Maria Teresa Rebelo

Torins are potent antimalarials that block replenishment of Plasmodium liver stage parasitophorous vacuole membrane proteins

Significance Plasmodium parasites have two distinct intracellular growth stages inside the mammalian host—the first stage, which is clinically silent, in liver hepatocytes, and the second, which causes the symptoms of malaria, in red blood cells. This study reports the discovery of a class of antimalarial compounds called torins, which are extremely potent inhibitors of both intracellular stages of Plasmodium. We show that torins block trafficking of liver stage parasite proteins to the physical host–parasite interface, called the parasitophorous vacuole membrane (PVM), and that without continuous trafficking of PVM-resident proteins, the parasite is subject to elimination by its host hepatocyte. Residence within a customized vacuole is a highly successful strategy used by diverse intracellular microorganisms. The parasitophorous vacuole membrane (PVM) is the critical interface between Plasmodium parasites and their possibly hostile, yet ultimately sustaining, host cell environment. We show that torins, developed as ATP-competitive mammalian target of rapamycin (mTOR) kinase inhibitors, are fast-acting antiplasmodial compounds that unexpectedly target the parasite directly, blocking the dynamic trafficking of the Plasmodium proteins exported protein 1 (EXP1) and upregulated in sporozoites 4 (UIS4) to the liver stage PVM and leading to efficient parasite elimination by the hepatocyte. Torin2 has single-digit, or lower, nanomolar potency in both liver and blood stages of infection in vitro and is likewise effective against both stages in vivo, with a single oral dose sufficient to clear liver stage infection. Parasite elimination and perturbed trafficking of liver stage PVM-resident proteins are both specific aspects of torin-mediated Plasmodium liver stage inhibition, indicating that torins have a distinct mode of action compared with currently used antimalarials.

Simple flow cytometric detection of haemozoin containing leukocytes and erythrocytes for research on diagnosis, immunology and drug sensitivity testing

BackgroundMalaria pigment (haemozoin, Hz) has been the focus of diverse research efforts. However, identification of Hz-containing leukocytes or parasitized erythrocytes is usually based on microscopy, with inherent limitations. Flow cytometric detection of depolarized Side-Scatter is more accurate and its adaptation to common bench top flow cytometers might allow several applications. These can range from the ex-vivo and in-vitro detection and functional analysis of Hz-containing leukocytes to the detection of parasitized Red-Blood-Cells (pRBCs) to assess antimalarial activity.MethodsA standard benchtop flow cytometer was adapted to detect depolarized Side-Scatter. Synthetic and Plasmodium falciparum Hz were incubated with whole blood and PBMCs to detect Hz-containing leukocytes and CD16 expression on monocytes. C5BL/6 mice were infected with Plasmodium berghei ANKA or P. berghei NK65 and Hz-containing leukocytes were analysed using CD11b and Gr1 expression. Parasitized RBC from infected mice were identified using anti-Ter119 and SYBR green I and were analysed for depolarized Side Scatter. A highly depolarizing RBC population was monitored in an in-vitro culture incubated with chloroquine or quinine.ResultsA flow cytometer can be easily adapted to detect depolarized Side-Scatter and thus, intracellular Hz. The detection and counting of Hz containing leukocytes in fresh human or mouse blood, as well as in leukocytes from in-vitro experiments was rapid and easy. Analysis of CD14/CD16 and CD11b/Gr1 monocyte expression in human or mouse blood, in a mixed populations of Hz-containing and non-containing monocytes, appears to show distinct patterns in both types of cells. Hz-containing pRBC and different maturation stages could be detected in blood from infected mice. The analysis of a highly depolarizing population that contained mature pRBC allowed to assess the effect of chloroquine and quinine after only 2 and 4 hours, respectively.ConclusionsA simple modification of a flow cytometer allows for rapid and reliable detection and quantification of Hz-containing leukocytes and the analysis of differential surface marker expression in the same sample of Hz-containing versus non-Hz-containing leukocytes. Importantly, it distinguishes different maturation stages of parasitized RBC and may be the basis of a rapid no-added-reagent drug sensitivity assay.

Identification of sarcosaprophagous Diptera species through DNA barcoding in wildlife forensics

In recent years, forensic entomology has been applied in wildlife crimes, such as neglect cases, animal cruelty and illegal poaching. Likewise in human death investigations, in which insects can help to provide information about postmortem interval (PMI) and corpse transfer, entomology may be an important source of information in animal murder suspicion. The use of insects in forensic context relies primarily on its identification at the species level. To overcome some problems of morphological determination, molecular identification has gained relevance and has been applied frequently in forensic areas. Cytochrome c oxidase I (COI) gene was adopted in DNA barcoding approach. This methodology intends to unify the DNA-based identification using a specific region of mitochondrial DNA. COI sequences have been collected into the BOLD online database, allowing the molecular identification of sequences from unknown specimens. Nonetheless, to achieve a correct identification of an unknown sample, it is necessary that sequences from species under study exist, for comparison, in online databases. Due to the geographic differences, it is of huge importance to have samples from a certain species from its distribution range. In that sense, the aim of this research is to contribute to the potential and accuracy improvement of such databases in identification of species commonly found in wildlife carcasses. A portion of COI was sequenced from 95 specimens of seven species belonging to two families of Diptera (Calliphoridae and Muscidae) found in wildlife carcasses-baited traps in Serra da Estrela (Portugal). All specimens were identified at species level with a high specimen similarity and maximum identity percentage (through BOLD Systems and GenBank online databases, respectively). We also demonstrate the correct discrimination of all species through phylogenic and sequence divergence analyses proposed in DNA barcoding studies, reinforcing the suitability of this marker.

Small Bait Traps as Accurate Predictors of Dipteran Early Colonizers in Forensic Studies

Abstract Insect carrion communities vary among habitats and over time. Concerning the dipteran early colonizers of carrion, the use of small bait traps should be accurate because the odors emitted from meat baits should contain many of the volatile organic compounds emitted from the freshly dead mammals. In addition, this kind of trap is easy to replicate and set in position in a given habitat. In the present study, small bait preferences of early Diptera carrion colonizers were examined in an urban biotope. Specifically, three baits were compared (pork muscle, pork liver, and fish flavored cat food) in respect to the number of specimens and species captured and the presence or absence of oviposition at high and low environmental temperatures. A total of 2371 specimens were trapped, primarily belonging to three insect orders, Diptera, Coleoptera, and Hymenoptera. Diptera was the predominant order, with blowflies (Calliphoridae) being the most representative family, followed by filth flies (Muscidae). The pork muscle bait was responsible for the highest number of captures and the highest diversity. The community of Diptera collected with the most efficient bait, pork muscle, was compared with the carrion communities reported in the literature from the Iberian Peninsula. Similar taxonomic species composition was found regarding Calliphoridae species. A specimen from all species morphologically identified were also identified at a molecular level using the cytochrome c oxidase I (COI) barcode region, and the sequences were submitted to online databases.

Isolation and characterization of fifteen polymorphic microsatellite loci for the citrus mealybug, Planococcus citri (Hemiptera: Pseudococcidae), and cross-amplification in two other mealybug species

1Computational Biology and Population Genomics Group, Centro de Biologia Ambiental, Departamento de Biologia Animal, Faculdade de Ciencias da Universidade de Lisboa, 1749-016 Lisboa, Portugal 2Centro de Estudos Florestais, Instituto Superior de Agronomia, 1349-017 Lisboa, Portugal 3Centro de Estudos do Ambiente e do Mar (CESAM), Departamento de Biologia Animal, Faculdade de Ciencias da Universidade de Lisboa, 1749-016 Lisboa, Portugal 4Centro de Engenharia dos Biossistemas, Instituto Superior de Agronomia, 1349-017 Lisboa, Portugal 5Department of Entomology, Agricultural Research Organization, The Volcani Center, P.O. Box 6, Bet Dagan 50250, Israel

Morphology and distribution of antennal sensilla in a mealybug parasitoid, Anagyrus sp. near pseudococci (Hymenoptera, Encyrtidae)

* Department of Terrestrial Ecology, Netherlands Institute of Ecology (NIOO-KNAW), 6700 AB Wageningen, The Netherlands ** Centro de Estudos Florestais, Instituto Superior de Agronomia, Universidade Técnica de Lisboa, 1349-017 Lisboa, Portugal *** Departamento de Biologia Animal (DBA)/Centro de Estudos do Ambiente e do Mar (CESAM), Faculdade de Ciências da Universidade de Lisboa, Campo Grande, 1749-016 Lisboa, Portugal

Chewing lice (Phthiraptera) from wild birds in southern Portugal

This study was carried out to determine chewing louse species of wild birds in the Ria Formosa Natural Park, located in southern Portugal. In addition, the hypothesis that bird age, avian migration and social behaviour have an impact on the louse prevalence was tested. Between September and December of 2013, 122 birds (belonging to 10 orders, 19 families, 31 genera and 35 species) captured in scientific ringing sessions and admitted to the Wildlife Rehabilitation and Investigation Centre of Ria Formosa were examined for lice. Twenty-six (21.3%) birds were found to be infested with at least one chewing louse species. The chewing lice identified include 18 species. Colonial birds (34.9%) and migratory birds (29.5%) had statistically significant higher prevalence than territorial birds (6.8%) and resident birds (13.1%), respectively. This paper records 17 louse species for the first time in southern Portugal: Laemobothrion maximum, Laemobothrion vulturis, Actornithophilus piceus lari, Actornithophilus umbrinus, Austromenopon lutescens, Colpocephalum heterosoma, Colpocephalum turbinatum, Eidmanniella pustulosa, Nosopon casteli, Pectinopygus bassani, Pseudomenopon pilosum, Trinoton femoratum, Trinoton querquedulae, Craspedorrhynchus platystomus, Degeeriella fulva, Falcolipeurus quadripustulatus, Lunaceps schismatus. Also a nymph of the genus Strigiphilus was collected from a Eurasian eagle-owl. These findings contribute to the knowledge of avian chewing lice from important birds areas in Portugal.

Molecular evidence of polyandry in the citrus mealybug, Planococcus citri (Hemiptera: Pseudococcidae).

The occurrence of polyandry in Planococcus citri , presumed by earlier observations of mating behavior, was confirmed using microsatellite genotyping of pools of over 400 eggs resulting from controlled crosses of one female with two males. The genetic contribution of both mated males was confirmed in 13 out of 43 crosses. In three crosses it was possible to determine that only the first male fertilized the eggs, which may be due to sperm competition or unviable sperm supply. The microsatellite analysis also allowed the confirmation of aspects of the chromosomal inheritance detected previously in cytogenetic studies in Planococcus citri , namely that only one of the alleles is transmitted by the male, indicating that the males are functionally haploid, supporting the observation of Paternal Genome Elimination (PGE) in these insects.

The Asian tiger mosquito, Aedes ( Stegomyia ) albopictus (Skuse), a vector of dengue, chikungunya and zika viruses, reaches Portugal (Diptera: Culicidae)

The Asian tiger mosquito, Aedes (Stegomyia) albopictus (Skuse), is a tropical species originally from south-eastern Asia (Hawley, 1988). It has experienced a rampant human-mediated range expansion since the 1970s to now occupy almost the whole of the tropical and subtropical areas of the world. As an eclectic haematophagous species, it attacks humans and is able to use a number of man-made and natural structures where stagnant water is present (Estrada-Franco Craig, 1995). Eggs are able to survive for extended periods of time in complete dryness and diapause over unsuitable cold season, making it an especially resilient species (Hawley, 1988).

The first record of feather mite Alloptes (Conuralloptes) calidridis Dubinin, 1951 (Acari: Alloptidae) from the Dunlin, Calidris alpina (L., 1758) (Passeriformes: Scolopacidae) in southern of Portugal

ABSTRACT A single individual of feather mite Alloptes (Conuralloptes) calidridis Dubinin, 1951 is reported from the covert feathers of a juvenile Dunlin Calidris alpina Linnaeus, 1758. A redescription of a male of this mite is given. To the authors’ knowledge, this is the first report of Alloptes (C.) calidridis in a wild bird from Portugal, expanding the geographical distribution range of this ectoparasite to Western Europe.