Susceptibility of the Non-Targeted Crustacean Eurytemora affinis to the Endocrine Disruptor Tebufenozide: A Transcriptomic Approach.

Copepods are zooplanktonic crustaceans ubiquitously widespread in aquatic systems. Although they are not the target, copepods are exposed to a wide variety of pollutants such as insect growth regulators (IGRs). The aim of this study was to investigate the molecular response of a non-targeted organism, the copepod Eurytemora affinis, to an IGR. Adult males and females were exposed to two sub-lethal concentrations of tebufenozide (TEB). Our results indicate a sex-specific response with a higher sensitivity in males, potentially due to a differential activation of stress response pathways. In both sexes, exposure to TEB triggered similar pathways to those found in targeted species by modulating the transcription of early and late ecdysone responsive genes. Among them were genes involved in cuticle metabolism, muscle contraction, neurotransmission, and gametogenesis, whose mis-regulation could lead to moult, locomotor, and reproductive impairments. Furthermore, genes involved in epigenetic processes were found in both sexes, which highlights the potential impact of exposure to TEB on future generations. This work allows identification of (i) potential biomarkers of ecdysone agonists and (ii) further assessment of putative physiological responses to characterize the effects of TEB at higher biological levels. The present study reinforces the suitability of using E. affinis as an ecotoxicological model.

Tadalafil impacts the mechanical properties of Plasmodium falciparum gametocyte-infected erythrocytes.

Plasmodium falciparum gametocytes modify the mechanical properties of their erythrocyte host to persist for several weeks in the blood circulation and to be available for mosquitoes. These changes are tightly regulated by the plasmodial phosphodiesterase delta that decreases both the stiffness and the permeability of the infected host cell. Here, we address the effect of the phosphodiesterase inhibitor tadalafil on deformability and permeability of gametocyte-infected erythrocytes. We show that this inhibitor drastically increases isosmotic lysis of gametocyte-infected erythrocytes and impairs their ability to circulate in an in vitro model for splenic retention. These findings indicate that tadalafil represents a novel drug lead potentially capable of blocking malaria parasite transmission by impacting gametocyte circulation.

Glucose-mediated proliferation of a gut commensal bacterium promotes Plasmodium infection by increasing mosquito midgut pH.

Plant-nectar-derived sugar is the major energy source for mosquitoes, but its influence on vector competence for malaria parasites remains unclear. Here, we show that Plasmodium berghei infection of Anopheles stephensi results in global metabolome changes, with the most significant impact on glucose metabolism. Feeding on glucose or trehalose (the main hemolymph sugars) renders the mosquito more susceptible to Plasmodium infection by alkalizing the mosquito midgut. The glucose/trehalose diets promote proliferation of a commensal bacterium, Asaia bogorensis, that remodels glucose metabolism in a way that increases midgut pH, thereby promoting Plasmodium gametogenesis. We also demonstrate that the sugar composition from different natural plant nectars influences A. bogorensis growth, resulting in a greater permissiveness to Plasmodium. Altogether, our results demonstrate that dietary glucose is an important determinant of mosquito vector competency for Plasmodium, further highlighting a key role for mosquito-microbiota interactions in regulating the development of the malaria parasite.

Prevalence of Glucose-6-Phosphate Dehydrogenase Deficiency and Gametocytemia in a Pre-Elimination, Low Malaria Transmission Setting in Southern Zambia.

The WHO recommends single low-dose (SLD) primaquine as a gametocytocide to reduce Plasmodium falciparum transmission in areas of low transmission. Despite this recommendation, uptake of SLD primaquine has been low because of concerns of glucose-6-phosphate dehydrogenase (G6PD) deficiency. Individuals with G6PD deficiency can experience hemolysis when exposed to primaquine. In Southern Province, Zambia, malaria transmission has declined significantly over the past decade. Single low-dose primaquine may be an effective tool, but there is limited information on G6PD deficiency. We screened 137 residents in Macha, Southern Province, Zambia, and the prevalence of G6PD (A-) was 15%. We also revisited data collected from 2008 to 2013 in the same area and found the highest gametocyte burden among those aged 5-15 years. The findings from this study suggest that SLD primaquine targeted to school-aged children may be an effective tool to help achieve malaria elimination in southern Zambia.

An intracellular membrane protein GEP1 regulates xanthurenic acid induced gametogenesis of malaria parasites.

Gametocytes differentiation to gametes (gametogenesis) within mosquitos is essential for malaria parasite transmission. Both reduction in temperature and mosquito-derived XA or elevated pH are required for triggering cGMP/PKG dependent gametogenesis. However, the parasite molecule for sensing or transducing these environmental signals to initiate gametogenesis remains unknown. Here we perform a CRISPR/Cas9-based functional screening of 59 membrane proteins expressed in the gametocytes of Plasmodium yoelii and identify that GEP1 is required for XA-stimulated gametogenesis. GEP1 disruption abolishes XA-stimulated cGMP synthesis and the subsequent signaling and cellular events, such as Ca2+ mobilization, gamete formation, and gametes egress out of erythrocytes. GEP1 interacts with GCα, a cGMP synthesizing enzyme in gametocytes. Both GEP1 and GCα are expressed in cytoplasmic puncta of both male and female gametocytes. Depletion of GCα impairs XA-stimulated gametogenesis, mimicking the defect of GEP1 disruption. The identification of GEP1 being essential for gametogenesis provides a potential new target for intervention of parasite transmission.

Review of the Reproductive Toxicity of T-2 Toxin.

T-2 toxin, an inevitable environmental pollutant, is the most toxic type A trichothecene mycotoxin. Reproductive disruption is a key adverse effect of T-2 toxin. Herein, this paper reviews the reproductive toxicity of T-2 toxin and its mechanisms in male and female members of different species. The reproductive toxicity of T-2 toxin is evidenced by decreased fertility, disrupted structures and functions of reproductive organs, and loss of gametogenesis in males and females. T-2 toxin disrupts the reproductive endocrine axis and inhibits reproductive hormone synthesis. Furthermore, exposure to T-2 toxin during pregnancy results in embryotoxicity and the abnormal development of offspring. We also summarize the research progress in counteracting the reproductive toxicity of T-2 toxin. This review provides information toward a comprehensive understanding of the reproductive toxicity mechanisms of T-2 toxin.

Deficient autophagy in epithelial stem cells drives aging in the freshwater cnidarian Hydra.

Hydra possesses three distinct stem cell populations that continuously self-renew and prevent aging in Hydra vulgaris However, sexual animals from the H. oligactis cold-sensitive strain Ho_CS develop an aging phenotype upon gametogenesis induction, initiated by the loss of interstitial stem cells. Animals stop regenerating, lose their active behaviors and die within 3 months. This phenotype is not observed in the cold-resistant strain Ho_CR To dissect the mechanisms of Hydra aging, we compared the self-renewal of epithelial stem cells in these two strains and found it to be irreversibly reduced in aging Ho_CS but sustained in non-aging Ho_CR We also identified a deficient autophagy in Ho_CS epithelial cells, with a constitutive deficiency in autophagosome formation as detected with the mCherry-eGFP-LC3A/B autophagy sensor, an inefficient response to starvation as evidenced by the accumulation of the autophagosome cargo protein p62/SQSTM1, and a poorly inducible autophagy flux upon proteasome inhibition. In the non-aging H. vulgaris animals, the blockade of autophagy by knocking down WIPI2 suffices to induce aging. This study highlights the essential role of a dynamic autophagy flux to maintain epithelial stem cell renewal and prevent aging.

Transcription pattern of reproduction relevant genes along the brain-pituitary-gonad axis of female, male and intersex thicklip grey mullets, Chelon labrosus, from a polluted harbor.

The reproductive cycle of teleost fishes is regulated by the brain-pituitary-gonad (BPG) axis. The transcription profile of genes involved in the reproduction signalling in the BPG-axis differs in females and males during the gametogenic cycle. Impacts of endocrine disrupting chemicals on these signalling pathways in fish are known, but the participation of the BPG-axis in the development of the intersex condition is not well understood. Intersex thicklip grey mullets (Chelon labrosus) have been identified in several estuaries from the SE Bay of Biscay, revealing the presence of feminizing contaminants in the area. In previous studies, transcription patterns of genes related with steroidogenesis and gamete growth have been shown to differ among female, male and intersex mullets. However, many components of the reproduction control have not been studied yet. The aim of this study was to assess the transcription levels of target BPG-axis genes in female, male and intersex mullets captured in the polluted harbour of Pasaia, during their gametogenic cycle. After histologically examining the gonads, the transcription levels of previously sequenced target genes were measured by qPCR: kiss2, gpr54 and gnrh1 in brain, fshß and lhß in pituitary and fshr and lhr in gonads. In both females and males, brain genes were most transcribed in early gametogenesis, proving their relevance in the onset of both oogenesis and spermatogenesis. Pituitary gonadotropins in females showed upregulation as oogenesis progressed, reaching the highest transcription levels at vitellogenic stage, while in males transcript levels were constant during spermatogenesis. Transcription levels of gonadotropin receptors showed different patterns in ovaries and testes, suggesting differing function in relation to gametogenesis and maturation. Intersex mullets showed transcription levels of brain target genes similar to those observed in females at cortical alveoli stage and to those in mid spermatogenic males. In intersex testes the transcription pattern of gonadotropin receptor fshr was downregulated in comparison to non-intersex testes.

Inhibition of Plasmepsin V Activity Blocks Plasmodium falciparum Gametocytogenesis and Transmission to Mosquitoes.

Plasmodium falciparum gametocytes infect mosquitoes and are responsible for malaria transmission. New interventions that block transmission could accelerate malaria elimination. Gametocytes develop within erythrocytes and activate protein export pathways that remodel the host cell. Plasmepsin V (PMV) is an aspartyl protease that is required for protein export in asexual parasites, but its function and essentiality in gametocytes has not been definitively proven, nor has PMV been assessed as a transmission-blocking drug target. Here, we show that PMV is expressed and can be inhibited specifically in P. falciparum stage I-II gametocytes. PMV inhibitors block processing and export of gametocyte effector proteins and inhibit development of stage II-V gametocytes. Gametocytogenesis in the presence of sublethal inhibitor concentrations results in stage V gametocytes that fail to infect mosquitoes. Therefore, PMV primes gametocyte effectors for export, which is essential for the development and fitness of gametocytes for transmission to mosquitoes.

Recent advances in transmission-blocking drugs for malaria elimination.

The scientific community worldwide has realized that malaria elimination will not be possible without development of safe and effective transmission-blocking interventions. Primaquine, the only WHO recommended transmission-blocking drug, is not extensively utilized because of the toxicity issues in G6PD deficient individuals. Therefore, there is an urgent need to develop novel therapeutic interventions that can target malaria parasites and effectively block transmission. But at first, it is imperative to unravel the existing portfolio of transmission-blocking drugs. This review highlights transmission-blocking potential of current antimalarial drugs and drugs that are in various stages of clinical development. The collective analysis of the relationships between the structure and the activity of transmission-blocking drugs is expected to help in the design of new transmission-blocking antimalarials.

Validation of the protein kinase PfCLK3 as a multistage cross-species malarial drug target.

The requirement for next-generation antimalarials to be both curative and transmission-blocking necessitates the identification of previously undiscovered druggable molecular pathways. We identified a selective inhibitor of the Plasmodium falciparum protein kinase PfCLK3, which we used in combination with chemogenetics to validate PfCLK3 as a drug target acting at multiple parasite life stages. Consistent with a role for PfCLK3 in RNA splicing, inhibition resulted in the down-regulation of more than 400 essential parasite genes. Inhibition of PfCLK3 mediated rapid killing of asexual liver- and blood-stage P. falciparum and blockade of gametocyte development, thereby preventing transmission, and also showed parasiticidal activity against P. berghei and P. knowlesi Hence, our data establish PfCLK3 as a target for drugs, with the potential to offer a cure-to be prophylactic and transmission blocking in malaria.

The Xenopus tropicalis Model for Studies of Developmental and Reproductive Toxicity.

The reproductive cycle encompasses processes such as sex organ differentiation and development in the early life stages and maturation of the gametes in the adult organism. During the early life stages, critical developmental programming of the endocrine and reproductive systems occurs, and exposure to chemicals during these critical developmental windows can result in impaired reproductive function later in life. It is therefore important to evaluate long-term consequences of early life stage exposure to endocrine-disrupting chemicals. The African clawed frog Xenopus tropicalis has several characteristics that facilitate studies of developmental and reproductive toxicity. Here I present a X. tropicalis life cycle test protocol including study design, exposure regimes, and endpoints for chemical disruption of sex differentiation, gonadal and Müllerian duct development, the thyroxin-regulated metamorphosis, estrogen synthesis (activity of the CYP19 aromatase enzyme), spermatogenesis, oogenesis, puberty and fertility.

Effects of Cobalt on Spore Germination, Gametophyte Growth and Gametogenesis of Undaria pinnatifida (Phaeophyceae).

With rapid development of the construction of nuclear power plants along the coast, the concern of negative effects of potentially unexpected release of nuclides on marine organisms has increased. Cobalt (Co) is one of the crucial nuclides in nuclear polluted seawater. The effect of its presence in seawater on life cycle of macroalgae has seldom been studied. In this investigation, a series of Co concentrations including 1, 10, 100 µg L-1, and 1, 10 mg L-1 (the background concentration of Co in culture seawater was determined to be at the level of 0.75 ± 0.11 µg L-1) were used to test the effects of their presence on spore germination, gametophyte growth and gametogenesis of the important brown macroalga Undaria pinnatifida. It was found that the spore germination rate of 10 mg L-1 group was significantly lower than that of the control group after 1- and 2 days exposure. The gametophyte sizes of 1 and 10 mg L-1 groups were much smaller than that of the control group after 6- and 12-days exposure. Oogonia and juvenile sporophytes were observed to appear in 1, 10 µg L-1 and the control groups after 12 and 15 days, respectively, but not in the higher concentration groups. In the recovery test, sporophytes appeared in the 100 µg L-1 group on the 5th day, but not in 1 and 10 mg L-1 groups. These results demonstrate that presence of Co at high concentrations in seawater disturbs the life cycle by suppressing both the gametophyte growth and gametogenesis in U. pinnatifida.

Thermal manipulation and GnRHa therapy applied to the reproduction of lambari-do-rabo-amarelo, Astyanax altiparanae females (Characiformes: Characidae) during the non-breeding season.

Lambari-do-rabo-amarelo Astyanax altiparanae in the wild reproduce during spring and summer, but females undergo vitellogenesis throughout the year, including the non-spawning winter period when water temperatures are low. The present study investigated the physiological role of temperature modulation on the hypothalamus-pituitary-gonads axis of lambari during winter, as well as the effects of gonadotropin releasing hormone agonist (GnRHa) therapy. Captive females were exposed to two different temperatures (20 °C and 27 °C) and were injected weekly with GnRHa for 21 days during winter (Control, CTR; Low dose; LD and high dose of GnRHa, HD). At the end of the 21-days period gonadosomatic index (GSI), oocyte stage of development and theoretical fecundity were evaluated, together with plasma levels of 17ß-estradiol (E2). Gene expression of the two pituitary gonadotropins follicle-stimulating hormone (fshß) and luteinizing hormone (lhß), as well as hepatic vitellogenin-A (vtgA) expression were also analyzed. At the end of the experimental period, females from the six different experimental conditions were induced to spawn using human chorionic gonadotropin (hCG). Spawning performance parameters and plasma levels of the maturation inducing steroid (MIS) were analyzed. Gene expression of fshß did not change with temperature manipulation, but females exposed to 27 °C and supplemented with a HD of GnRHa exhibited an increased fshß gene expression, associated with higher E2 levels. The higher water temperature alone was able to increase E2 levels. At both water temperatures GnRHa injections induced a decrease in E2 levels. GnRHa injected females had a lower vtgA gene expression levels at 20 °C. Even with differences in the gene expression of gonadotropins among the various temperature/GnRHa treatments, GSI and oocyte diameter did not change, but GnRHa enhanced the number of vitellogenic oocytes at 20 °C. The reproductive performance of lambari induced to spawn with hCG was better after the combined treatment with GnRHa and summer temperature.

Environmental exposure to oestrogenic endocrine disruptors mixtures reflecting on gonadal sex steroids and gametogenesis of the neotropical fishAstyanax rivularis.

Discharge of municipal wastewater promotes the entry of diverse oestrogenic compounds into the water bodies. This complex mixture of substances interferes in the steroidogenic pathway, being able to promote severe reproductive impairment in freshwater fish populations. The purpose of the present study was to evaluate the effects of oestrogenic endocrine disruptors (EDCs) mixture on gonadal sex steroids (testosterone, T; 11-ketotestosterone, 11-KT; 17ß-oestradiol, E2; 17-hydroxyprogesterone, 17-OHP) in the peak of the reproductive season of Astyanax rivularis, correlating the results obtained with the proportion of germ cells and gonadal histopathology. Three sampling sites were chosen to conduct the study, one reference site (S1), without contamination by municipal wastewater and two sites (S2 and S3) receiving discharge of municipal wastewater. Males of A. rivularis presented higher concentrations of E2, lower androgens (T and 11-KT) in gonads when compared to males from site S1. Concentrations of 17-OHP did not present significant difference among sites. In sites S2 and S3, the proportion of early spermatocytes, spermatids and Leydig cells increased while spermatozoa decreased compared to fish from S1. The following gonadal histopathologies were detected in the male fishes: intersex gonads (28% in S3) and testicular degeneration with germinal epithelium exhibiting agglutinated germ cells masses and empty cysts (57% in S2 and 71% in S3). In females, concentrations of T, E2 and 17-OHP did not present significant difference among the sites, however higher 11-KT concentrations were detected in females from sites S2 and S3. A lower proportion of perinucleolar follicles and a higher incidence of vitellogenic follicles, besides, aged oocytes and the presence of eosinophilic proteinaceous fluid in the interstitial compartment were also found in females from impacted sites. These results indicate that the urbanization and consequent release of municipal wastewater containing oestrogenic compounds in the headwater creeks are altering the levels of sex hormones and gametogenesis of A. rivularis. Further studies should be performed to determine whether oestrogenic endocrine disrupters are disrupting the reproduction of A. rivularis.

Identification of Antimalarial Inhibitors Using Late-Stage Gametocytes in a Phenotypic Live/Dead Assay.

Malaria remains a major cause of morbidity and mortality worldwide with ~3.3 billion people at risk of contracting malaria and an estimated 450,000 deaths each year. While tools to reduce the infection prevalence to low levels are currently under development, additional efforts will be required to interrupt transmission. Transmission between human host and vector by the malaria parasite involves gametogenesis in the host and uptake of gametocytes by the mosquito vector. This stage is a bottleneck for reproduction of the parasite, making it a target for small-molecule drug discovery. Targeting this stage, we used whole Plasmodium falciparum gametocytes from in vitro culture and implemented them into 1536-well plates to create a live/dead phenotypic antigametocyte assay. Using specialized equipment and upon further validation, we screened ~150,000 compounds from the NIH repository currently housed at Scripps Florida. We identified 100 primary screening hits that were tested for concentration response. Additional follow-up studies to determine specificity, potency, and increased efficacy of the antigametocyte candidate compounds resulted in a starting point for initial medicinal chemistry intervention. From this, 13 chemical analogs were subsequently tested as de novo powders, which confirmed original activity from the initial analysis and now provide a point of future engagement.

Critical examination of approaches exploited to assess the effectiveness of transmission-blocking drugs for malaria.

In the absence of clinically proven vaccines and emerging resistance to common antimalarials and insecticides, the onus of interrupting the life cycle of Plasmodium falciparum, is upon the transmission-blocking drugs. Current transmission-blocking drug primaquine finds its use restricted because of associated hemolytic toxicity issues in Glucose-6-Phosphate-Dehydrogenase deficient individuals. This article provides an extensive review of the assays used by the investigators to evaluate the transmission-blocking activity of drugs. Furthermore, limitations in existing transmission-blocking assessment approaches/studies are also covered in detail. This review is expected to help in the identification of lacunae in current understanding of transmission-blocking strategies, which are hindering our efforts to develop sustainable and effective transmission-blocking interventions.

Morphological alterations in the ovaries of Amblyomma cajennense semi-engorged ticks exposed to ethanolic extract of Acmella oleracea.

The present study analyzed the effects of different concentrations of Acmella oleracea crude ethanolic extract (EEAO) on the development of germ cells from semi-engorged Amblyomma cajennense females in order to evaluate the potential of this natural chemical as a strategy to control these important ectoparasites. A hundred semi-engorged females were divided into five groups (duplicates) (10 animals/group): Control 1 (distilled water); Control 2 (solvent ethanol 50% and DMSO 1%); and Treatment I to III (3.1, 6.2, and 12.5 mg/mL of EEAO, respectively). For the exposure of the ticks to the extract was used the Adult Immersion Test. After the exposition, the ovaries were removed and submitted to histological analysis using Harris hematoxylin and aqueous eosin. The histochemical tests were performed using PAS and Bromophenol blue staining techniques, for the detection of total polysaccharides and total protein, respectively. The extract caused significant alterations in the oocytes, including changes in the shape of the cells, disorganization, and cytoplasmic vacuolation, decrease in the number of yolk granules and germ vesicle fragmentation. These alterations were more intense in the oocytes in initial developmental stages (I and II). The results obtained in this study confirm the cytotoxic potential of the ethanolic extract of A. oleracea on the germ cells of A. cajennense females, opening up the possibility to use this extract as an alternative to control these ectoparasites.

Replacement Bisphenols Adversely Affect Mouse Gametogenesis with Consequences for Subsequent Generations.

20 years ago, accidental bisphenol A (BPA) exposure caused a sudden increase in chromosomally abnormal eggs from our control mice [1]. Subsequent rodent studies demonstrated developmental effects of exposure with repercussions on adult health and fertility (e.g., [2-9]; reviewed in [10-17]). Studies in monkeys, humans, fish, and worms suggest BPA effects extend across species (e.g., [18-30]; reviewed in [31-33]). Widespread use has resulted in ubiquitous environmental contamination and human BPA exposure. Consumer concern resulted in "BPA-free" products produced using structurally similar bisphenols that are now detectable environmental and human contaminants (e.g., [34-41]). We report here studies initiated by meiotic changes mirroring our previous BPA experience and implicating exposure to BPS (a common BPA replacement) from damaged polysulfone cages. Like with BPA [1, 2, 5], our data show that exposure to common replacement bisphenols induces germline effects in both sexes that may affect multiple generations. These findings add to growing evidence of the biological risks posed by this class of chemicals. Rapid production of structural variants of BPA and other EDCs circumvents efforts to eliminate dangerous chemicals, exacerbates the regulatory burden of safety assessment, and increases environmental contamination. Our experience suggests that these environmental contaminants pose a risk not only to reproductive health but also to the integrity of the research environment. EDCs, like endogenous hormones, can affect diverse processes. The sensitivity of the germline allows us to detect effects that, although not immediately apparent in other systems, may induce variability that undermines experimental reproducibility and impedes scientific advancement.

A high throughput screen for next-generation leads targeting malaria parasite transmission.

Spread of parasite resistance to artemisinin threatens current frontline antimalarial therapies, highlighting the need for new drugs with alternative modes of action. Since only 0.2-1% of asexual parasites differentiate into sexual, transmission-competent forms, targeting this natural bottleneck provides a tangible route to interrupt disease transmission and mitigate resistance selection. Here we present a high-throughput screen of gametogenesis against a ~70,000 compound diversity library, identifying seventeen drug-like molecules that target transmission. Hit molecules possess varied activity profiles including male-specific, dual acting male-female and dual-asexual-sexual, with one promising N-((4-hydroxychroman-4-yl)methyl)-sulphonamide scaffold found to have sub-micromolar activity in vitro and in vivo efficacy. Development of leads with modes of action focussed on the sexual stages of malaria parasite development provide a previously unexplored base from which future therapeutics can be developed, capable of preventing parasite transmission through the population.