Developmental and cell cycle progression defects in Drosophila hybrid males.

Matings between D. melanogaster females and males of sibling species in the D. melanogaster complex yield hybrid males that die prior to pupal differentiation. We have reexamined a previous report suggesting that the developmental defects in these lethal hybrid males reflect a failure in cell proliferation that may be the consequence of problems in mitotic chromosome condensation. We also observed a failure in cell proliferation, but find in contrast that the frequencies of mitotic figures and of nuclei staining for the mitotic marker phosphohistone H3 in the brains of hybrid male larvae are extremely low. We also found that very few of these brain cells in male hybrids are in S phase, as determined by BrdU incorporation. These data suggest that cells in hybrid males are arrested in either the G(1) or G(2) phases of the cell cycle. The cells in hybrid male brains appear to be particularly sensitive to environmental stress; our results indicate that certain in vitro incubation conditions induce widespread cellular necrosis in these brains, causing an abnormal nuclear morphology noted by previous investigators. We also document that hybrid larvae develop very slowly, particularly during the second larval instar. Finally, we found that the frequency of mitotic figures in hybrid male larvae mutant for Hybrid male rescue (Hmr) is increased relative to lethal hybrid males, although not to wild-type levels, and that chromosome morphology in Hmr(-) hybrid males is also not completely normal.

The control of anterior foregut motility during a larval molt of the moth Manduca sexta involves the modulation of presynaptic activity.

In the moth, Manduca sexta, anterior foregut motility is modulated during the larval-larval molts in order to control the timing of molting fluid (MF) ingestion. MF is the enzymatic mixture that destroys the outer cuticle so that it can be shed at the end of the molt. The onset of the larval-larval molt is characterized by a dramatic decline in the amplitude of the anterior foregut contractions so that MF is not prematurely ingested. As the end of the molt approaches, the robust contractions of the anterior foregut return and the MF is ingested, enabling the larva to free itself from its old cuticle. In the present study we examine possible mechanisms involved in modulating anterior foregut motility during a larval-larval molt. Our results reveal that the release of a blood-borne factor plays a role in the decline in anterior foregut peristaltic activity during the molt. This blood-borne factor reduces the efficacy of the presynaptic endings of the motorneurons, resulting in a reduction in the amplitude of the excitatory junctional potential (EJP) recorded from the anterior foregut musculature. We also present evidence that crustacean cardioactive peptide (CCAP) targets the motorneuron terminals and its actions are sufficient to trigger the dramatic increase in EJP amplitude and anterior foregut contractions. Finally, the surgical ablation of the subesophageal ganglion, which has been previously described to be a source of CCAP neurons and the CCAP projections to the anterior foregut region, blocks both the increase in anterior foregut motility and the ingestion of MF that normally occur at the end of a larval-larval molt.

Inhibitors of ubiquitin-dependent proteolysis can delay programmed cell death of adult intersegmental muscles in the moth Manduca sexta.

In the moth Manduca sexta, intersegmental muscles (ISMs) undergo rapid programmed cell death (PCD) within 48 hr of adult emergence. ISM PCD involves ubiquitin-dependent proteasomal degradation accompanied by the down-regulation of expression of actin genes and the up-regulation of degradative gene expression such as ubiquitin. Hemin chloride and N-acetyl-leu-leu-norleucinal (ALLN), both inhibitors of proteasomal activity, administered before adult emergence delayed PCD for up to 5 days in ISMs maintained from the larval stage, such as the dorsal internal medial muscle in abdominal segment 4 (DIM-A4). ISMs that developed during metamorphosis from respecified larval muscles such as the DIM-A2 were less dramatically affected. The increase in polyubiquitinated proteins and the decrease in actin mRNA expression accompanying maintained ISM PCD were delayed after inhibitor application. No changes were detected in respecified ISMs. These results reveal a regulatory role for proteasomal activity in an early stage of maintained ISM cell death.

Modulation of foregut synaptic activity controls resorption of molting fluid during larval molts of the moth Manduca sexta.

We examined the role of the foregut in the resorption of molting fluid (MF) from the exuvial space during the last larval-larval molt of the moth Manduca sexta. In intermolt larvae, the activity of the foregut is characterized by robust peristaltic contractions. With the onset of the molt, MF is secreted into the exuvial space where it digests and weakens the old cuticle. The appearance of MF in the exuvial space is accompanied by a dramatic reduction in the amplitude of the foregut contractions. Foregut peristalsis returned about halfway through the molt, followed shortly by the appearance of MF in the gut. These observations suggested that larvae use their foreguts to remove MF from the exuvial space. Animals whose foreguts were surgically inactivated did not resorb their MF and most failed to successfully shed their old cuticles. The reduction in foregut motility at the onset of the molt was correlated with a sharp decline in the amplitude of the excitatory junctional potentials. With the onset of the molt there was also a decline in the number of presynaptic terminals on the foregut that loaded with the activity-dependent dye FM1-43. In the second half of the molt, the appearance of MF in the foregut and the return of foregut motility was correlated with an increase in FM1-43 loading. These data reveal that during a larval-larval molt, vesicle release and/or recycling of the presynaptic endings on the foregut muscles is modulated to assure the proper timing of MF resorption.