Cell size uniformity in healthy tissues suggests that control mechanisms might coordinate cell growth and division. We derived a method to assay whether cellular growth rates depend on cell size, by monitoring how variance in size changes as cells grow. Our data revealed that, twice during the cell cycle, growth rates are selectively increased in small cells and reduced in large cells, ensuring cell size uniformity. This regulation was also observed directly by monitoring nuclear growth in live cells. We also detected cell-size-dependent adjustments of G1 length, which further reduce variability. Combining our assays with chemical/genetic perturbations confirmed that cells employ two strategies, adjusting both cell cycle length and growth rate, to maintain the appropriate size. Additionally, although Rb signaling is not required for these regulatory behaviors, perturbing Cdk4 activity still influences cell size, suggesting that the Cdk4 pathway may play a role in designating the cell’s target size.

Animal cells within a tissue typically display a striking regularity in their size. To date, the molecular mechanisms that control this uniformity are still unknown. We have previously shown that size uniformity in animal cells is promoted, in part, by size-dependent regulation of G1 length. To identify the molecular mechanisms underlying this process, we performed a large-scale small molecule screen and found that the p38 MAPK pathway is involved in coordinating cell size and cell cycle progression. Small cells display higher p38 activity and spend more time in G1 than larger cells. Inhibition of p38 MAPK leads to loss of the compensatory G1 length extension in small cells, resulting in faster proliferation, smaller cell size and increased size heterogeneity. We propose a model wherein the p38 pathway responds to changes in cell size and regulates G1 exit accordingly, to increase cell size uniformity.

The cytoplasmically inherited microorganism Wolbachia pipientis behaves like a sexually selected trait in its host, the flour beetle Tribolium confusum, enhancing male fertility at the expense of female fecundity. Here we show that infected females have fewer offspring than uninfected females but infected males have a large fertility advantage over uninfected males within multiply-inseminated infected or uninfected females. The male fertility effect accelerates the spread of the Wolbachia through the host population and expands the initial opportunity for hitch-hiking of host nuclear genes. Sperm competition in a host, mediated by endosymbionts, has not been previously described.

We report the existence of postmating but prezygotic reproductive isolation within flour beetles of the genus Tribolium. Specifically, when a female of either T castaneum or T freemani is paired simultaneously with both a conspecific and a heterospecific male, virtually all of the offspring are sired by the conspecific male. In contrast, when a female of either species is paired only with a heterospecific male, she produces near normal numbers of offspring. Mate choice experiments rule out the possibility that premating reproductive isolation accounts for this phenomenon. A number of different mechanisms could explain this phenomenon of postmating but prezygotic reproductive isolation.

The uniformity of cell size in healthy tissues suggests that control mechanisms might coordinate cell growth and division. We derived a method to assay whether growth rates of individual cells 6Monitoring nuclear growth in live cells confirmed that these decreases in variance reflect a process that selectively inhibits the growth of large cells while accelerating growth of small cells. 8We also detected cell-size-dependent adjustments of G1 length, which further reduce variability.Combining our assays with chemical and genetic perturbations confirmed that cells employ two 10 strategies, adjusting both cell cycle length and growth rate, to maintain the appropriate size.Introduction:

We report a protocol for transferring the cytoplasmic endosymbiont Wolbachia pipientis from infected to uninfected hosts in the flour beetle, Tribolium confusum, by microinjection of cytoplasm from infected donor eggs into uninfected host eggs. Postinjection survivorship was limited by the halocarbon oil, which was lethal to hatching larvae but necessary to stem the backflow of cytoplasm out of injected eggs. In test crosses, approximately 39% of surviving and maturing larvae exhibited the complete reproductive incompatibility typically associated with W. pipientis in this species. However, some transfected females failed to transmit the W. pipientis to their progeny and the incompatibility could be lost in the generation following transfection. Conversely, in some cases, females that did not themselves exhibit the incompatibility had some offspring that did exhibit partial incompatibilities.Key words: microinjection, Wolbachia, flour beetle, Tribolium confusum.

We report the existence of partial reproductive isolation between two natural populations of the flour beetle, Tribolium confusum, collected in Kaduna, Nigeria and Zagreb, Croatia. When a female from either population is paired with a single sympatric or allopatric male, she produces near normal numbers of semifertile offspring. However, when females are multiply mated with allopatric and sympatric males, the sympatric males sire the large majority of offspring. When measured in offspring numbers, the mean relative fitness of allopatric males is 0.425 with Nigerian females and 0.085 with Croatian females. Thus, the reproductive isolation is reciprocal but asymmetric. Behavioural observations indicate that only a fraction of mating attempts by allopatric males are successful because females do not become quiescent as often when mounted by allopatric males. The premating isolation is also reciprocal but asymmetric: Nigerian females are more accepting of allopatric males as mates than are Croatian females. The prezygotic behavioural isolation between these two populations is different from the postmating, prezygotic isolation observed between two other species in the genus, T castaneum and T freemani. Furthermore, the T confusum interpopulation hybrids are fertile although they exhibit a weak female bias.

The lethality of halocarbon and other oils to hatching larvae of the flour beetle Tribolium confusum limits existing microinjection protocols, because postinjection survivorship is only 5.6% of the eggs injected. We report the development of an oil-free protocol that improves survivorship fivefold. We used this protocol to transfect the cytoplasmic endosymbiont Wolbachia pipientis from infected donor eggs to uninfected host eggs and observed reproductive incompatibility in 40% of the surviving, injected eggs. Compared with mock injected controls (35.9% survival) or microinjection of uninfected cytoplasm (32.3% survival), injection of Wolbachia-infected cytoplasm into host eggs significantly reduced egg-to-adult survival (25.1%).