Australian mosquito species significantly impact human health through nuisance biting and the transmission of endemic and exotic pathogens. Surveillance programmes designed to provide an early warning of mosquito-borne disease risk require reliable identification of mosquitoes. This study aimed to investigate the viability of Matrix-Assisted Laser Desorption/Ionization–Time-of-Flight Mass Spectrometry (MALDI-TOF MS) as a rapid and inexpensive approach to the identification of Australian mosquitoes and was validated using a three-step taxonomic approach. A total of 300 mosquitoes representing 21 species were collected from south-eastern New South Wales and morphologically identified. The legs from the mosquitoes were removed and subjected to MALDI-TOF MS analysis. Fifty-eight mosquitoes were sequenced at the cytochrome c oxidase subunit I (cox1) gene region and genetic relationships were analysed. We create the first MALDI-TOF MS spectra database of Australian mosquito species including 19 species. We clearly demonstrate the accuracy of MALDI-TOF MS for identification of Australian mosquitoes. It is especially useful for assessing gaps in the effectiveness of DNA barcoding by differentiating closely related taxa. Indeed, cox1 DNA barcoding was not able to differentiate members of the Culex pipiens group, Cx. quinquefasciatus and Cx. pipiens molestus, but these specimens were correctly identified using MALDI-TOF MS.

The anthropogenic modification of natural landscapes, and the consequent changes in the environmental conditions and resources availability at multiple spatial scales can affect complex species interactions involving key-stone species such as bat–parasite interactions. In this study, we aimed to identify the drivers potentially influencing host–bat fly interactions at different spatial scales (at the host, vegetation stand and landscape level), in a tropical anthropogenic landscape. For this purpose, we mist-netted phyllostomid and moormopid bats and collected the bat flies (streblids) parasitizing them in 10 sites representing secondary and old growth forest. In general, the variation in fly communities largely mirrored the variation in bat communities as a result of the high level of specialization characterizing host–bat fly interaction networks. Nevertheless, we observed that: (1) bats roosting dynamics can shape bat–streblid interactions, modulating parasite prevalence and the intensity of infestation; (2) a degraded matrix could favor crowding and consequently the exchange of ectoparasites among bat species, lessening the level of specialization of the interaction networks and promoting novel interactions; and (3) bat–fly interaction can also be shaped by the dilution effect, as a decrease in bat diversity could be associated with a potential increase in the dissemination and prevalence of streblids.

In the present paper, the phylogenetic relationships between genera, subfamilies and families of the Hemiuroidea are explored. Twelve new sequences of 28 rDNA and data taken from GenBank (NSBI) on 43 species affiliated to 34 genera were included in the analysis. Most of the hemiuroidean trematodes form two highly supported clades (A and B), which are sister groups to each other. Hemipera manteri joined with Gonocerca spp. with moderate statistical support. This clade is basal relative to the clades A and B. Сlade A is polytomic and contains representatives of the families Accacoeliidae, Syncoeliidae, Didymozoidae, Hirudinellidae and Sclerodistomidae, and derogenid subfamilies Derogeninae and Halipeginae. At the same time, the Syncoeliidae, Hirudinellidae and Accacoeliidae form a well-supported monophyletic group. The phylogenetic relationship between Derogeninae and Halipeginae is poorly resolved. Сlade B unites the isoparorchiid, bunocotylid, lecithasterid and hemiurid trematodes. Our data re-establishes the family Bunocotylidae, which consists of two subfamilies, Opisthadeninae and Bunocotylinae, and the Machidatrema chilostoma + Hysterolecithoides frontilatus group. The Bunocotylidae is the sister group to the Hemiuridae + Lecithasteridae group and the Isoparorchiidae is a basal relative to the representatives of these three hemiuroid families.

Angiostrongylus cantonensis is a pathogenic nematode and the cause of neuroangiostrongyliasis, an eosinophilic meningitis more commonly known as rat lungworm disease. Transmission is thought to be primarily due to ingestion of infective third stage larvae (L3) in gastropods, on produce, or in contaminated water. The gold standard to determine the effects of physical and chemical treatments on the infectivity of A. cantonensis L3 larvae is to infect rodents with treated L3 larvae and monitor for infection, but animal studies are laborious and expensive and also raise ethical concerns. This study demonstrates propidium iodide (PI) to be a reliable marker of parasite death and loss of infective potential without adversely affecting the development and future reproduction of live A. cantonensis larvae. PI staining allows evaluation of the efficacy of test substances in vitro, an improvement upon the use of lack of motility as an indicator of death. Some potential applications of this assay include determining the effectiveness of various anthelmintics, vegetable washes, electromagnetic radiation and other treatments intended to kill larvae in the prevention and treatment of neuroangiostrongyliasis.

To date, there are no recent studies identifying the prevalence of parasites of human and veterinary importance in dogs and cats in Ireland. The interaction between pets and wildlife species in the environment is an important source of parasite exposure to canids and felines, and one likely to be heightened in the stray animal population. This study aimed to establish the prevalence of endoparasites in unowned dogs and cats in County Dublin, Ireland. Feces from stray dogs (n = 627) and cats (n = 289) entering a rehoming centre were collected immediately after defecation. The main parasitic agents detected were ascarids (15.52 and 30.26%), Cystoisospora (3.27 and 3.69%), Giardia spp. (6.02 and 1.84%) and lungworms (0.64 and 2.08%), in dogs and cats respectively. Animals younger than 3 months of age were more likely to be infected with ascarids (P < 0.001) and Cystoisospora spp. (P = 0.008 and P = 0.014) than older animals. All lungworms were morphologically identified and dogs were infected with Angiostrongylus vasorum (0.48%) and Crenosoma vulpis (0.16%) whereas cats were only infected with Aelurostrongylus abstrusus (2.08%). This represents the first prevalence study of stray animals in Ireland. Data collected will inform the treatment and in addition, the future monitoring and control studies of parasite populations.

Antibodies at gastrointestinal mucosal membranes play a vital role in immunological protection against a range of pathogens, including helminths. Gastrointestinal health is central to efficient livestock production, and such infections cause significant losses. Fecal samples were taken from 114 cattle, across three beef farms, with matched blood samples taken from 22 of those animals. To achieve fecal antibody detection, a novel fecal supernatant was extracted. Fecal supernatant and serum samples were then analysed, using adapted enzyme-linked immunosorbent assay protocols, for levels of total immunoglobulin (Ig)A, IgG, IgM, and Teladorsagia circumcincta-specific IgA, IgG, IgM and IgE (in the absence of reagents for cattle-specific nematode species). Fecal nematode egg counts were conducted on all fecal samples. Assays performed successfully and showed that IgA was the predominant antibody in fecal samples, whereas IgG was predominant in serum. Total IgA in feces and serum correlated within individuals (0.581, P = 0.005), but other Ig types did not. Results support the hypothesis that the tested protocols are an effective method for the non-invasive assessment of cattle immunology. The method could be used as part of animal health assessments, although further work is required to interpret the relationship between results and levels of infection and immunity.

Taenia hydatigena, is a common parasite, lived mostly in dogs and wild carnivores in its mature stage, and the larvae, Cysticercus tenuicollis, is found on ruminants and pigs. The aim of the current study was to determine the genetic diversity in 20 isolates of the sheep and goats. After the isolation of total genomic DNA from C. tenuicollis isolates, genetic characterization of the mitochondrial NADH dehydrogenase subunit 1 gene region was amplified using specific JB11–JB12 primers in PCR and the PCR products were sequenced and haplotype and genetic diversity analyses were utilized. As a result, multiple nucleotide changes were determined in the sequence analyses of the isolates leading to detection of 16 and 15 different haplotypes in sheep and goat samples, respectively. These findings are important in terms of showing the diversity of nucleotide variation in C. tenuicollis in Turkey.

Extensive insecticide use has led to the resistance of mosquitoes to these insecticides, posing a major barrier to mosquito control. Previous Solexa high-throughput sequencing of Culex pipiens pallens in the laboratory has revealed that the abundance of a novel microRNA (miRNA), miR-13664, was higher in a deltamethrin-sensitive (DS) strain than a deltamethrin-resistant (DR) strain. Real-time quantitative PCR revealed that the miR-13664 transcript level was lower in the DR strain than in the DS strain. MiR-13664 oversupply in the DR strain increased the susceptibility of these mosquitoes to deltamethrin, whereas inhibition of miR-13664 made the DS strain more resistant to deltamethrin. Results of bioinformatic analysis, quantitative reverse-transcriptase polymerase chain reaction, luciferase assay and miR mimic/inhibitor microinjection revealed CpCYP314A1 to be a target of miR-13664. In addition, downregulation of CpCYP314A1 expression in the DR strain reduced the resistance of mosquitoes to deltamethrin. Taken together, our results indicate that miR-13664 could regulate deltamethrin resistance by interacting with CpCYP314A1, providing new insights into mosquito resistance mechanisms.

The kinetoplastid parasite Trypanosoma brucei causes African trypanosomiasis in both humans and animals. Infections place a significant health and economic burden on developing nations in sub-Saharan Africa, but few effective anti-parasitic treatments are currently available. Hence, there is an urgent need to identify new leads for drug development. The T. brucei neutral sphingomyelinase (TbnSMase) was previously established as essential to parasite survival, consequently being identified as a potential drug target. This enzyme may catalyse the single route to sphingolipid catabolism outside the T. brucei lysosome. To obtain new insight into parasite sphingolipid catabolism, the substrate specificity of TbnSMase was investigated using electrospray ionization tandem mass spectrometry (ESI-MS/MS). Recombinant TbnSMase was shown to degrade sphingomyelin, inositol-phosphoceramide and ethanolamine-phosphoceramide sphingolipid substrates, consistent with the sphingolipid complement of the parasites. TbnSMase also catabolized ceramide-1-phosphate, but was inactive towards sphingosine-1-phosphate. The broad-range specificity of this enzyme towards sphingolipid species is a unique feature of TbnSMase. Additionally, ESI-MS/MS analysis revealed previously uncharacterized activity towards lyso-phosphatidylcholine despite the enzyme's inability to degrade phosphatidylcholine. Collectively, these data underline the enzyme's importance in choline homoeostasis and the turnover of sphingolipids in T. brucei.

Trichomonas vaginalis induces cellular damage to the host cells (cytotoxicity) through the proteolytic activity of multiple proteinases of the cysteine type (CPs). Some CPs are modulated by environmental factors such as iron, zinc, polyamines, etc. Thus, the goal of this study was to assess the effect of glucose on T. vaginalis cytotoxicity, proteolytic activity and the particular role of TvCP2 (TVAG_057000) during cellular damage. Cytotoxicity assays showed that glucose-restriction (GR) promotes the highest HeLa cell monolayers destruction (~95%) by trichomonads compared to those grown under high glucose (~44%) condition. Zymography and Western blot using different primary antibodies showed that GR increased the proteolytic activity, amount and secretion of certain CPs, including TvCP2. We further characterized the effect of glucose on TvCP2. TvCP2 increases in GR, localized in vesicles close to the plasma membrane and on the surface of T. vaginalis. Furthermore, pretreatment of GR-trichomonads with an anti-TvCP2r polyclonal antibody specifically reduced the levels of cytotoxicity and apoptosis induction to HeLa cells in a concentration-dependent manner. In conclusion, our data show that GR, as a nutritional stress condition, promotes trichomonal cytotoxicity to the host cells, increases trichomonad proteolytic activity and amount of CPs, such as TvCP2 involved in cellular damage.

Hexokinase (HK) is a core glycolytic enzyme of Microsporidia which regulates host cell metabolic processes. The goal of the present study was to test for the utility of HK for molecular phylogenetics, species identification and molecular detection of microsporidia in infected insects. HK sequence-based reconstructions were essentially similar to those based upon largest subunit RNA polymerase (RPB1) gene sequences, as well as previously published rRNA gene and genome-based trees. Comparing HK sequences allowed clear differentiation of closely related taxa, such as Nosema bombycis and Nosema pyrausta. In Nosema ceranae, unique SNPs were found for an isolate from wild colonies of the Burzyan dark honey bee as compared with the isolates from domesticated European honey bee. Similarly, in Encephalitozoon cuniculi, HK was as effective as RPB1 for discrimination of isolates belonging to different ITS genotypes. Amplification using species-specific primers flanking short fragments at the 3′-end of HK gene showed the presence of infection in insect tissues infected with N. pyrausta, Nosema ceranae and Paranosema (Antonospora) locustae. For the latter parasite species, HK expression was also demonstrated at early stages of infection using total mRNA extracts of locust larvae. These results indicate the suitability of HK as a novel tool for molecular genetic studies of Microsporidia.

In the current study, phage-exposed mimotopes as targets against tegumentary leishmaniasis (TL) were selected by means of bio-panning cycles employing sera of TL patients and healthy subjects, besides the immune stimulation of peripheral blood mononuclear cells (PBMCs) collected from untreated and treated TL patients and healthy subjects. The clones were evaluated regarding their specific interferon-γ (IFN-γ) and interleukin-4 (IL-4) production in the in vitro cultures, and selectivity and specificity values were calculated, and those presenting the best results were selected for the in vivo experiments. Two clones, namely A4 and A8, were identified and used in immunization protocols from BALB/c mice to protect against Leishmania amazonensis infection. Results showed a polarized Th1 response generated after vaccination, being based on significantly higher levels of IFN-γ, IL-2, IL-12, tumour necrosis factor-α (TNF-α) and granulocyte-macrophage colony-stimulating factor (GM-CSF); which were associated with lower production of specific IL-4, IL-10 and immunoglobulin G1 (IgG1) antibodies. Vaccinated mice presented significant reductions in the parasite load in the infected tissue and distinct organs, when compared with controls. In conclusion, we presented a strategy to identify new mimotopes able to induce Th1 response in PBMCs from TL patients and healthy subjects, and that were successfully used to protect against L. amazonensis infection.

An aurantiactinomyxon type is described from the marine naidid Tubificoides pseudogaster (Dahl, 1960), collected from the lower estuary of a Northern Portuguese River. This type constitutes the first of its collective group to be reported from Portugal, and only the fourth described from a marine oligochaete worldwide. Extensive morphological comparisons of new aurantiactinomyxon isolates to all known types without available molecular data are proposed to be unnecessary, given the artificiality of the usage of morphological criteria for actinosporean differentiation and the apparent strict host specificity of the group. Recognition of naidid oligochaetes as the hosts of choice for marine types of aurantiactinomyxon and other collective groups, suggests that the family Naididae played a preponderant role in the myxosporean colonization of estuarine communities. Molecular analyses of the type in study further infer its involvement in the life cycle of Paramyxidium giardi (Cépède, 1906) Freeman and Kristmundsson, 2018, a species that infects the kidney of European eel Anguilla anguilla (Linnaeus, 1758) and that has been reported globally, including from Portuguese waters. The low intraspecific difference registered in relation to Icelandic isolates of P. giardi (0.6%) is hypothesized to result from the emergence of genotypically different subspecies due to geographic isolation.

Why some parasites evolve and maintain extreme levels of virulence is a question that remains largely unanswered. A body of theory predicts that parasites that form long-lived spores able to persist in the environment evolve higher virulence, known as the sit and wait hypothesis. Such parasites can obliterate their local host population and wait in the environment for further hosts to arrive, reducing some of the costs of high virulence. On the other hand, some models predict the opposite to be true, that virulence and environmental persistence are both costly and traded off, the resource allocation hypothesis. I conducted a meta-analysis on published data on the relationship between environmental persistence and virulence collected to date. I first examined all data available to date and then conducted a smaller analysis focussing on just those studies testing the specific predictions of the sit and wait hypothesis. Empirical work supports both hypotheses; however, the direction of the effect is largely associated with parasite type. In both analyses, viruses tend to show evidence of resource allocation trade-offs, these traits are positively correlated in bacterial and fungal parasites.

Parasitological analysis of coprolites has allowed exploring ecological relationships in ancient times. Ancient DNA analysis contributes to the identification of coprolites and their parasites. Pleistocene mammalian carnivore coprolites were recovered from paleontological and archaeological site Peñas de las Trampas 1.1 in the southern Puna of Argentina. With the aim of exploring ancient ecological relationships, parasitological analysis was performed to one of them, dated to 16 573–17 002 calibrated years BP, with 95.4% probability. Parasite eggs attributed to Toxascaris sp. by morphological characters were isolated. DNA of coprolite and eggs was extracted to molecular identification. Ancient mitochondrial DNA analysis confirmed the zoological origin of the coprolite as Puma concolor and that of parasite eggs as Toxascaris leonina. This is the oldest molecular parasite record worldwide, and it supports the presence of this parasite since the Pleistocene in America. These findings have implications for the biogeographic history of parasites and for the natural history of the region.

Apicomplexan parasites are amongst the most prevalent and morbidity-causing pathogens worldwide. They are responsible for severe diseases in humans and livestock and are thus of great public health and economic importance. Until the sequencing of apicomplexan genomes at the beginning of this century, the occurrence of N- and O-glycoproteins in these parasites was much debated. The synthesis of rudimentary and divergent N-glycans due to lineage-specific gene loss is now well established and has been recently reviewed. Here, we will focus on recent studies that clarified classical O-glycosylation pathways and described new nucleocytosolic glycosylations in Toxoplasma gondii, the causative agents of toxoplasmosis. We will also review the glycosylation of proteins containing thrombospondin type 1 repeats by O-fucosylation and C-mannosylation, newly discovered in Toxoplasma and the malaria parasite Plasmodium falciparum. The functional significance of these post-translational modifications has only started to emerge, but the evidence points towards roles for these protein glycosylation pathways in tissue cyst wall rigidity and persistence in the host, oxygen sensing, and stability of proteins involved in host invasion.

Host condition depends in large part on the quality and quantity of available food and heavily influences the outcome of parasite infection. Although parasite fitness traits such as growth rate and size may depend on host condition, whether host food quality or quantity is more important to parasite fitness and within-host interactions is poorly understood. We provided individual mosquito hosts with a standard dose of a gregarine parasite and reared mosquitoes on two food types of different quality and two quantities. We measured host size, total parasite count and area, and average size of parasites within each treatment. Food quality significantly influenced the number of parasites in a host; hosts fed a low-quality diet were infected with more parasites than those provided a high-quality diet. In addition, we found evidence of within-host competition; there was a negative relationship between parasite size and count though this relationship was dependent on host food quality. Host food quantity significantly affected total parasite area and parasite size; lower food quantity resulted in smaller parasites and reduced overall parasite area inside the host. Thus both food quality and quantity have the potential to influence parasite fitness and population dynamics.

Habitat avoidance is an anti-parasite behaviour exhibited by at-risk hosts that can minimize exposure to parasites. Because environments are often heterogeneous, host decision-making with regards to habitat use may be affected by the presence of parasites and habitat quality simultaneously. In this study we examine how the ovipositing behaviour of a cactiphilic fruit fly, Drosophila nigrospiracula, is affected by the presence of an ectoparasitic mite, Macrocheles subbadius, in conjunction with other environmental factors – specifically the presence or absence of conspecific eggs and host plant tissue. We hypothesized that the trade-off between site quality and parasite avoidance should favour ovipositing at mite-free sites even if it is of inferior quality. We found that although flies avoided mites in homogeneous environments (86% of eggs at mite-free sites), site quality overwhelmed mite avoidance. Both conspecific eggs (65% of eggs at infested sites with other Drosophila eggs) and host plant tissue (78% of eggs at infested sites with cactus) overpowered mite avoidance. Our results elucidate the context-dependent decision-making of hosts in response to the presence of parasites in variable environments, and suggest how the ecology of fear and associated trade-offs may influence the relative investment in anti-parasite behaviour in susceptible hosts.

Mesomycetozoean-induced infections (order Dermocystida, genus Amphibiocystidium) in European and North American amphibians are causing alarm. To date, the pathogenicity of these parasites in field conditions has been poorly studied, and demographic consequences on amphibian populations have not been explored. In this study, an Amphibiocystidium sp. infection is reported in a natural population of the Italian stream frog (Rana italica) of Central Italy, over a 7-year period from 2008 to 2014. Light and electron microscope examinations, as well as partial 18S rDNA sequence analysis were used to characterize the parasite. Moreover, a capture-mark-recapture study was conducted to assess the frog demographics in response to infection. Negative effects of amphibiocystidiosis on individual survival and population fitness were absent throughout the sampling period, despite the high estimates of disease prevalence. This might have been due to resistance and/or tolerance strategies developed by the frogs in response to the persistence of Amphibiocystidium infection in this system. We hypothesized that in the examined R. italica population, amphibiocystidiosis is an ongoing endemic/epidemic infection. However, ecological and host-specific factors, interacting in a synergistic fashion, might be responsible for variations in the susceptibility to Amphibiocystidium infection of both conspecific populations and heterospecific individuals of R. italica.