[Development of prospective diapause-germs (Bombyx mori L.)in vitro without dormancy : III. Their competence and...

In a foreign protein medium (LTS), naked prospective diapause-germs have a tendency to develop into fully segmented germ bands (stage of dormancy): however, deposition of chorion, serosa or yolk will stimulate themvia the medium to pass through organogenesis without delay (non-dormancy =Nd). The question remained if a germ from a non-diapausing egg would stimulate the uninterrupted development of such a test-germ (Tk). Using 2 different or 2 to 3 equal germs as well asTk alone (control) in hanging drops, experimental evidence was obtained permitting conclusions not only as to the competence and sensitivity of the embryonic reaction system, but also as to factors of the extraembryonicNd-action systems in eggs with and without predetermined diapause regulatory mechanism. 1. Control explants (1 prospective diapause-Tk inLYS) have aNd-level of 10% (= average ofNd-rates in operative stages). This difference from former controls (0%Nd) can be explained by the longer operative procedure in the cups when several test-germs were used. Seven minutes after opening an egg one can reckon on aNd-stimulationvia LYS medium from egg-residues in the operation cup. While transfering the test-germs, yolk-derived substances related to theNd-stimulation can be brought into the hanging drop. The difference betweenNd-controls and the results ofNd-stimulation have to be statistically significant. 2. In experiments with two different germs in LYS-droplets no influence of a non-diapause germ (polyvoltine) on a prospective diapause germ (univoltine) could be noted. Accordingly, the naturally prospectiveNd-germs, have a tendency to develop to dormant germs only, as do the 2Tk and 1Tk controls. In combination with artificially prospectiveNd-germs (univoltine, HCl-treated) no stimulation of the test germ occcurs; nor after explantation with an older germ-band or a young embryo. 3. Combination with two prospective diapause germs of equal age in a drop, having the same minor contamination ofNd-factors in the operation-cup, will result in a threefold increase of theNd-level as compared to the 1Tk-controls. The competence of the embryonic reaction system ranges from a coatlike germ-anlage to a germ with thoracic segmentation. In the dish-like germ-anlage at diapause onsetin ovo maximalNd-rate (70%) is foundin vitro, i.e. highest sensibility towardsNd-stimulatory factors. Also at this operative stage, bothTk of a drop, most frequently developed beyond the dormancy stage. 4. Experiments with three prospective diapause germs of equal age in a drop, having the same minor contamination ofNd-factors in the operation-cup, show a sevenfold increase of theNd-level as compared to controls, showing an almost similar stage-range in competence and stage-specificity in sensibility. The increase of theNd-stimulation rate in relation with the number of test germs in the drop corresponds to a morphogenetic crowding-effect, the influence of the test germs on each other being called "interference". 5. Experiments with LYS-media which was slightly contaminated with bacteria and then refiltered had a 42%Nd-level in the 1Tk experiments. This significant difference from the 10% control level can be related to a sensitivity increasing factor or to aNd-stimulating "masterkey"-effect of the bacterial products in addition to the yolk factors already present. The 65%Nd-level in the 2Tk experiments clearly indicates "interference". 6. Interference was also shown inexperiments with artificially prospective non-diapause germ of the same age. Test-germs isolated immediately after the HCl-treatment had a very highNd-rate (78% in 2Tk and 100% in 3Tk). Interference also occurred in germ-bands that were removed at a later stage, i.e.in ovo prior to organogenesis (2Tk, 60%Nd-rate instead of 1Tk, 20%Nd-rate). Consequently, interference may be caused by a factor promoting an overall increase of tissue activity. 7. Critical evaluation of the methods and discussion of the resultsin vitro permit the following conclusions as to the developmentin ovo. Up to organogenesis, the continuous development of the germ-anlage (non-dormancy), is stimulated by an extra-embryonic system. In particular, the yolk-cell system contains factors, without which all germ-bands (including nondiapausal eggs fromBombyx and presumably other insect eggs) can only pass autonomously through the already programmed and/or determined gastrulation and segmentation. In pre-determined egg-diapause, an extra-embryonic dormancy regulatory mechanism inactivates thisNd-action system. Maternal hormonal depots may influence the continuous stimulation of cell division and the information-transfer for the programming of organogenesis. The activation of enzyme-systems inNd-stimulation can be imitatedin vitro by bacterial contamination and by interference from test germs of the same age, and may be appear as increased sensibility of the test germs.