Combined proton NMR imaging and spectral analysis of locust embryonic development

Abstract

Images and spectra of desert locust[Schistocerca gregaria (Forskal)] embryos were collected in vivo using a 4.7-tesla proton ('H) NMR imaging spectrometer. The 100 x 100 x 500 ,um inmage resolution and 125-nl localized spectral volumes were obtained within minutes of each other. The dynamics of embryonic development were slow enough that the time delay between imaging and spectral measurements is negligible. Thus, image and spectral data correspond closely and approximate real-time observations of embryonic changes. The above procedure was applied every 12 hr during the entire course of development. This analytical approach demonstrates that imaging and localized spectroscopy can be used to visualize and assess changes in embryonic water and lipid content in concert with the development of a living embryo.NMR studies of developing plants and animals have scientific merit because they offer the opportunity to carry out research on living organisms in a nondestructive way. Developmental geneticists need to understand the sequential and compartmental processes in early embryonic development (1-6).Cryopreservationists need to identify chemical and structural changes that are critical to maintaining the integrity of the organism during freezing and thawing (7). Physiologists need to track relevant nuclei through plant (8) and animal (9) vascular systems. These and many other areas of in vivo research could benefit from the nondestructive methods available using NMR technology.Here we show that proton NMR spectroscopy designed to combine imaging and selected volume spectral analysis offers great promise for research on developmental dynamics. We use this NMR method [image-selected in vivo spectroscopy, ISIS (10)] to follow the course of development of the locust Schistocerca gregaria (Forskal) from a few hours after fertilization to the formation and hatching of the first nymphal stage. This technique demonstrates that the near real-time dynamics of a developmental system can be visualized and chemically analyzed noninvasively. Embryonic movements, the formation of anatomical structures, and the dynamics of water and lipid in selected submicroliter volumes can be evaluated.Locust embryos develop in a centrolecithal ooplasm where the yolk is distributed within a cytoplasmic matrix that is continuous with a peripheral layer of yolk-free cytoplasm. The first embryonic divisions take place in a substrate that consists of yolk elements that differ in kind and size. After several energid divisions, nuclei with their associated cytoplasms migrate to the surface to form the presumptive embryonic germ band and embryonic membranes; nuclei remaining in (or returning to) the interior give rise to vitellophags. In the locust, the germ band forms as a 300-,um disc on the yolk periphery near the posterior dorsal margin. The remaining energids contribute to (i) membranes that envelop the embryo, (ii) yolk bodies, or (iii) lytic cells that digest the yolk. These cytological details and the subsequent features of the locust emb...

Optical coherence tomography as a noninvasive 3D real time imaging tool for the rapid evaluation of phenotypic variations in insect embryonic development

“…The yolk in the egg on day 3 appeared to be coarser than that in the first 2 days, probably resulting from the enhancement of scattering signal in the OCT image ( Figure 4C). On day 3, the locust egg began to increase its size by water intake ( Figure 4C) from air or soil, in consistent with prior knowledge [8]. In the next 3 days ( Figure 4D-F), the water content of the egg increased dramatically and finally the egg expanded to twice of its original volume on day 6 ( Figure 4F).…”

“…With the advances in medical imaging, microscopy and imaging probes, a variety of noninvasive imaging methods emerged to advance embryonic development studies [1,[8][9][10][11][12]32]. As described in the Introduction, these methods may have one or more limitations associated with image acquisition time, resolution and the costeffectiveness to own and operate.…”

“…Observation of Katatrepsis: "Katatrepsis" in this paper refers to the combination of two embryonic movements as an embryo develops, one is the translational movement describing the embryo moving toward the anterior end of the egg and the other rotational movement of the embryo around the long axis of the egg [5,33,36], although there are some different definitions in the literatures of this term [8,27]. As shown in Figure 9A-F, one can clearly see that from the 22nd hour on day 7 to the 20th hour on day 8, the embryo gradually move from middle of the egg toward the anterior end of the egg while growing larger by consuming yolk.…”

“…In order to minimize the damage to embryo tissues, a variety of noninvasive in vivo imaging techniques, such as nuclear magnetic resonance imaging, X-ray imaging, magnetic resonance imaging, magnetic resonance microscopy, light sheet-based one-photon and multiphoton simultaneous multiview (SiMView), confocal microscopy and light-sheet-based fluorescence microscopy, have been developed and applied to exam the internal structure of insects [1,[8][9][10][11][12]. However, these methods have one or more limitations, such as long image acquisition time, low resolution, high cost, complicated to operate, demanding clean-room operation conditions and requiring dedicated operators.…”

Non-invasive lipid measurement in living insects using NMR microscopy

“…Since then, a new dimension of investigating animals opened up and many different species have been characterized by NMR microscopy: e.g. the development of a locust embryo (Gassner and Lohman, 1987), pH metabolism of living insects (Skibbe et al, 1995), development and metamorphosis of lepidopteran pupae (Behr et al, 2011;Goodman et al, 1995), metamorphosis of the silkworm (Mapelli et al, 1997) and the morphology of diving beetles (Wecker et al, 2002) and bees (Haddad et al, 2004;Tomanek et al, 1996). However, because of the high cost of MRI machines, only few zoological studies have been performed.…”