Tsetse flies (genus Glossina), the sole vectors of African trypanosomiasis, are distinct from other disease vectors, and most other insects, due to dramatic evolutionary adaptations required to support their unique life history. These morphological and physiological adaptations are driven by demands associated with their strict dietary and reproductive requirements. Tsetse reproduce by obligate viviparity which entails obligate intrauterine larval development and provisioning of nutrients for the developing larvae. Viviparous reproduction reduces reproductive capacity/rate which also drives increased inter- and intra-sexual competition. This work describes three-dimensional (3D) analysis of viviparity associated morphological adaptations of tsetse female reproductive tract as well as that of male seminal secretions by phase contrast microcomputed tomography (pcMicroCT). Structural features of note include abdominal modifications facilitating the extreme abdominal distention required during blood feeding and pregnancy; abdominal and uterine musculature required for parturition of developed larvae; reduction of ovarian structure and capacity; structural features of the male seminal spermatophore that enhance sperm delivery and inhibition of insemination by competing males; uterine morphological features facilitating expansion and contraction before, during and after pregnancy; analysis of structural optimizations of the milk gland facilitating nutrient incorporation and transfer into the uterus. The use of pcMicroCT provides unprecedented opportunities for examination and discovery of internal morphological features not possible with traditional microscopy techniques and new opportunities for comparative morphological analyses over time and between species.

Biological materials

Tsetse flies (Glossina morsitans) utilized in this analysis were obtained as pupae from the colony maintained at the Institute of Zoology at the Slovak Academy of Sciences in Bratislava, Slovakia. Flies were reared in the Tupper Hall arthropod containment level 2 insectary in the UC Davis School of Veterinary Medicine. Flies are maintained in an environmental chamber at 25ºC and 75% relative humidity with 12:12 light/dark photoperiod. Flies receive defibrinated bovine blood meals via an artificial feeding system Mondays, Wednesdays and Fridays as described ²⁶. Sterile defibrinated bovine blood for feeding is obtained from Hemostat Laboratories (Dixon, CA).

Sample collection

Tsetse pupae were placed into an eclosion cage and monitored for teneral females daily. Teneral flies were anesthetized on ice and sorted into cages by sex. At five days post eclosion, individual virgin females were combined with males in mating cages. Cages were observed for mate pairing. If pairing was not observed within 10 minutes the male was removed from the cage and a new male introduced. Tsetse flies require at least one hour of pairing for completion of the transfer and formation of the spermatophore ²⁷. Mate pairs lasting for less than 60 minutes were removed from the sample pool. Within an hour of mating completion, females were anesthetized on ice for sample preparation.

Sample preparation, fixation and staining

Chilled flies were prepared for fixation by removal of the legs and wings to allow the fixative to permeate into the haemocoel. The fly was then placed into Bouins fixative solution (acetic acid 5%, formaldehyde 9% and picric acid 0.9%) and incubated overnight at room temperature. Following fixation, flies were dehydrated using a graded series of ethanol washes (10%, 30%, 50%, 70% and 95%). Each wash was performed for 1 hour at room temperature. Flies were then stained in 1% iodine in 100% ethanol for 24 hours. After staining, flies were washed 3 times in 100% EtOH for 30 mins per wash.

Phase contrast micro computed tomography

During imaging, samples must remain in a fixed position with no movement during the scanning process to ensure proper alignment of the image stack. In preparation for imaging, fixed and stained flies were transferred into a 1.5 mL Eppendorf tube containing unscented Purell hand sanitizer (Gojo Industries, Akron OH). The specimen was gently pushed down to the bottom of the sample tube using a pipette tip. To ensure samples remained immobilized during scanning, the bottom of another 1.5 mL Eppendorf tube was cut off and pushed into the sample tube for use as a wedge. The wedge was gently pushed down into the sample tube until the specimen was secured between the wall of the container and the wedge. Once the specimen was secured, the remaining volume of the sample tube was filled with Purell. The sample tube was modified to attach to the sample holder (chuck) in the MicroCT imaging hutch by hot gluing a wooden dowel (3 mm diameter, 20 mm length) vertically to the flat surface of the Eppendorf tube cap. The sample was imaged using a monochromatic beam of 20 kilo electron volts (keV). Images were captured through a 4x optique peter lens system and a PCO.edge CMOS detector with a 150 mm sample to scintillator distance. The resulting size of the individual sections was 2560 x 2560 px with a resolution of 72 pixels/inch and a 32-bit depth. During the imaging process, 1596 images were captured across 180° of rotation. Image stacks (TIFF format) were produced using Xi‐CAM ²⁸ with the gridrec algorithm as implemented in TomoPy ²⁹. The resulting image stack has reconstructed voxel resolution of 1.6 microns.

26    Aksoy S. Establishment and Maintenance of Small Scale Tsetse Colonies. In: Maramorosch K, Mahmood F, editors. Maintenance of Human, Animal and Plant Pathogen Vectors. New Hampshire: Science Publishers, Inc; 1999. p. 123–36.

27    Saunders DS, Dodd CWH. Mating, Insemination, and Ovulation in Tsetse Fly, Glossina morsitans. J Insect Physiol 1972;18:187-.

28    Pandolfi RJ, Allan DB, Arenholz E, Barroso-Luque L, Campbell SI, Caswell TA, et al. Xi-cam: a versatile interface for data visualization and analysis. J Synchrotron Radiat 2018;25:1261–70. https://doi.org/10.1107/S1600577518005787.

29    Gürsoy D, De Carlo F, Xiao X, Jacobsen C. TomoPy: a framework for the analysis of synchrotron tomographic data. J Synchrotron Radiat 2014;21:1188–93. https://doi.org/10.1107/S1600577514013939.

This dataset includes phase-contrast MicroCT volumes from female tsetse flies. The samples include:

3 volumes from 5 day old unmated female abdomens - GMM_5day_PE_V (1-3)

3 volumes from 5 day old mated female abdomens (1 hour post mating) - GMM_5day_1hr_PM (1-3)

3 volumes from 6 day old unmated female abdomens - GMM_6Day_PE_V (1-3)

3 volumes from 6 day old mated female abdomens (24 hours post mating) - GMM_6Day_24Hr_PM (1-3)

3 volumes from 8 day old unmated female abdomens - GMM_8day_PE_V (1-3)

3 volumes from 8 day old mated female abdomens (72 hours post mating) - GMM_8day_72hr_PM (1-3)

Each dataset includes an array of 32 bit tiff images representing tsetse fly reproductive tract tissues. Some samples include multiple volumes which will need to be stitched together. All related scans for each sample are packaged within each zip file.

For use, the tiff files will need to be imported into an application which can import, view and manipulate 3D image data. The analyses in this work were performed using the Dragonfly software package version 4.0 developed by ORS (Object Research Systems) in Montreal, Canada. Software available at http://www.theobjects.com/dragonfly. Other 3D data analysis packages are available and the data in tiff format is non-proprietary so it can be used in most applications.