
Figure 4. Three dimensional surface image of female Pseudocarcinus gigas
reconstructed from CT scans. Both MRI and CT scans can be processed into three dimensional images and “sliced” along any plane.
Discussion
The thick exoskeleton of P. gigas impaired the use of both biopsy sampling and ultrasound. No entry location for the biopsy could be found other than via the gonopore, which was unsuitable, since the gonoduct tore when a biopsy tube was introduced. Ultrasound has been used in research of organisms with soft bodies, such as marine mammals and fish (Gales and Burton, 1987; Bonar et al., 1989), but it appears to be unsuited to crustaceans.
Conventional x-radiography produced relatively poor images, since the boundaries between the spermathecae and surrounding tissue were blurred. Consequently, the exact size and form of the spermathecae could not be determined. Pseudocarcinus gigas have particularly thick carapaces, around 3.0 mm in relatively x-ray opaque larger females. The definition of spermathecae by conventional x-radiography may be greater in species with a thinner carapace. If higher resolution can be achieved, x-radiography may have application where the objective is simply to determine whether insemination has occurred. This can be important in experiments such as those reported by Koga et al. (1993) and Sainte-Marie and Lovrich (1994), where female crabs were dissected to confirm mating. The technique may also have application in studies assessing repeated spawning, using stored sperm where
radiography is relatively inexpensive (around $US10 per exposure and several crabs can be x-rayed on each plate), and more readily available than the other feasible techniques, CT and MRI scans.
Of the methods tested, only CT and MRI produced clear images of the spermathecae so that size could be measured and some internal structure viewed. The calcified exoskeleton did not interfere with imaging in either method. The shell was effectively transparent to MRI, since this technique relies on resonance of hydrogen nuclei (protons) which tend to be in low concentration within calcified tissues (Young, 1984). Resolution of internal structure was greater with MRI than with CT, and separate regions could be discerned which may relate to separate sperm deposits. Several authors have made observations based on separate sperm deposits in work investigating sperm storage and competition in brachyurans (e.g., Paul, 1984; Diesel, 1989; Sévigny and Sainte-Marie, 1996). The
technique of MRI provides the option of measuring or viewing contents of the
spermathecae before, or perhaps even during, mating. Although logistically difficult, MRI may also have application in observing changes to spermathecae during the process of extrusion and fertilisation. An additional benefit from computerised scanning methods is that 3-dimensional plots may be generated, either of the external surface structure (CT), or of the internal boundary between shell and soft tissues (MRI).
Although CT scans were of lower resolution than MRI, additional information is gained as calcified structures are detected. Where calcified structures are the subject of research, such as in the examination of skeletal growth of corals, CT scans are valuable (Logan and Anderson, 1991). In crustaceans, a benefit of imaging internal calcified structures is in the location of tissues, as calcified plates in the stomach and between limb muscles can serve as landmarks.
Practical considerations in the use of MRI and CT imagery include the costs of the procedures and the restraint of the crabs to prevent movement during imaging. Both
processing. In CT-scanning undertaken subsequently to this project, groups of 3 giant crabs were scanned simultaneously to examine ovarian development, and it was possible to process over 50 crabs per hour. Crabs must be motionless during scanning so paralysing agents such as xylazine-HCl, ketamine-HCL, procaine-HCl, or clove oil should be used (Oswald, 1977; Gardner, 1997).
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