Timing of sub-lethal insecticide exposure determines parasite establishment success in an insect-helminth model.

Environmental toxicants are pervasive in nature, but sub-lethal effects on non-target organisms and their parasites are often overlooked. Particularly, studies on terrestrial hosts and their parasites exposed to agricultural toxicants are lacking. Here, we studied the effect of sequence and timing of sub-lethal exposures of the pyrethroid insecticide alpha-cypermethrin on parasite establishment using the tapeworm Hymenolepis diminuta and its intermediate insect host Tenebrio molitor as a model system. We exposed T. molitor to alpha-cypermethrin (LD20) before and after experimental H. diminuta infection and measured the establishment success of larval tapeworms. Also, we conducted in vitro studies quantifying the direct effect of the insecticide on parasite viability. Our results showed that there was no direct lethal effect of alpha-cypermethrin on H. diminuta cysticercoids at relevant concentrations (LD10 to LD90 of the intermediate host). However, we observed a significantly increased establishment of H. diminuta in beetles exposed to alpha-cypermethrin (LD20) after parasite infection. In contrast, parasite establishment was significantly lower in beetles exposed to the insecticide before parasite infection. Thus, our results indicate that environmental toxicants potentially impact host-parasite interactions in terrestrial systems, but that the outcome is context-dependent by enhancing or reducing parasite establishment depending on timing and sequence of exposure.

A parasitological evaluation of edible insects and their role in the transmission of parasitic diseases to humans and animals.

From 1 January 2018 came into force Regulation (EU) 2015/2238 of the European Parliament and of the Council of 25 November 2015, introducing the concept of "novel foods", including insects and their parts. One of the most commonly used species of insects are: mealworms (Tenebrio molitor), house crickets (Acheta domesticus), cockroaches (Blattodea) and migratory locusts (Locusta migrans). In this context, the unfathomable issue is the role of edible insects in transmitting parasitic diseases that can cause significant losses in their breeding and may pose a threat to humans and animals. The aim of this study was to identify and evaluate the developmental forms of parasites colonizing edible insects in household farms and pet stores in Central Europe and to determine the potential risk of parasitic infections for humans and animals. The experimental material comprised samples of live insects (imagines) from 300 household farms and pet stores, including 75 mealworm farms, 75 house cricket farms, 75 Madagascar hissing cockroach farms and 75 migrating locust farms. Parasites were detected in 244 (81.33%) out of 300 (100%) examined insect farms. In 206 (68.67%) of the cases, the identified parasites were pathogenic for insects only; in 106 (35.33%) cases, parasites were potentially parasitic for animals; and in 91 (30.33%) cases, parasites were potentially pathogenic for humans. Edible insects are an underestimated reservoir of human and animal parasites. Our research indicates the important role of these insects in the epidemiology of parasites pathogenic to vertebrates. Conducted parasitological examination suggests that edible insects may be the most important parasite vector for domestic insectivorous animals. According to our studies the future research should focus on the need for constant monitoring of studied insect farms for pathogens, thus increasing food and feed safety.

Efficacy of condensed tannins against larval Hymenolepis diminuta (Cestoda) in vitro and in the intermediate host Tenebrio molitor (Coleoptera) in vivo.

Natural anti-parasitic compounds in plants such as condensed tannins (CT) have anthelmintic properties against a range of gastrointestinal nematodes, but for other helminths such effects are unexplored. The aim of this study was to assess the effects of CT from three different plant extracts in a model system employing the rat tapeworm, Hymenolepis diminuta, in its intermediate host, Tenebrio molitor. An in vitro study examined infectivity of H. diminuta cysticercoids (excystation success) isolated from infected beetles exposed to different concentrations of CT extracts from pine bark (PB) (Pinus sps), hazelnut pericarp (HN) (Corylus avellana) or white clover flowers (WC) (Trifolium repens), in comparison with the anthelmintic drug praziquantel (positive control). In the in vitro study, praziquantel and CT from all three plant extracts had dose-dependent inhibitory effects on cysticercoid excystation. The HN extract was most effective at inhibiting excystation, followed by PB and WC. An in vivo study was carried out on infected beetles (measured as cysticercoid establishment) fed different doses of PB, HN and praziquantel. There was a highly significant inhibitory effect of HN on cysticercoid development (p=0.0002). Overall, CT showed a promising anti-cestodal effect against the metacestode stage of H. diminuta.

Parasitization by Scleroderma guani influences expression of superoxide dismutase genes in Tenebrio molitor.

Superoxide dismutase (SOD) is an antioxidant enzyme involved in detoxifying reactive oxygen species. In this study, we identified genes encoding the extracellular and intracellular copper-zinc SODs (ecCuZnSOD and icCuZnSOD) and a manganese SOD (MnSOD) in the yellow mealworm beetle, Tenebrio molitor. The cDNAs for ecCuZnSOD, icCuZnSOD, and MnSOD, respectively, encode 24.55, 15.81, and 23.14 kDa polypeptides, which possess structural features typical of other insect SODs. They showed 20-94% identity to other known SOD sequences from Bombyx mori, Musca domestica, Nasonia vitripennis, Pediculus humanus corporis, and Tribolium castaneum. Expression of these genes was analyzed in selected tissues and developmental stages, and following exposure to Escherichia coli and parasitization by Scleroderma guani. We recorded expression of all three SODs in cuticle, fat body, and hemocytes and in the major developmental stages. Relatively higher expressions were detected in late-instar larvae and pupae, compared to other developmental stages. Transcriptional levels were upregulated following bacterial infection. Analysis of pupae parasitized by S. guani revealed that expression of T. molitor SOD genes was significantly induced following parasitization. We infer that these genes act in immune response and in host-parasitoid interactions.

Sophisticated adaptations of Gregarina cuneata (Apicomplexa) feeding stages for epicellular parasitism.

BACKGROUND: Gregarines represent a very diverse group of early emerging apicomplexans, parasitising numerous invertebrates and urochordates, and are considered of little practical significance. Recently, they have gained more attention since some analyses showed that cryptosporidia are more closely related to the gregarines than to coccidia. METHODOLOGY/PRINCIPAL FINDINGS: Using a combined microscopic approach, this study points out the spectacular strategy of Gregarina cuneata for attachment to host tissue and nutrient acquisition while parasitising the intestine of yellow mealworm larvae, and reveals the unusual dynamics of cellular interactions between the host epithelium and parasite feeding stages. Trophozoites of G. cuneata develop epicellularly, attached to the luminal side of the host epithelial cell by an epimerite exhibiting a high degree of morphological variability. The presence of contractile elements in the apical region of feeding stages indicates that trophozoite detachment from host tissue is an active process self-regulated by the parasite. A detailed discussion is provided on the possibility of reversible retraction and protraction of the eugregarine apical end, facilitating eventual reattachment to another host cell in better physiological conditions. The gamonts, found in contact with host tissue via a modified protomerite top, indicate further adaptation of parasite for nutrient acquisition via epicellular parasitism while keeping their host healthy. The presence of eugregarines in mealworm larvae even seems to increase the host growth rate and to reduce the death rate despite often heavy parasitisation. CONCLUSIONS/SIGNIFICANCE: Improved knowledge about the formation of host-parasite interactions in deep-branching apicomplexans, including gregarines, would offer significant insights into the fascinating biology and evolutionary strategy of Apicomplexa. Gregarines exhibit an enormous diversity in cell architecture and dimensions, depending on their parasitic strategy and the surrounding environment. They seem to be a perfect example of a coevolution between a group of parasites and their hosts.

GpMyoF, a WD40 repeat-containing myosin associated with the myonemes of Gregarina polymorpha.

This study presents the first characterization of a WD40 repeat-containing myosin identified in the apicomplexan parasite Gregarina polymorpha. This 222.7 kDa myosin, GpMyoF, contains a canonical myosin motor domain, a neck domain with 6 IQ motifs, a tail domain containing short regions of predicted coiled-coil structure, and, most notably, multiple WD40 repeats at the C-terminus. In other proteins such repeats assemble into a beta-propeller structure implicated in mediating protein-protein interactions. Confocal microscopy suggests that GpMyoF is localized to the annular myonemes that gird the parasite cortex. Extraction studies indicate that this myosin shows an unusually tight association with the cytoskeletal fraction and can be solubilized only by treatment with high pH (11.5) or the anionic detergent sarkosyl. This novel myosin and its homologs, which have been identified in several related genera, appear to be unique to the Apicomplexa and represent the only myosins known to contain the WD40 domain. The function of this myosin in G. polymorpha or any of the other apicomplexan parasites remains uncertain.

Lack of preference for infective faeces in Hymenolepis diminuta-infected beetles (Tenebrio molitor).

The beetle-tapeworm life cycle provides a convenient system to study how host behaviour influences the probability of re-infection because initial and secondary infections can be tracked. The beetle, Tenebrio molitor, is infected with the tapeworm Hymenolepis diminuta when it ingests rat faeces containing tapeworm eggs, which upon hatching undergo five morphologically distinct stages while developing inside the beetle. In a series of preference trials, both individual and groups of previously infected beetles were exposed to baits of infective (faeces with eggs) and uninfective faeces. Beetles did not differ in the amount of time spent or in the number of occurrences at each bait type, suggesting that infected beetles show no preference for infective faeces. This may be a host adaptation to avoid further infection, parasite manipulation to avoid competition for host resources, or both. Further, once infected, beetles are no more or no less likely to become re-infected than uninfected beetles. An analysis of the mean and variance of infection suggests that some individuals are highly susceptible to and some are highly resistant to infection, with males being more variable than females. This could explain the higher load of cysticercoids observed in males.

A behavioral study of the beetle Tenebrio molitor infected with cysticercoids of the rat tapeworm Hymenolepis diminuta.

The host-parasite relationship, Tenebrio molitor-Hymenolepis diminuta, was analyzed. The learning behavior of infected and uninfected (control) beetles in a T-maze was compared. The infected beetles moved much slower in the T-maze than the controls. The infected beetles reached the same level of learning as the controls. However, they needed more trials than the controls. The effect of the infection was already distinct after the first week and even higher after the second week. This indicates that the initial phase of infection caused stress in the beetles. Longer infection did not worsen their ability to learn. Thus, the parasites clearly changed the behavior of their intermediate host and probably made them more susceptible to their final host, the rat.

The effect of Hymenolepis diminuta (Cestoda) cysticercoids on the weight change, frass production, and food intake of the intermediate host, Tenebrio molitor (Coleoptera).

Parasitism results in nutritionally related changes in hosts, often leading to altered feeding behavior. Infected hosts that increase their feeding also increase their probability of reinfection. To study this, I used a beetle (Tenebrio molitor)-tapeworm (Hymenolepis diminuta) system. Infected and uninfected male and female beetles were individually housed in vials with food. Each beetle's weight change, food intake, and frass production were measured over 24-h periods at 3, 7, 12, and 16 days postinfection. Treatment (infection) had no effect on weight change, but males lost more weight and produced more frass than females. Additionally, treatment had no effect on food consumption, but males had a higher food intake than females. These results suggest that infection status will not alter the probability of reinfection, but males will be more susceptible to infection than females. However, despite the male's greater food intake during the experimental infection period, parasite loads did not differ between males and females.

Life cycle and paratenesis of American gordiids (Nematomorpha: Gordiida).

To complete their life cycle, gordiids must make a transition from aquatic to terrestrial environments. However, epibenthic aquatic larvae and their terrestrial definitive hosts do not overlap in habitat. This has led many investigators to suggest that infections are acquired through the ingestion of insects, which become infected as aquatic larvae with gordiid cysts and subsequently carry gordiids to land. This proposed life cycle was experimentally tested using 3 common American species of gordiids: Gordius robustus, Paragordius varius, and Chordodes morgani. Cysts of all 3 species survived the metamorphosis of Tanytarsus sp., a midge. Subsequent infection trials of definitive hosts with cysts from imagos show that cysts surviving the metamorphosis of insects remained viable and free of host internal defense reaction. Data from naturally infected mayflies, Callibaetus sp., show that encystment and survival of gordiids within aquatic insects occur in nature. Paratenesis between paratenic hosts was also shown to be possible in these 3 species. This latter finding appears to indicate that cysts formed in spurious paratenic hosts may not be lost but may eventually transfer to normal paratenic hosts.

The life cycle of a horsehair worm, Gordius robustus (Nematomorpha: Gordioidea).

Aspects of the life cycle of the nematomorph Gordius robustus were investigated. Gordius robustus larvae fed to Tenebrio molitor (Coleoptera: Tenebrionidae) readily penetrated and subsequently encysted in the posterior portion of the midgut wall. Parasite development was then arrested in each infected beetle. Upon feeding these cysts to laboratory-reared Gryllus firmus, worm larvae developed to adults in the cricket hemocoel. In an additional experiment, G. robustus larvae fed to G. firmus did not develop to adults. These findings are in contrast to other studies that found direct infection to be an alternative mode of infection. This experiment is the first employing a laboratory-maintained stock of hosts to rear nematomorph worms.

Impairment of the chemical defence of the beetle, Tenebrio molitor, by metacestodes (cysticeroids) of the tapeworm, Hymenolepis diminuta.

The defensive glands of beetles, Tenebrio molitor, infected with metacestodes (cysticercoids) of Hymenolepis diminuta are everted less frequently upon stimulation, and contain less toluquinone (methylbenzoquinone) and m-cresol, than glands of uninfected controls. These differences, as shown in predation trials with wild rats, increase the likelihood that both cysticercoids and beetles will be ingested by the tapeworm's definitive host. This is the first documented case of a parasite inhibiting the chemical defence of an intermediate host, and one of only a few reports of parasite-induced manipulation of host biology supported by empirical evidence implicating facilitated parasite transmission between host species.

A NMR study of parasitized Tenebrio molitor and Hymenolepis diminuta cysticercoids.

In vivo NMR spectra of uninfected and Hymenolepis diminuta-infected Tenebrio molitor fed D-(1-13C)glucose showed that infected beetles of both sexes had a significantly higher ratio for (glycogen C1/lipid (CH2)n) than the corresponding controls. Quantitative metabolic profiles and the per cent 13C-label in metabolites, based on NMR of perchloric acid extracts, are presented for control and infected beetles fed D-(1-13C)glucose and for H. diminuta cysticercoids. Female beetles, both control and infected, contained more glycogen than their male counterparts and infected beetles of both sexes possessed less glycerophos-phocholine, but more glycogen and a higher percentage label in glucose and trehalose than their respective controls. Label was also incorporated into glycogen, succinate, acetate, alanine and lactate. Extracts of cysticercoids from beetles fed D-(1-13C)glucose contained the following labelled compounds, in order of decreasing per cent 13C label: glucose, trehalose, alanine, succinate, lactate, glycogen and acetate. In vitro cultivation experiments, employing D-(1-13C)glucose, revealed that trehalose found in cysticercoids was of parasite, and not beetle, origin.

Hymenolepis diminuta metacestodes (Cysticeroids) are unable to utilize exogenous trehalose.

Cysticercoids (metacestodes) of Hymenolepis diminuta were incubated in Tyrode's salt solution (pH 7.2) containing 1.25 mM or 40 mM trehalose for 5 or 18 hr, and the amounts of trehalose remaining in the media were determined using high-pressure liquid chromatography. No differences were detected in the amounts of trehalose remaining in control (no cysticercoids) or experimental incubations under any of the experimental conditions. Thus, cysticercoids are apparently unable to utilize trehalose present in the external medium.

Phosphate metabolites of Tenebrio molitor (Coleoptera: tenebrionidae) infected with metacestodes of Hymenolepis diminuta.

In vivo phosphorus-31 nuclear magnetic resonance (31P NMR) spectra and those of extracts of Tenebrio molitor L. infected with metacestodes of Hymenolepis diminuta R. showed modifications in the amounts of phosphorous-containing metabolites when compared with those of uninfected beetles. Infected females were more affected than infected males, having significantly more glucose-6-phosphate (Glu-6-P), glycerol-3-phosphate (Gly-3-P), and phosphorylethanolamine (PE), but less inorganic orthophosphate (Pi), glycerophosphorylethanolamine (GPE), gylcerolphosphorylcholine (GPC), phosphoarginine (PAr), and adenosine diphosphate (ADP). Infected males had more Glu-6-P and PE, but less Pi, GPE, and GPC. These changes directly reflected the phosphorylation potential of infected hosts, which increased approximately 100% and 50% in infected females and males, respectively.

A study on the life-cycle and epidemiology of Pseudanoplocephala crawfordi Baylis, 1927.

A study of the life-cycle and epidemiology of Pseudanoplocephala crawfordi Baylis, 1927 is reported. Intermediate hosts are Tenebrio obscurus, T. molitor, Carpophilus dimidiatus and Tribolium castaneum. The first three species are new host records. Development in the intermediate host takes 1-1.5 months. The completion of the life-cycle takes 2-2.5 months. Surveys of intermediate hosts, pigs, humans and rats were carried out. In Rattus norvegicus, the prevalence was as high as 22%. Epidemiological factors and suggestions for control measures are discussed.

The rostellar tegumentary cytoplasm of the metacestode of Hymenolepis diminuta (Cyclophyllidea: Cestoda).

Prior to scolex-retraction, the tegumentary syncytial cytoplasm of the presumptive rostellar region of Hymenolepis diminuta (from Tenebrio molitor kept at 26 degrees C) is indistinguishable from that of the rest of the cysticercoid. At 1 day after scolex-retraction differentiation of the rostellum has commenced, the tegumentary cytoplasm containing a small number of membrane-bound, ovoid, electron-dense granules which are absent from all other tegumentary regions of the metacestode. By 3 days after scolex-withdrawal there is a substantial increase in the number of ovoid granules within the rostellar tegumentary syncytium and the Golgi systems of the rostellar cytons are highly secretory. At this stage, although the cyst wall tissues are not fully developed, the metacestode is infective. This early and rapid development of the rostellar region presumably enables the 'adult' condition to be readily attained. In older metacestodes there is a progressive accumulation of ovoid granules within the rostellar tegumentary cytoplasm, accompanied by a decrease within the rostellar cytons. At 23 days following scolex-retraction the rostellar syncytium has the appearance of that of the adult tapeworm. The rostellar cytons also produce tegumentary discs and vesicles and are therefore regarded as homologous to the other tegumentary cytons of the metacestode.

Ultrastructure of the cyst of Hymenolepis diminuta larvae.

Electron microscopical studies of the cyst of 1-month-old H. diminuta larvae revealed considerable thickening of the superficial syncytium of tegument and formation of a large number of cavities and canals. The cytoplasm of the tegument is filled with microtubules produced by cytons of parenchymal layer which are analogs of tonofibrils. These peculiarities, together with numerous processes of the external fibrous layer, are regarded as adaptative to the changes of the hydrostatic pressure of the hemocoel of the intermediate host. The superficial syncytium is covered with microvilli and keeps polycellular cytoplasmatic bonds with cytons, which ensures energetic and plastic requirements for the stabilization of the hypertrophied syncytium and its physiological regeneration. Accumulations of fibres identical with those of the connective tissue were found in the cytons of the tegument. The cyst parenchyma consists mostly of cells with widened canals of granular endoplasmic reticulum associated with the microfibrils and production of fibrous filaments. The internal fibrous layer is produced by typical fibroblasts. The tegument of the cercomer is thinner and is considered to be less differentiated.

[Development of cysticercoid larva of Hymenolepis nana var. fraterna in Tenebrio molitor and Leucophaea maderae haemoceles (author's transl)]. / Le développement du cysticercoïde d'Hymenolepis nana var. fraterna (Cestoda: Hymenolepididae) dans la cavité générale de Tenebrio molitor (Coleoptera: Tenebrionidae) et de Leucophaea maderae (Dictyoptera: Blattidae).

When embryos of Hymenolepis nana var. fraterna are injected abdominally, they are able to reach the cysticercoid stage in the haemocele of Leucophaea maderae which naturally resist to infection by ingestion of the eggs. The haemocytic defence reaction of the cockroach and the structure of the surface of larvae are examined and compared with development in a natural host Tenebrio molitor.