DOI: 10.25338/b8z33b
title: Interpreting morphological adaptations associated with viviparity in the Tsetse fly (Glossina morsitans) by three-dimensional analysis

description: 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.
description: Biological materials Tsetse flies (<i>Glossina
 morsitans</i>) 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 <sup>26</sup>.
 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
 <sup>27</sup>. 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
 <sup>28</sup> with the gridrec algorithm as implemented in
 TomoPy <sup>29</sup>. 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. <i>Maintenance of Human, Animal
 and Plant Pathogen Vectors</i>. New Hampshire: Science Publishers,
 Inc; 1999. p. 123–36. 27    Saunders
 DS, Dodd CWH. Mating, Insemination, and Ovulation in Tsetse Fly,
 <i>Glossina morsitans</i>. <i>J Insect Physiol</i>
 1972;<b>18</b>:187-. 28    Pandolfi
 RJ, Allan DB, Arenholz E, Barroso-Luque L, Campbell SI, Caswell TA,
 <i>et al.</i> Xi-cam: a versatile interface for data
 visualization and analysis. <i>J Synchrotron Radiat</i>
 2018;<b>25</b>: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. <i>J Synchrotron Radiat</i>
 2014;<b>21</b>:1188–93.
 https://doi.org/10.1107/S1600577514013939.
description: 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.