Postmitotic centriole disengagement and maturation leads to centrosome amplification in polyploid trophoblast giant cells.

DNA replication is normally coupled with centriole duplication in the cell cycle. Trophoblast giant cells (TGCs) of the placenta undergo endocycles resulting in polyploidy but their centriole state is not known. We used a cell culture model for TGC differentiation to examine centriole and centrosome number and properties. Before differentiation, trophoblast stem cells (TSCs) have either two centrioles before duplication or four centrioles after. We find that the average nuclear area increases approximately eight-fold over differentiation, but most TGCs do not have more than four centrioles. However, these centrioles become disengaged, acquire centrosome proteins, and can nucleate microtubules. In addition, some TGCs undergo further duplication and disengagement of centrioles, resulting in substantially higher numbers. Live imaging revealed that disengagement and separation are centriole autonomous and can occur asynchronously. Centriole amplification, when present, occurs by the standard mechanism of one centriole generating one procentriole. PLK4 inhibition blocks centriole formation in differentiating TGCs but does not affect endocycle progression. In summary, centrioles in TGC endocycles undergo disengagement and conversion to centrosomes. This increases centrosome number but to a limited extent compared with DNA reduplication.

The nucleus serves as the pacemaker for the cell cycle.

Mitosis is a dramatic process that affects all parts of the cell. It is driven by an oscillator whose various components are localized in the nucleus, centrosome, and cytoplasm. In principle, the cellular location with the fastest intrinsic rhythm should act as a pacemaker for the process. Here we traced the waves of tubulin polymerization and depolymerization that occur at mitotic entry and exit in Xenopus egg extracts back to their origins. We found that mitosis was commonly initiated at sperm-derived nuclei and their accompanying centrosomes. The cell cycle was ~20% faster at these initiation points than in the slowest regions of the extract. Nuclei produced from phage DNA, which did not possess centrosomes, also acted as trigger wave sources, but purified centrosomes in the absence of nuclei did not. We conclude that the nucleus accelerates mitotic entry and propose that it acts as a pacemaker for cell cycle.

A popular Indian clove-based mosquito repellent is less effective against Culex quinquefasciatus and Aedes aegypti than DEET.

Insect repellents are widely used as the first line of defense against mosquito bites and transmission of disease-causing agents. However, the cost of daily applications of even the most affordable and the gold standard of insect repellents, DEET, is still high for low-income populations where repellents are needed the most. An Indian clove-based homemade recipe has been presented as a panacea. We analyzed this homemade repellent and confirmed by behavioral measurements and odorant receptor responses that eugenol is the active ingredient in this formulation. Prepared as advertised, this homemade repellent is ineffective, whereas 5x more concentrated extracts from the brand most enriched in eugenol showed moderate repellency activity against Culex quinquefasciatus and Aedes aegypti. DEET showed higher performance when compared to the 5x concentrated formulation and is available in the same market at a lower price than the cost of the ingredients to prepare the homemade formulation.

Toxic Effect of Blood Feeding in Male Mosquitoes.

Blood- and sugar feeding of female mosquitoes has been frequently observed in the laboratory and in the field, but only sugar feeding of males has been reported. Here, we describe for the first time that Culex quinquefasciatus males feed on blood as well. Blood feeding easily happened on a blood-soaked cotton roll and, to a lesser extent, through a thin artificial layer. Mating history of a male specimen does not affect his blood feeding behavior. Male mosquitoes feed on blood even when they have a readily available sugar source. Nevertheless, feeding on blood reduces the survival rate of males to just a few days, as compared to more than a month for mosquitoes fed only on sugar. Comparing survival of male mosquitoes fed on blood only, sugar only, and a combination of both clearly demonstrated that mortality is not affected by malnutrition (reduced sugar levels), but rather due to ingested blood. On average male mosquitoes ingested ca. 0.5 µl of blood, i.e., about 10% of the amount of blood ingested by an engorged female. Although this unexpected observation of blood feeding in the laboratory by male mosquitoes is interesting, structural impairment prevents male feeding on vertebrate blood. In agreement with the literature, male and female proboscises and stylets were in general of similar size, but male mandibles were significantly shorter than female counterparts, thus explaining their inability to pierce through skin layers.

Multitasking roles of mosquito labrum in oviposition and blood feeding.

Reception of odorants by two main head appendages, antennae and maxillary palps, is essential for insects' survival and reproduction. There is growing evidence in the literature suggesting that the proboscis is also an olfactory appendage and its function as an additional "antenna" has been previously proposed. We surmised that movements of the labrum toward a blood vessel might be chemically oriented and, if so, there should be odorant receptors expressed in the labrum. To test this hypothesis, we first compared by quantitative PCR expression of odorant receptors (OR) from the Southern house mosquito, Culex quinquefasciatus in antennae and proboscis and, subsequently compared OR expression in various proboscis parts. Our data suggested that a receptor for the oviposition attractant, skatole, CquiOR21, was not expressed in proboscis, whereas a receptor for another oviposition attractant, 4EP (4-ethylphenol), CquiOR99, and a receptorf for the insect repellent DEET, CquiOR136, were expressed in the stylet of the proboscis, particularly in the tip of the labrum. In a dual-choice olfactometer, mosquitoes having the stylet coated with nail polish were attracted to 4EP in the same manner as the untreated mosquitoes. By contrast, in an oviposition assay, the stylet-treated mosquitoes did not discriminate 4EP from control oviposition cups, whereas the untreated mosquitoes (as well as mosquitoes having the labella coated) laid significantly more egg rafts in cups treated with 4EP. Ablation experiments confirmed that 4EP was sensed by the labrum where CquiOR99 is highly expressed. Stylet-coated, labella-coated, and untreated mosquitoes laid significantly more egg rafts in skatole-treated cups than in control cups. Likewise, coating of proboscis structures with nail polish had no effect on DEET-mediated oviposition deterrence. In a behavioral arena designed to mimic a human arm, mosquitoes showed significantly reduced probing time when blood was impregnated with 4EP, i.e., they engaged more rapidly in continuous blood feeding as compared to untreated blood. The time of engagement for feeding in skatole-containing blood vs. untreated blood did not differ significantly. Taken together, these data suggest that 4EP reception by the labrum is important not only for oviposition decisions, but also for reducing probing and initiation of blood feeding.

1-Octen-3-ol - the attractant that repels.

Since the discovery in the early 1980s that 1-octen-3-ol, isolated from oxen breath, attracts tsetse fly, there has been growing interest in exploring the use of this semiochemical as a possible generic lure for trapping host-seeking mosquitoes. Intriguingly, traps baited with 1-octen-3-ol captured significantly more females of the malaria mosquito, Anopheles gambiae, and the yellow fever mosquito, Aedes aegypti, than control traps, but failed to attract the southern house mosquito, Culex quinquefasciatus. Additionally, it has been demonstrated that this attractant is detected with enantioselective odorant receptors (ORs) expressed only in maxillary palps. On the basis of indoor behavioral assays it has even been suggested that 1-octen-3-ol might be a repellent to the southern house mosquito. Our approach was two-prong, i.e., to isolate 1-octen-3-ol-sensitive ORs expressed in maxillary palps and antennae of southern house female mosquito, and test the hypothesis that this semiochemical is a repellent. An OR with high transcript levels in maxillary palps, CquiOR118b, showed remarkable selectivity towards ( R)-1-octen-3-ol, whereas an OR expressed in antennae, CquiOR114b, showed higher preference for ( S)-1-octen-3-ol than its antipode. Repellency by a surface landing and feeding assay showed that not only racemic, but enantiopure ( R)- and ( S)-1-octen-3-ol are repellents at 1% dose thus suggesting the occurrence of other ( S)-1-octen-3-ol-sensitive OR(s). Female mosquitoes with ablated maxillary palps were repelled by 1-octen-3-ol, which implies that in addition to OR(s) in the maxillary palps, antennal OR(s) are essential for repellency activity.