A Novel Flow Cytometric Hemozoin Detection Assay for Real-Time Sensitivity Testing of Plasmodium falciparum

Resistance of Plasmodium falciparum to almost all antimalarial drugs, including the first-line treatment with artemisinins, has been described, representing an obvious threat to malaria control. In vitro antimalarial sensitivity testing is crucial to detect and monitor drug resistance. Current assays have been successfully used to detect drug effects on parasites. However, they have some limitations, such as the use of radioactive or expensive reagents or long incubation times. Here we describe a novel assay to detect antimalarial drug effects, based on flow cytometric detection of hemozoin (Hz), which is rapid and does not require any additional reagents. Hz is an optimal parasite maturation indicator since its amount increases as the parasite matures. Due to its physical property of birefringence, Hz depolarizes light, hence it can be detected using optical methods such as flow cytometry. A common flow cytometer was adapted to detect light depolarization caused by Hz. Synchronized in vitro cultures of P. falciparum were incubated for 48 hours with several antimalarial drugs. Analysis of depolarizing events, corresponding to parasitized red blood cells containing Hz, allowed the detection of parasite maturation. Moreover, chloroquine resistance and the inhibitory effect of all antimalarial drugs tested, except for pyrimethamine, could be determined as early as 18 to 24 hours of incubation. At 24 hours incubation, 50% inhibitory concentrations (IC50) were comparable to previously reported values. These results indicate that the reagent-free, real-time Hz detection assay could become a novel assay for the detection of drug effects on Plasmodium falciparum.     

Bloodmeal analysis in Culicoides midges collected near horses,donkeys and zebras in the Eastern Cape,South Africa

An upsurge in African horse sickness (AHS) in the Eastern Cape, South Africa, from 2006 led to an epidemiological reassessment of the disease there. Light trapping surveys carried out near horses, donkeys and zebras in 2014–2016 collected 39 species of Culicoides midge (Diptera: Ceratopogonidae) that are potential vectors of AHS. To establish if these midges fed on equids, DNA sequences were obtained from the gut contents of 52 female midges (35 freshly blood‐fed, 13 gravid and four parous), representing 11 species collected across 11 sites. Culicoides leucostictus fed on all three equids. Culicoides bolitinos, Culicoides imicola and Culicoides magnus fed on both horses and donkeys. Culicoides onderstepoortensis fed on donkeys, and Culicoides similis and Culicoides pycnostictus fed on zebras. Bloodmeals from cows, pigs, warthogs, impalas and a domestic dog were also identified in various species, but none of the midges tested had fed on birds. These results contribute to knowledge of the vectorial capacity of several species of Culicoides with regard to AHS in the Eastern Cape and point to potential reservoir hosts, of which donkeys, zebras and domestic dogs have previously been found to harbour AHS. Blood‐fed midges were also obtained throughout winter, indicating the potential for endemic AHS in the province.     

Systematic analysis of the long-chain components of Eubacterium lentum

The cellular long-chain component patterns of 33 strains of Eubacterium lentum were determined by gas chromatography. Two main types of long-chain component patterns were distinguished. The first (26 strains) was characterized by saturated branched-chain fatty acids (br14:0, br15:0, br16:0 and br17:0). The second (7 strains) did not contain branched-chain fatty acids and was characterized by saturated straight-chain fatty acids (11:0, 12:0, 14:0 and 16:0). Both types contained fatty aldehydes and their respective dimethyl acetals (14ald and 14dma, 16ald and 16dma). br16dma was only found in the first type. The G + C content of the DNA (Tm) of the 33 strains varied between 63.7 and 69.1 mol %. Canonical correlation analysis distinguished three subtypes within the first main type.     

Phase determination of the circadian rhythm of conidiation in heterocaryons between two out-of-phase mycelia in Neurospora crassa

Neurospora grows vegetatively as a syncytium in which multiple nuclei exist within a connected cytoplasm. Because of the ability of separate and distinct mycelia to fuse, the possibility exists of generating heterocaryotic cultures in which the nuclei and cytoplasms of two different strains are comingled into the same syncytium. We have used such heterocaryons, in which the component parts differed with respect to their circadian clock phase, to examine whether or not clock-dominant phases exist in the circadian cycle. To this end, the phase subsequent to the formation of heterocaryons by pairs of mycelial discs that are initially at different circadian phases was examined in Neurospora crassa. The resulting phase was an average of the parent phases in many cases, but was sometimes observed to correspond more closely to just one of the original parental phases. In these cases, we did not observe any dominant phases in the circadian cycle; the phase of a particular parent disc was more dominant in the heterocaryon when the proportion of the nuclei from that parent was greater in the heterocaryon. In some instances, which occurred mostly when the difference in phase of the parental discs was large, the resultant phase could not be related in a simple way to the parental phases. An interpretation based on a limit cycle model of the circadian oscillation is possible.     

Interacting forces of predation and fishing affect species’ maturation size

1. Fishing is a strong selective force and is supposed to select for earlier maturation at smaller body size. However, the extent to which fishing‐induced evolution is shaping ecosystems remains debated. This is in part because it is challenging to disentangle fishing from other selective forces (e.g., size‐structured predation and cannibalism) in complex ecosystems undergoing rapid change.
2. Changes in maturation size from fishing and predation have previously been explored with multi‐species physiologically structured models but assumed separation of ecological and evolutionary timescales. To assess the eco‐evolutionary impact of fishing and predation at the same timescale, we developed a stochastic physiologically size‐structured food‐web model, where new phenotypes are introduced randomly through time enabling dynamic simulation of species'' relative maturation sizes under different types of selection pressures.
3. Using the model, we carried out a fully factorial in silico experiment to assess how maturation size would change in the absence and presence of both fishing and predation (including cannibalism). We carried out ten replicate stochastic simulations exposed to all combinations of fishing and predation in a model community of nine interacting fish species ranging in their maximum sizes from 10 g to 100 kg. We visualized and statistically analyzed the results using linear models.
4. The effects of fishing on maturation size depended on whether or not predation was enabled and differed substantially across species. Fishing consistently reduced the maturation sizes of two largest species whether or not predation was enabled and this decrease was seen even at low fishing intensities (F = 0.2 per year). In contrast, the maturation sizes of the three smallest species evolved to become smaller through time but this happened regardless of the levels of predation or fishing. For the four medium‐size species, the effect of fishing was highly variable with more species showing significant and larger fishing effects in the presence of predation.
5. Ultimately our results suggest that the interactive effects of predation and fishing can have marked effects on species'' maturation sizes, but that, at least for the largest species, predation does not counterbalance the evolutionary effect of fishing. Our model also produced relative maturation sizes that are broadly consistent with empirical estimates for many fish species.