Female Parasites Research Articles

No pre-zygotic isolation mechanisms between Schistosoma haematobium and Schistosoma bovis parasites: From mating interactions to differential gene expression.

Species usually develop reproductive isolation mechanisms allowing them to avoid interbreeding. These preventive barriers can act before reproduction, "pre-zygotic barriers", or after reproduction, "post-zygotic barriers". Pre-zygotic barriers prevent unfavourable mating, while post-zygotic barriers determine the viability and selective success of the hybrid offspring. Hybridization in parasites and the underlying reproductive isolation mechanisms maintaining their genetic integrity have been overlooked. Using an integrated approach this work aims to quantify the relative importance of pre-zygotic barriers in Schistosoma haematobium x S. bovis crosses. These two co-endemic species cause schistosomiasis, one of the major debilitating parasitic diseases worldwide, and can hybridize naturally. Using mate choice experiments we first tested if a specific mate recognition system exists between both species. Second, using RNA-sequencing we analysed differential gene expression between homo- and hetero-specific pairing in male and female adult parasites. We show that homo- and hetero-specific pairing occurs randomly between these two species, and few genes in both sexes are affected by hetero-specific pairing. This suggests that i) mate choice is not a reproductive isolating factor, and that ii) no pre-zygotic barrier except spatial isolation "by the final vertebrate host" seems to limit interbreeding between these two species. Interestingly, among the few genes affected by the pairing status of the worms, some can be related to pathways affected during male and female interactions and may also present interesting candidates for species isolation mechanisms and hybridization in schistosome parasites.

Transcriptional profiles in Strongyloides stercoralis males reveal deviations from the Caenorhabditis sex determination model

The human and canine parasitic nematode Strongyloides stercoralis utilizes an XX/XO sex determination system, with parasitic females reproducing by mitotic parthenogenesis and free-living males and females reproducing sexually. However, the genes controlling S. stercoralis sex determination and male development are unknown. We observed precocious development of rhabditiform males in permissive hosts treated with corticosteroids, suggesting that steroid hormones can regulate male development. To examine differences in transcript abundance between free-living adult males and other developmental stages, we utilized RNA-Seq. We found two clusters of S. stercoralis-specific genes encoding predicted transmembrane proteins that are only expressed in free-living males. We additionally identified homologs of several genes important for sex determination in Caenorhabditis species, including mab-3, tra-1, fem-2, and sex-1, which may have similar functions. However, we identified three paralogs of gld-1; Ss-qki-1 transcripts were highly abundant in adult males, while Ss-qki-2 and Ss-qki-3 transcripts were highly abundant in adult females. We also identified paralogs of pumilio domain-containing proteins with sex-specific transcripts. Intriguingly, her-1 appears to have been lost in several parasite lineages, and we were unable to identify homologs of tra-2 outside of Caenorhabditis species. Together, our data suggest that different mechanisms control male development in S. stercoralis and Caenorhabditis species.

Reproductive biology of the bopyrid isopod Robinione overstreeti, a branchial parasite of the ghost shrimp Callichirus islagrande (Decapoda: Callichiridae) in the Gulf of Mexico

ABSTRACT Robinione overstreeti is a bopyrid isopod endemic to the Gulf of Mexico that inhabits the branchial chamber of the ghost shrimp Callichirus islagrande. Information on this host–parasite association is scant. This study describes population and reproductive features of R. overstreeti and its host. A total of 90 (9.97%) out of 903 individuals of C. islagrande collected from Tamiahua lagoon, Veracruz, Mexico, between April 2012 and October 2013, were parasitized by R. overstreeti. Parasite prevalence in male and female ghost shrimps was similar. However, no parasitized ovigerous females of C. islagrande were recorded, suggesting that R. overstreeti is a parasitic castrator. Host and parasite body sizes were positively correlated suggesting early host infestation by parasites and simultaneous growth of the host and parasite. Parasitic isopods frequently inhabited the same branchial chamber (n = 77) of ghost shrimps as adult male-female pairs. Females were occasionally found with supernumerary juveniles and cryptoniscus larvae in the same host individual, and rarely solitarily. Furthermore, the body size of paired adult male and female parasites was tightly correlated suggesting long-term ‘social’ monogamy in R. overstreeti. Fecundity in R. overstreeti varied between 1668 and 9884 embryos female−1 and increased with female body size.

Previously unknown behavior in parasitic cuckoo females: male-like vocalization during migratory activity

BackgroundIn the last decade, enigmatic male-like cuckoo calls have been reported several times in East Asia. These calls exhibited a combination of vocal traits of both Oriental Cuckoo (Cuculus optatus) and Common Cuckoo (Cuculus canorus) advertising calls, and some authors therefore suggested that the enigmatic calls were produced by either Common × Oriental Cuckoo male hybrids or Common Cuckoo males having a gene mutation. However, the exact identity of calling birds are still unknown.MethodsWe recorded previously unknown male-like calls from three captive Oriental Cuckoo females, and compared these calls with enigmatic vocalizations recorded in the wild as well as with advertising vocalizations of Common and Oriental Cuckoo males. To achieve this, we measured calls automatically. Besides, we video-recorded captive female emitting male-like calls, and compared these recordings with the YouTube recordings of calling males of both Common and Oriental Cuckoos to get insight into the mechanism of call production.ResultsThe analysis showed that female male-like calls recorded in captivity were similar to enigmatic calls recorded in the wild. Therefore, Oriental Cuckoo females might produce the latter calls. Two features of these female calls appeared to be unusual among birds. First, females produced male-like calls at the time of spring and autumn migratory activity and on migration in the wild. Because of this, functional significance of this call remained puzzling. Secondly, the male-like female call unexpectedly combined features of both closed-mouth (closed beak and simultaneous inflation of the ‘throat sac’) and open-mouth (prominent harmonic spectrum and the maximum neck extension observed at the beginning of a sound) vocal behaviors.ConclusionsThe Cuculus vocalizations outside the reproductive season remain poorly understood. Here, we found for the first time that Oriental Cuckoo females can produce male-like calls in that time. Because of its rarity, this call might be an atavism. Indeed, female male-like vocalizations are still known in non-parasitic tropical and apparently more basal cuckoos only. Therefore, our findings may shed light on the evolution of vocal communication in avian brood parasites.

Plasmodium transcription repressor AP2-O3 regulates sex-specific identity of gene expression in female gametocytes.

Male and female gametocytes are sexual precursor cells essential for mosquito transmission of malaria parasite. Differentiation of gametocytes into fertile gametes (known as gametogenesis) relies on the gender‐specific transcription program. How the parasites establish distinct repertoires of transcription in the male and female gametocytes remains largely unknown. Here, we report that an Apetala2 family transcription factor AP2‐O3 operates as a transcription repressor in the female gametocytes. AP2‐O3 is specifically expressed in the female gametocytes. AP2‐O3‐deficient parasites produce apparently normal female gametocytes. Nevertheless, these gametocytes fail to differentiate into fully fertile female gametes, leading to developmental arrest in fertilization and early development post‐fertilization. AP2‐O3 disruption causes massive upregulation of transcriptionally dormant male genes and simultaneously downregulation of highly transcribed female genes in the female gametocytes. AP2‐O3 targets a substantial proportion of the male genes by recognizing an 8‐base DNA motif. In addition, the maternal AP2‐O3 is removed after fertilization, which is required for the zygote to ookinete development. Therefore, the global transcriptional repression of the male genes in the female gametocytes is required for safeguarding female‐specific transcriptome and essential for the mosquito transmission of Plasmodium.

Genetic homogeneity coupled with morphometric variability suggests high phenotypic plasticity in the sea louse Caligus rogercresseyi (Boxshall and Bravo, 2000), infecting farmed salmon (Salmo salar) along a wide latitudinal range in southern Chile.

The sea louse Caligus rogercresseyi is the most important pathogen causing "caligidosis" in the Chilean salmon industry. In this study, using cox1 gene, we evaluate the genetic variation of C.rogercresseyi from farmed Salmo salar along a latitudinal range (40°-52°S) in south Chile to determine whether morphological differences are explained by genetic or environmental factors. Female parasites were randomly collected from S.salar at five farms. Body variation was examined using multivariate analyses and genetic heterogeneity was explored with AMOVA. C.rogercresseyi exhibited significant morphometric variability among sites and parasites collected from >54°S were the longest ones. Parasites did not show genetic structure among farms. Thus, C.rogercresseyi infesting salmons is panmictic along an extensive latitudinal range in south Chile. The same genetic pattern can be explained by the frequent movement of parasitized S.salar among farms in that region. Phenotypic plasticity in parasites could be explained by natural or aquaculture-mediated environment variability. C.rogercreseyi from 54°S could favor the local spread of this disease, suggesting an immediate health risk for the recent salmon industry in that region. Further research is required to confirm genetic homogeneity of this parasite along its geographical distribution using more powerful markers (e.g. SNPs).

STRONGYLOIDIASIS IN PINE BARRENS TREEFROGS (HYLA ANDERSONII).

Over a period of 4 mo, an entire collection of seven Pine Barrens treefrogs (Hyla andersonii) died or were euthanized after developing pallor, generalized edema, and coelomic effusion. Necropsy revealed large numbers of strongyloidid nematodes within the small intestines associated with a moderate mucosal hyperplasia. Strongyloides sp. parasitic females, representing a novel species, were isolated from the fixed intestinal tract. This case report represents the first full description of strongyloidiasis in a tree frog army and highlights the potential of Strongyloides spp. as a cause of rapid mortality events associated with protein-losing enteropathy in frogs.

Female common cuckoo calls dampen the mobbing intensity of great reed warbler hosts

AbstractTo avoid mobbing attacks by their hosts during egg laying, some avian brood parasites have evolved traits to visually and/or acoustically resemble predator(s) of their hosts. Prior work established that reed warblers (Acrocephalus scirpaceus), a small host species of the brood parasitic common cuckoo (Cuculus canorus), delayed returning to the nest when confronted by either the calls of the female cuckoo or that of the predatory sparrowhawk (Accipiter nisus). It remains less clear, however, whether female cuckoo calls also suppress the nest defences of larger and more aggressive hosts. Such hosts typically attack vigorously, and can even hurt the brood parasitic intruders, instead of fleeing in the face of danger. Here, we tested whether the female cuckoo calls dampen mobbing intensity in a much larger Acrocephalus host of the common cuckoo, the great reed warbler (A. arundinaceus). We presented great reed warbler pairs with female common cuckoo models at their nests without and then with playing back the female‐specific bubbling calls of the cuckoo. As controls, we tested the hosts’ responses to harmless collared dove (Streptopelia decaocto) models, also without and then with the playbacks of dove calls. We found that the playback of female brood parasite calls reduced the aggression of hosts towards the cuckoo models as compared to model presentations without female calls, but we detected no such effect of the control calls with dove models. Our results revealed that female cuckoo calls effectively suppress the antiparasitic responses of great reed warbler hosts, which could aid parasites to approach the nest undiscovered and to evade the costly attacks of this large host. Therefore, the female call can be regarded as a general part of the cuckoo's trickery repertoire for successful parasitism.

Sex-specific Separation of Plasmodium falciparum Gametocyte Populations.

Plasmodium falciparum is a unicellular eukaryotic parasite that causes malaria in humans. The parasite is spread by Anopheles mosquitoes after ingestion of sexual stage parasites known as gametocytes. Malaria transmission depends on parasites switching from the disease-causing asexual blood forms to male and female gametocytes. The current protocol allows the simultaneous isolation of male and female parasites from the same population to study this critical lifecycle stage in a sex-specific manner. We have generated a transgenic P. falciparum cell line that expresses a GFP-tagged parasite protein in female, but not male, parasites. Gametocyte production is stress induced and, through a series of steps, sexual stage parasites are enriched relative to uninfected red blood cells or red blood cells infected with asexual stage parasites. Finally, male and female gametocytes are separated by fluorescence-activated cell sorting. This protocol allows for the separation of up to 12 million live male and female parasites from the same population, which are amenable to further analysis.

An Enzyme-Linked Immunosorbent Assay for the Detection of Antibodies to Linguatula serrata in Experimentally Infected Dogs.

Background:Linguatula serrata is a causative agent of visceral and nasopharyngeal linguatulosis in humans and animals. The aim of the present study was to investigate the immune response of dogs experimentally infected by L. serrata with ELISA.Methods:Five puppies were infected by inserting the L. serrata nymphs in their nasal cavities (infected group) in the Department of Parasitology of Shahid Chamran University of Ahvaz, during 2018–2019. Three animals were kept as the non-infected control group. Blood samples were collected from the animals for seven months at approximately monthly intervals for serum preparation. Nasal samples were taken weekly from the fourth month. ELISA was designed and performed on 64 sera (24 negatives, and 40 positives) using somatic (S), and excretory-secretory (ES) antigens.Results:Overall, 100% of the animals were infected with the parasite. Based on the results of ELISA, the ES antigen (sensitivity 95% and specificity 92%) was more preferred than the S antigen (sensitivity 95% and specificity 85%). Female parasites had significant effects on the immune response. There was a significant correlation between the clinical symptoms and the presence of female parasites (P<0.05).Conclusion:The results showed a practical method for dogs’ experimental infection. ELISA method is suitable for the detection of infection at different stages of development, especially before the maturation stage of the parasite. In this regard, the ES antigen of the parasite was more immunogenic. Therefore, ELISA can be used as a serological method in the early detection and epidemiological studies of infection with L. serrata in dogs.

Comparison of some immunological parameters between untreated and ivermectin-treated onchocerciasis patients in the Nkwanta North District of Ghana

Background: Onchocerciasis (river blindness) is a neglected tropical disease that affects the skin and the eye. It is caused by a nematode parasite, Onchocerca volvulus. The disease pathology is largely attributed to host immune responses to the microfilariae. The efficacy of ivermectin (IVM) is defined by its microfilaricidal effects (clearing >60% of microfilariae within 24 h, ∼100% within 7 days) and its ability to temporarily retard the embryogenesis of the adult female parasite at least within 90 days. We sought to profile and compare immune parameters of untreated microfilaremic and IVM-treated amicrofilaremic onchocerciasis patients to identify immune factors differentially associated with microfilaremia. This will help understand the disease mechanism. Methods & Materials: Four bloodless skin biopsies were taken from the two iliac crests and two calves using the corneoscleral punch. The microfilariae emerging from the skin biopsies following overnight incubation in physiological saline in 96-well plate was quantified under the inverted light microscope. We measured white blood cell differentials for 124 adults; 53 microfilaremic (MF) and 71 ivermectin treated (1–5 rounds) seropositive amicrofilaremic (AMF) individuals 3 months following last treatment at the Nkwanta North District of Ghana. Subgroups (16 ≤ n ≤ 20) of both MF and first-time IVM-treated AMF were assayed for plasma cytokines, total serum IgE and urine histamine. Comparisons were made by Mann-Whitney U tests. Results: The geometric mean microfilariae density/milligram skin snip was 22.5 (range 5.8–56.3). Basophil count was lower (p < 0.0001) while eosinophil counts were higher (p = 0.02) for MF compared to AMF. Out of 14 cytokines (IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12, IL-13, IFN-γ, TNF-α, GM-CSF and TGFβ); three, Interleukin (IL)-13 (p = 0.002), IL-8 (p = 0.03) and Interferon-gamma (p = 0.003) were higher among MF compared to first time IVM-treated AMF. Histamine and IgE were not different between the two groups. Conclusion: IL-13, IL-8 and interferon-gamma, basophils and eosinophils differentiated between untreated and treated patients and hence these immune parameters might be contributing to the disease pathophysiology among microfilaremic onchocerciasis patients in the Nkwanta North District of Ghana.

A new paraprobiotic-based treatment for control of Haemonchus contortus in sheep

Haemonchus contortus is a critical parasite of goats and sheep. Infection by this blood-feeding gastrointestinal nematode (GIN) parasite has significant health consequences, especially in lambs and kids. The parasite has developed resistance to virtually all known classes of small molecule anthelmintics used to treat it, giving rise in some areas to multidrug resistant parasites that are very difficult to control. Thus, new anthelmintics are urgently needed. Bacillus thuringiensis (Bt) crystal protein 5B (Cry5B), a naturally occurring protein made by a bacterium widely and safely used around the world as a bioinsecticide, represents a new non-small molecule modality for treating GINs. Cry5B has demonstrated anthelmintic activities against parasites of monogastric animals, including some related to those that infect humans, but has not yet been studied in a ruminant. Here we show that H. contortus adults are susceptible to Cry5B protein in vitro. Cry5B produced in its natural form as a spore-crystal lysate against H. contortus infections in goats had no significant efficacy. However, a new Active Pharmaceutical Ingredient (API) paraprobiotic form of Cry5B called IBaCC (Inactivated Bacterium with Cytosolic Crystals), in which Cry5B crystals are encapsulated in dead Bt cell wall ghosts, showed excellent efficacy in vitro against larval stages of H. contortus and relative protein stability in bovine rumen fluid. When given to sheep experimentally infected with H. contortus as three 60 mg/kg doses, Cry5B IBaCC resulted in significant reductions in fecal egg counts (90%) and parasite burdens (72%), with a very high impact on female parasites (96% reduction). These data indicate that Cry5B IBaCC is a potent new treatment tool for small ruminants in the battle against H. contortus.

A new species of Pseudovaigamus Amado, Ho Rocha, 1984 (Cyclopoida: Ergasilidae) from the teleost Pimelodus maculatus Lacepède, 1803.

A new species of the copepod genus Pseudovaigamus Amado, Ho Rocha, 1984 is described herein based on parasitic adult females found attached to the gills of the freshwater teleost Pimelodus maculatus Lacepède, 1803, sampled in two tributaries (Veados and Paranapananema Rivers) of the Jurumirim Reservoir, Upper Paranapanema River, São Paulo State, Brazil. The new copepod was identified as an undescribed species of Pseudovaigamus because it shares with the type-species, Pseudovaigamus spinicephalus (Thatcher Robertson, 1984), the combination of first leg with 2-segmented endopod, fourth leg with 3-segmented endopod and 2-segmented exopod, and cephalothorax armed with dorsolateral stylets (or retrostylets). However, the new copepod differs from its congener in having a trifid rostral spine, retrostylets with long spatulate process, 5-segmented antennule, and caudal rami simple or lacking any distal lobe. We erected a new species, Pseudovaigamus tridentatus n. sp.. It is the first report of a Pseudovaigamus found on a freshwater fish in Brazil.

Exposure Time and age Links Parasitism, Emergence and Development of Pupal Parasitoid Dirhinus giffardii Against Bactrocera zonata

The parasitism, emergence and development of pupal parasitoid, Dirhinus giffardii (Silvestri) was assessed against the pupae of the fruit fly, Bactrocera zonata (Saunders), under laboratory conditions. The fruit fly and D. giffardii were reared in glass cages on the artificial diet, and a known number of different 1-hour (fresh), 1-day, 2-day, 3-day and 4-day old pupae were offered to the respective parasitoids for a period of 6, 12, 18 and 24 hours. It was noted that the parasitism was increased gradually with an increase in pupal age and exposure time. The highest parasitism occurred on 3-day old pupae followed by 4-day, 2-day, 1-day and 1 hour (fresh) old pupae. The studies also manifested that exposure time and host age have a significant effect on the oviposition, per female parasitism, percent parasitism, emergence and development of pupal parasitoid, D. giffardii. The average developmental time of parasitoid was recorded significantly longer in 1-hour (fresh) old pupae than in the older pupae. The study revealed that D. giffardii is a virtuous candidate for the biological control of B. zonata and the pupae of B. zonata might be the perfect host for laboratory rearing of this parasitoid.

Molecular identification of Stylops advarians (Strepsiptera: Stylopidae) in western Canada.

Strepsiptera are an enigmatic order of insects with extreme sexual dimorphism which makes it difficult to "match-up" free-living adult males with parasitic conspecific females of the Stylopidia, and free-living females of the Mengenillidae using morphological characters. Species identification is further complicated for the Stylopidia because adult females are endoparasitic and neotenic. Therefore, we used DNA sequencing of the mitochondrial cytochrome c oxidase subunit 1 gene (cox1) to confirm the species identity of adult strepsipterans that were morphologically identified as Stylops advarians. These specimens, collected from Saskatoon (Saskatchewan, Canada), included one adult male, and eight females, the latter of which had been collected from solitary bees (Andrena milwaukeensis). Also included in the analyses were three pools of first-instar larvae that had emerged from three of the females. The results of the molecular analyses revealed that all specimens had an identical cox1 sequence, and belonged to a clade, with total statistical support (bootstrap value of 100%), that contained specimens of S. advarians from New York and Maine (USA). Hence, the results were consistent with the morphological identification of S. advarians. This study demonstrates the usefulness of a molecular approach for the identification of endoparasitic adult female and larval strepsipterans, life cycle stages that lack significant morphological characters for species identification.

The Prevalence and Infectivity of Entamoebahistolytica in Baghdad Province

The present study aimed to explore the Entamoebahistolytica in 500 patients (human stool) selected from aprivate laboratory in Baghdad/Iraq during the period from 1st of April 2018 till the 31th of August, by usingthe iodine stain to detect the stages of parasite in all samples, evaluate the prevalence of the parasite betweenmales and females and to evaluate the relationship of the parasite in selected months of this study. Thisstudy showed that the total number of infected patients is 394 from 500 taken sample. The high incidence(81.13%) is occurred in August and the low incidence (76.84%)is occurred in April. This result showed nosignificant differences appeared between the chosen months of the study (P&lt;0.01). The result of this studyshowed no significant differences (p&lt;0.01) appeared between the males and females of the study, the numberof infected patients are (192 males out of 240 and 202 females out of 260).

Female Salamanders Experience Higher Parasitism Compared to Males: A Cost of Female Reproduction?

Males tend to experience higher rates of parasitism compared to females, a phenomenon associated with ecological factors, the fact that males engage in risky behaviors, and because testosterone is known to be immunosuppressive. However, females could experience higher rates of parasitism if energy is allocated from costly immune responses towards producing eggs. We used pooled data sets from laboratory experiments to investigate sex-specific differences in salamander (Plethodon cinereus) resistance to the emerging fungal pathogen Batrachochytrium dendrobatidis (“Bd”). Contrary to our predictions, we found that female salamanders had a higher prevalence of infection (∼56%) and carried a higher Bd infection burden (455 zoospores equivalents per sample) compared to male salamanders (which had a Bd infection prevalence of ∼24% and an average infection burden of 58 zoospore equivalents per sample). We also found that female reproductive investment (i.e., mass of eggs) positively correlated with Bd infection burden, suggesting that females who previously invested more into reproduction carried a higher Bd infection burden. Collectively, our findings might indicate that female salamanders experience a cost of reproduction in the form of decreased disease resistance.

Marcação, dispersão e capacidade de parasitismo de Cotesiaflavipes (CAMERON) (HYMENOPTERA: BRACONIDAE)

The objective of this study was to evaluate the biological aspects of C. flavipesyour markup, and the development of a methodology for adult capture. Sugarcane borers fed on artificial diet containing the dyes SudanRed7B and Solvent Blue 35 in concentrations of 200 and 400 ppm, vegetable oil at 200 and 400 ppm and without oil. Later, the caterpillars were parasitized in the above treatments. We evaluated the period between parasitism and larvae emergence, pupation, weight of the masses and pupae, sex ratio, adult longevity and marking. After the setting of the dye / marker suitable concentration for adult C. flavipes, we evaluated the dispersibility and parasitism of adult females under field conditions. We used the Dunnett test for the variables related to the development of larvae, pupae and adults of C. flavipesand the GLM procedure for analysis of variance and the LSMEANS to fit the media in each factor tested. Set up The SudanRed 7B dye at a concentration of 200 ppm is the best dye / concentration to mark the larval parasitoid C. flavipes after developed the methodology for capturing C. flavipes, which associated with marking, allowed evaluating the flight capacity by distance and capture these individuals accurately. It was defined bottle trap 600 as the best pet for evaluation capture and parasitism. The maximum flight distance field in individuals was 17 meters, and the captured number was increased up to five meters.80% of the traps with captured females were parasitized.

Seasonal dynamics of parasite Lernanthropus kroyeri (van Beneden, 1851) on cultured sea bass Dicentrarchus labrax (Linnaeus, 1758) from the Adriatic Sea

Due to the great economic importance of sea bass Dicentrarchus labrax (Linnaeus, 1758) in Mediterranean mariculture, the aim of this study was to explore the seasonal dynamics of the copepod parasite Lernanthropus kroyeri (van Beneden, 1851) on the gills of cultured sea bass. This is the first study of L. kroyeri on cultured sea bass from the Adriatic Sea. Sea bass (N = 457) samples were taken monthly from October 2012 to October 2014, from the same floating net cage. A significant positive correlation (r = 0.24; P < 0.0001), was found between the sea bass length and the total number of parasites, and between the fish length and egg string length of L. kroyeri (r = 0.647; P < 0.0001). Prevalence of L. kroyeri ranged from 94.4% to 100%. Female parasites dominated the population throughout the research period (72.6%), followed by males (25%) and immature parasites (2.4%). Gravid females were most dominant during the summer season, with their numbers significantly dropping in the winter. Observed seasonal dynamics of L. kroyeri reproductive patterns suggest that the winter season may be the best period for control and treatment of the infection. Furthermore, this new data may provide a basis for the development of epidemiological models with a focus on preventive measures in order to improve health management of mariculture in the Adriatic Sea, as well as in the whole Mediterranean area.

Novel Method for the Separation of Male and Female Gametocytes of the Malaria Parasite Plasmodium falciparum That Enables Biological and Drug Discovery.

We developed a flow-cytometry-based method to separate and collect cocultured male and female Plasmodium falciparum gametocytes responsible for malaria transmission. The purity of the collected cells was estimated at >97% using flow cytometry, and sorted cells were observed by Giemsa-stained thin-smear and live-cell fluorescence microscopy. The expression of validated sex-specific markers corroborated the sorting strategy. Collected male and female gametocytes were used to confirm three novel sex-specific markers by quantitative real-time PCR that were more enriched in sorted male and female gametocyte populations than existing sex-specific markers. We also applied the method as a proof-of-principle drug screen that allows the identification of drugs that kill gametocytes in a sex-specific manner. Since the developed method allowed for the separation of male and female parasites from the same culture, we observed for the first time a difference in development time between the sexes: females developed faster than males. Hence, the ability to separate male and female gametocytes opens the door to a new field of sex-specific P. falciparum gametocyte biology to further our understanding of malaria transmission.IMPORTANCE The protozoan Plasmodium falciparum causes the most severe form of human malaria. The development of sexual forms (so-called gametocytes) is crucial for disease transmission. However, knowledge of these forms is severely hampered by the paucity of sex-specific markers and the inability to extract single sex gametocytes in high purity. Moreover, the identification of compounds that specifically affect one sex is difficult due to the female bias of the gametocytes. We have developed a system that allows for the separation of male and female gametocytes from the same population. Applying our system, we show that male and female parasites mature at different rates, which might have implications for transmission. We also identified new sex-specific genes that can be used as sex markers or to unravel sex-specific functions. Our system will not only aid in the discovery of much needed gametocidal compounds, but it also represents a valuable tool for exploring malaria transmission biology.