Synthesis and Anthelmintic Activity of New Thiosemicarbazide Derivatives-A Preliminary Study.

Parasitic infections caused by different species of intestinal helminths still poses a threat to public health. There is a need to search for new, effective anthelmintic drugs. A series of novel thiosemicarbazides were synthesized and evaluated for their in vitro anthelmintic activity. The preliminary results showed that the most of synthesized compounds were very active. 4-Phenyl-1-[(1-methyl-4-nitroimidazol-2-yl)carbonyl]thiosemicarbazide and 4-(3-chlorophenyl)-1-[(1-methyl-4-nitroimidazol-2-yl)carbonyl]thiosemicarbazide showed a 100% mortality of nematodes and a high anthelmintic activity in both tested concentrations.

Photorhabdus: a tale of contrasting interactions.

Different model systems have, over the years, contributed to our current understanding of the molecular mechanisms underpinning the various types of interaction between bacteria and their animal hosts. The genus Photorhabdus comprises Gram-negative insect pathogenic bacteria that are normally found as symbionts that colonize the gut of the infective juvenile stage of soil-dwelling nematodes from the family Heterorhabditis. The nematodes infect susceptible insects and release the bacteria into the insect haemolymph where the bacteria grow, resulting in the death of the insect. At this stage the nematodes feed on the bacterial biomass and, following several rounds of reproduction, the nematodes develop into infective juveniles that leave the insect cadaver in search of new hosts. Therefore Photorhabdus has three distinct and obligate roles to play during this life-cycle: (1) Photorhabdus must kill the insect host; (2) Photorhabdus must be capable of supporting nematode growth and development; and (3) Photorhabdus must be able to colonize the gut of the next generation of infective juveniles before they leave the insect cadaver. In this review I will discuss how genetic analysis has identified key genes involved in mediating, and regulating, the interaction between Photorhabdus and each of its invertebrate hosts. These studies have resulted in the characterization of several new families of toxins and a novel inter-kingdom signalling molecule and have also uncovered an important role for phase variation in the regulation of these different roles.

Description, molecular characterization and life cycle of Serpentirhabdias mussuranae n. sp. (Nematoda: Rhabdiasidae) from Clelia clelia (Reptilia: Colubroidea) in Brazil.

Serpentirhabdias mussuranae n. sp. is described from the lungs of the mussurana, Clelia clelia (Daudin, 1803), from vicinities of Lábrea, Amazonas State, Brazil. The species is characterized by the triangular oral opening, the presence of teeth (onchia) in the oesophastome, the excretory glands longer than the oesophagus and the tail abruptly narrowing in its anterior half and gradually tapering in posterior half. Among the Neotropical representatives of the genus, three species are known to possess the onchia in the oesophastome: S. atroxi, S. moi and S. viperidicus. Serpentirhabdias mussuranae n. sp. differs from S. atroxi and S. viperidicus by its triangular shape of the oral opening and the oesophastome in apical view, vs. round in the latter two congeners. Additionally, S. viperidicus has a larger oesophastome, 13-22 micrometers wide and 13-23 micrometers deep. The new species has relatively longer excretory glands than S. moi. The new species is morphologically and genetically close to S. atroxi, S. moi and S. viperidicus, all parasitic in Brazilian snakes, based on the presence of onchia and the comparison of nucleotide sequences of nuclear ribosomal DNA and mitochondrial cox1 gene (differences varied between 3.8% and 7.1%). Data on the life cycle of S. mussuranae n. sp. is provided, and the life cycle is typical of the genus Serpentirhabdias, with the combination of direct development and heterogony. Free-living larval stages and the adults of amphimictic free-living generation are described. The results of molecular phylogenetic analysis based on nuclear ribosomal internal transcribed spacer (ITS) + partial 28S region and partial mitochondrial cox1 gene are provided.

Evaluation and entomopathogenicity of gut bacteria associated with dauer juveniles of Oscheius chongmingensis (Nematoda: Rhabditidae).

The nematodes of genus Oscheius are insect parasites with a potential role as biological control agents. The composition of gut microbiota and its potential assistant role in the complex pathogenic mechanism of nematodes have been poorly illustrated. In this study, the intestinal bacteria associated with dauer juveniles of the nematode Oscheius chongmingensis Tumian were classified by 16S rDNA high-throughput sequencing. The raw reads were assigned to 845 operational taxonomic units (OTUs) after quality filtering. The results showed that the genus Ochrobactrum, with a proportion of 59.82%, was the most abundant genus, followed by 7.13% Bacillus, 4.7% Albidiferax, 4.26% Acinetobacter, and 3.09% Rhodococcus. The two dominant bacteria, Ochrobactrum and Bacillus, were further isolated by culturing on NBTA and LB medium respectively, and then identified as Ochrobactrum tritici and Bacillus cereus by morphological and 16S rDNA sequence analysis. Furthermore, the entomopathogenicity of these two bacterial species was studied. The results showed that O. tritici caused 93.33% mortality within 144 hr in the 4th -instar larvae of Galleria mellonella treated with 2 × 109  CFU/ml, whereas B. cereus showed 100% mortality at a concentration of 3.3 × 107  CFU/ml within 48 hr. These findings, especially the presence of O. tritici, which had not been found in other nematode species in the genus Oscheius, indicate that the associated nematode O. chongmingensis may have particular utility as a biocontrol agent.

Development of Phasmarhabditis hermaphrodita (and members of the Phasmarhabditis genus) as new genetic model nematodes to study the genetic basis of parasitism.

The genetic mechanisms of how free-living nematodes evolved into parasites are unknown. Current genetic model nematodes (e.g. Caenorhabditis elegans) are not well suited to provide the answer, and mammalian parasites are expensive and logistically difficult to maintain. Here we propose the terrestrial gastropod parasite Phasmarhabditis hermaphrodita as a new alternative to study the evolution of parasitism, and outline the methodology of how to keep P. hermaphrodita in the lab for genetic experiments. We show that P. hermaphrodita (and several other Phasmarhabditis species) are easy to isolate and identify from slugs and snails from around the UK. We outline how to make isogenic lines using 'semi-natural' conditions to reduce in-lab evolution, and how to optimize growth using nematode growth media (NGM) agar and naturally isolated bacteria. We show that P. hermaphrodita is amenable to forward genetics and that unc and sma mutants can be generated using formaldehyde mutagenesis. We also detail the procedures needed to carry out genetic crosses. Furthermore, we show natural variation within our Phasmarhabditis collection, with isolates displaying differences in survival when exposed to high temperatures and pH, which facilitates micro and macro evolutionary studies. In summary, we believe that this genetically amenable parasite that shares many attributes with C. elegans as well as being in Clade 5, which contains many animal, plant and arthropod parasites, could be an excellent model to understand the genetic basis of parasitism in the Nematoda.

Heterorhabditis pakistanense n. sp. (Nematoda: Heterorhabditidae) a new entomopathogenic nematode from Pakistan.

A new entomopathogenic nematode species of Heterorhabditis, described as H. pakistanense n. sp., was isolated from soil samples around the roots of grass at Malir, Karachi, Sindh, Pakistan. The new species is characterized morphologically by features of males: body size 819 µm (720-1013 µm), D% ((distance from anterior end to excretory pore divided by pharynx length) × 100) 119 (110-126), SW% ((spicule length divided by anal body diameter) × 100) 156 (144-191), GS% ((gubernaculum length divided by spicule length) × 100) 58 (48-65) and variations in the number of bursal papillae of the terminal group: 8th and 9th papillae sometimes absent on both sides, sometimes eight papillae present on the right side whereas six papillae present on the left side. On the right side the arrangement of papillae is 1 + 2 + 3 + 2 whereas on the left side it is 1 + 2 + 3. The hermaphrodite has a prominent post-anal swelling and a conoid tail 82 µm (64-95 µm) long with a pointed terminus. Hermaphrodites of H. pakistanense n. sp. can be distinguished from all species of Heterorhabditis except H. downesi by having a mucronate tail. Infective juveniles have a medium-sized body (581 µm (558-624 µm)), long pharynx (117 µm (113-125 µm)), ensheathed tail (99 µm (95-110 µm)) and E% ((distance from anterior end to excretory pore divided by tail length) × 100) 100 (95-107). The new species can be distinguished from all species of Heterorhabditis by the absence of the 7th, 8th and 9th bursal papillae. Heterorhabditis pakistanense is further characterized by the internal transcribed spacer (ITS) and the D2D3 region of the 28S rDNA gene. The closest species H. indica, H. gerrardi, H. amazonensis and H. noenieputensis being separated by 9, 7, 66 and 15 bp, respectively, in the ITS region. Molecular phylogenetic trees based on sequences of ITS rDNA, D2D3 regions and the mitochondrial 12S rRNA gene support the description of H. pakistanense as a new species.

Alarmone (p)ppGpp regulates the transition from pathogenicity to mutualism in Photorhabdus luminescens.

The enteric gamma-proteobacterium Photorhabdus luminescens kills a wide range of insects, whilst also maintaining a mutualistic relationship with soil nematodes from the family Heterorhabditis. Pathogenicity is associated with bacterial exponential growth, whilst mutualism is associated with post-exponential (stationary) phase. During post-exponential growth, P. luminescens also elaborates an extensive secondary metabolism, including production of bioluminescence, antibiotics and pigment. However, the regulatory network that controls the expression of this secondary metabolism is not well understood. The stringent response is a well-described global regulatory system in bacteria and mediated by the alarmone (p)ppGpp. In this study, we disrupted the genes relA and spoT, encoding the two predicted (p)ppGpp synthases of P. luminescens TTO1, and we showed that (p)ppGpp is required for secondary metabolism. Moreover, we found the (p)ppGpp is not required for pathogenicity of P. luminescens, but is required for bacterial survival within the insect cadaver. Finally, we showed that (p)ppGpp is required for P. luminescens to support normal nematode growth and development. Therefore, the regulatory network that controls the transition from pathogenicity to mutualism in P. luminescens requires (p)ppGpp. This is the first report outlining a role for (p)ppGpp in controlling the outcome of an interaction between a bacteria and its host.

Characterization of an isolate of Heterorhabditis bacteriophora (Nematoda: Heterorhabditidae) from the Northern Territory, Australia, using morphology and molecular data.

An entomopathogenic nematode, Heterorhabditis H39, was found in Darwin, Australia. Based on morphological and morphometric similarities, and molecular characterisation, it is an isolate of Heterorhabditis bacteriophora. Males, hermaphrodites, females and juveniles showed important similarities on most characters that define H. bacteriophora. The morphometrics of the infective juvenile of Heterorhabditis H39 are similar to those of H. bacteriophora, including average body length (562 (537-587) vs 570 (520-600) µm), maximum body width (21 (19-22) vs 24 (21-31) µm), distance from the anterior end to the EP (96 (87-104) vs 104 (94-109) µm) and tail length (101 (94-111) vs 91 (83-99) µm). The morphology of the spicules and gubernaculum of male Heterorhabditis H39 are indistinguishable from those of H. bacteriophora. The biology and life cycle of Heterorhabditis H39 are similar to those of other Heterorhabditis species. The Neighbour-Joining Tree based on 475 nucleotides of the SSU rRNA gene showed that Heterorhabditis H39 formed a monophyletic group with other H. bacteriophora isolates with a bootstrap value of 100. Thus, phylogenetic study of SSU sequence data provided strong evidence that Heterorhabditis H39 is an isolate of H. bacteriophora. This is the first record of H. bacteriophora in northern Australia.

In vivo and in vitro rearing of entomopathogenic nematodes (Steinernematidae and Heterorhabditidae).

Entomopathogenic nematodes (EPN) (Steinernematidae and Heterorhabditidae) have a mutualistic partnership with Gram-negative Gamma-Proteobacteria in the family Enterobacteriaceae. Xenorhabdus bacteria are associated with steinernematids nematodes while Photorhabdus are symbionts of heterorhabditids. Together nematodes and bacteria form a potent insecticidal complex that kills a wide range of insect species in an intimate and specific partnership. Herein, we demonstrate in vivo and in vitro techniques commonly used in the rearing of these nematodes under laboratory conditions. Furthermore, these techniques represent key steps for the successful establishment of EPN cultures and also form the basis for other bioassays that utilize these organisms for research. The production of aposymbiotic (symbiont-free) nematodes is often critical for an in-depth and multifaceted approach to the study of symbiosis. This protocol does not require the addition of antibiotics and can be accomplished in a short amount of time with standard laboratory equipment. Nematodes produced in this manner are relatively robust, although their survivorship in storage may vary depending on the species used. The techniques detailed in this presentation correspond to those described by various authors and refined by P. Stock's Laboratory, University of Arizona (Tucson, AZ, USA). These techniques are distinct from the body of techniques that are used in the mass production of these organisms for pest management purposes.

The effect of initial dose on the recovery and final yields of Heterorhabditis megidis (Rhabditida: Heterorhabditidae) in larvae of the great wax moth, Galleria mellonella.

The aim of this study was to determine the effect of different initial doses of the infective juveniles (IJs) (50 IJs, 200 IJs, 1000 IJs) of Heterorhabditis megidis Poinar (Rhabditida: Heterorhabditidae) strain IsM15/09 on recovery, final yields and percent final yields in larvae Galleria mellonella ( L.). Percent recovery was not directly related to initial dose. Final yields also did not change with the initial dose. However, percent yields was highly negatively correlated with initial dose of nematodes and was the highest with the 50 IJs dose. Additional point of the study was to investigate whether the nematodes are able to produce progeny from one hermaphroditic individual. The results showed that the invasive larvae resumed growth and transformed into hermaphroditic individuals that reproduced without cross-fertilisation.

Molecular characterisation of the recovery process in the entomopathogenic nematode Heterorhabditis bacteriophora.

In Heterorhabditis bacteriophora, an insect-parasitic nematode, the third juvenile is the infective, developmentally arrested form. When it infects a suitable host, the infective juvenile recovers from developmental arrest and resumes growth and development. This process is called recovery and it is the first outcome of the host-parasite interaction. Recovery is also very important from a commercial point of view. To characterise the recovery in H. bacteriophora, we sought to identify genes involved in this process. A large-scale bioassay for recovery was established and subtraction libraries of recovering infective juvenile from arrested infective juvenile transcripts were constructed at different time points. Most of the genes identified as differentially expressed between recovering and developmentally arrested infective juveniles belonged to metabolic pathways. Elevated expression levels of 23 selected genes during recovery were confirmed by quantitative PCR. For eight of these genes, transcription silencing in H. bacteriophora resulted in a significant decline in infective juvenile recovery rates, suggesting that these genes are critical to the recovery process. Two of the genes were associated with the insulin-like growth factor-1 (insulin/IGF-1) pathway, known to regulate dauer formation in the free-living nematode Caenorhabditis elegans, whereas the other six genes were associated with pathways not previously associated with recovery in nematodes. These results suggest that although little is known about parasitism-unique genes, the pathways regulating recovery in H. bacteriophora include those activated in C. elegans and those that might be unique to parasitic nematodes; the latter may be activated in response to host signals and enable the parasite to recognise its host.

Selective breeding for desiccation tolerance in liquid culture provides genetically stable inbred lines of the entomopathogenic nematode Heterorhabditis bacteriophora.

The entomopathogenic nematode (EPN) Heterorhabditis bacteriophora is used in biological plant protection to control pest insects. In the past, several attempts targeted at an enhancement of the desiccation tolerance of EPN by genetic selection in order to improve their storage stability. The subsequent loss of improved beneficial traits after release of selection pressure has often been reported. In order to stabilize progress of selective breeding, selection during liquid culturing was tested against propagation in host insects. After release of the selection pressure, the tolerance was monitored over additional reproductive cycles in vivo and in vitro to compare the stability of the trait. Furthermore, it was tested whether the virulence of the selected strains would be impaired. Exposure to desiccation stress prior to propagation, in vivo or in vitro, both resulted in increasing desiccation tolerance. When selection pressure was released, the gained tolerance was lost again during in vivo production, whereas the tolerance was maintained at a high level when EPNs were cultured in liquid culture. In Heterorhabditis sp., liquid culture conditions produce highly homozygous, genetically stable inbred lines. The investigation provides easily applicable methods to improve and stabilize beneficial traits of heterorhabditid EPNs through selective breeding in liquid culture. Compared to nematodes from in vivo propagation, production in liquid media yielded EPN of higher virulence.

Review of Rhabdiasidae (Nematoda) from the Holarctic.

The review deals with the species of Rhabdiasidae Railliet, 1915 occurring in the Holarctic. Descriptions of 26 species from the genera Rhabdias Stiles et Hassal, 1905 (21 species), Entomelas Travassos, 1930 (4 species), and KurolonemaSzczerbak et Sharpilo, 1969 (1 species) are presented. Additionally, 5 species of Rhabdias are listed as species inquirenda, and 4 species are included into the review, since they have been reported close to south-eastern border of Palaearctic (the exact distribution is unknown). Keys to the Holarctic species and diagnoses of the genera are provided. An overview of the information on the biology, morphology and taxonomy of Rhabdiasidae is also presented, as well as the host-parasite list of Holarctic Rhabdiasidae.

Regulation of sexual plasticity in a nematode that produces males, females, and hermaphrodites.

The mechanisms by which new modes of reproduction evolve remain important unsolved puzzles in evolutionary biology. Nematode worms are ideal for studying the evolution of mating systems because the phylum includes both a large range of reproductive modes and large numbers of evolutionarily independent switches [1, 2]. Rhabditis sp. SB347, a nematode with sexual polymorphism, produces males, females, and hermaphrodites [3]. To understand how the transition between mating systems occurs, we characterized the mechanisms that regulate female versus hermaphrodite fate in Rhabditis sp. SB347. Hermaphrodites develop through an obligatory nonfeeding juvenile stage, the dauer larva. Here we show that by suppressing dauer formation, Rhabditis sp. SB347 develops into females. Conversely, larvae that under optimal growth conditions develop into females can be respecified toward hermaphroditic development if submitted to dauer-inducing conditions. These results are of significance to understanding the evolution of complex mating systems present in parasitic nematodes.

Submerged monoxenic culture medium development for Heterorhabditis bacteriophora and its symbiotic bacterium Photorhabdus luminescens: protein sources.

Most medium formulations for improving culture of entomopathogenic nematodes (EPN) based on protein sources have used enriched media like animal feed such as dried egg yolk, lactalbumin, and liver extract, among other ingredients. Most results, however, showed unstable yields and longer production time. Many of the results do not show the detailed parameters of fermentation. Soy flour, cotton seed flour, corn gluten meal, casein powder, soytone, peptone, casein hydrolysates, and lactalbumin hydrolysate as protein sources were tested to determine the source to support optimal symbiotic bacteria and nematode growth. The protein hydrolysates selected did not improve bacterial cell mass compared with the yeast extract control, but soy flour was the best, showing 75.1% recovery and producing more bacterial cell number (1.4×109/ml) than all other sources. The highest yield (1.85×105 IJs/ml), yield coefficient (1.67×106 IJs/g medium), and productivity (1.32×107 IJs/l/day) were also achieved at enriched medium with soybean protein.

Effects of barium and cadmium on the population development of the marine nematode Rhabditis (Pellioditis) marina.

Offshore oil and gas drilling often involves the use of fluids containing barium and traces of other heavy metals. These may affect the environment, but information on their toxicity to benthic biota remains scant. Here, we present results of a 10-day bioassay with the marine nematode Rhabditis (Pellioditis) marina at different loads of barium (0-10 ,000 ppm nominal concentrations) and cadmium (0-12 ppm) in the range of concentrations reported from drilling-impacted sediments. Barium did not affect the fitness and population development of R. (P.) marina at concentrations up to 300 ppm, but did cause a decrease in population abundance and an increase in development time from concentrations of 400-2000 ppm onwards. Increased mortality occurred at 4800 ppm Ba. For cadmium, LOEC and EC50 values for total population abundance were 2.95 and 8.82 ppm, respectively. Cd concentrations as low as 2.40 to 2.68 caused a decrease in the abundance of adult nematodes, indicating that assays covering more generations would likely demonstrate yet more pronounced population-level effects. Our results indicate that oil and gas drilling activities may potentially have important implications for the meiobenthos through the toxicity of barium and associated metals like cadmium.

Assessing the influence of the entomopathogenic nematode Heterorhabditis baujardi LPP7 (Rhabiditina) on embryogenesis and hatching of the plant-parasitic nematode Meloidogyne mayaguensis (Tylenchina).

Two assays were conducted to assess the influence of infective juveniles (IJs) of Heterorhabditis baujardi LPP7 on the embryogenesis and hatching of Meloidogyne mayaguensis. In the first assay, eggs were incubated in water alone or in the presence of infective juveniles, and completion of embryogenesis was evaluated 14days later. In the second assay, unhatched second-stage juveniles were incubated in distilled water alone or in the presence of infective juveniles. Cumulative hatching was compared at various time intervals. Embryogenesis was not affected, whereas second-stage juveniles hatching was delayed probably because of the eggs permeability to noxious metabolites released by Photorhabdus luminescens, which is the bacterial symbiont of H. baujardi.

Rhabdias lacertae n. sp. (Nematoda: Rhabdiasidae), the first rhabdiasid species parasitising lizards in Europe.

A new nematode species, Rhabdias lacertae n. sp. (Rhabdiasidae), is described from the body-cavity of the common lizard Lacerta vivipara Jacquin (Lacertidae) from the Ridge of Malá Fatra (Sokolie Hill), north-western Slovakia. The new species differs from its congeners mainly in possessing 3 min cuticular spikes at the tail tip and some other features. This is the first species of Rhabdias Stiles & Hassall, 1905 described from lizards in Europe and the first species of this genus parasitising hosts belonging to the Lacertidae.

Photorhabdus phase variants express a novel fimbrial locus, mad, essential for symbiosis.

Fimbriae are adhesive organelles known to enable pathogens to colonize animal tissue, but little is known of their function in mutualistic symbioses. Photorhabdus colonization of Heterorhabditis bacteriophora nematodes is essential for the pair's insect pathogenic lifestyle. Maternal nematodes acquire Photorhabdus symbionts as a persistent intestinal biofilm prior to transmission to infective juvenile (IJ) stage offspring developing inside the maternal body. Screening 8000 Photorhabdus mutants for defects in IJ colonization revealed that a single fimbrial locus, named mad for maternal adhesion defective, is essential. The mad genes encode a novel usher/chaperone assembled fimbria regulated by an ON/OFF invertible promoter switch. Adherent Photorhabdus cells in maternal nematode intestines had the switch ON opposite to the OFF orientation of most other cells. A ΔmadA mutant failed to adhere to maternal intestines and be transmitted to the IJs. Mad fimbriae were detected on TT01 phase ON cells but not on ΔmadA phase ON cells. Also required for transmission is madJ, predicted to encode a transcriptional activator related to GrlA. Expression of madA-K or madIJK restored the ability of madJ mutant to adhere. The Mad fimbriae were not required for insect pathogenesis, indicating the specialized function of Mad fimbriae for symbiosis.

Comparative life cycles and life histories of North American Rhabdias spp. (Nematoda: Rhabdiasidae): lungworms from snakes and anurans.

The present study used experimental infections to compare the life cycles and life histories of 6 Rhabdias spp. infecting snakes and anurans. Free-living development of anuran lungworms was primarily limited to heterogonic reproduction, and females utilized matricidal endotoky exclusively, whereas snake lungworms primarily reproduced homogonically and, when heterogonic reproduction occurred, females used a combination of releasing eggs and matricidal endotoky. Infective snake lungworms survived for longer periods in fresh water compared to anuran worms. Infective anuran lungworms penetrated into the skin of frogs and toads; few infections resulted from per os infections. In contrast, snake lungworms were unable to penetrate skin; instead, infective juveniles penetrated into snake esophageal tissue during per os infections. Despite separate points of entry, anuran and snake lungworms both migrated and developed in the fascia, eventually penetrating into the body cavity of the host. Worms molted to adulthood inside the body cavity and subsequently penetrated into the host's lungs, where they fed on blood while becoming gravid. Adult lungworm survival varied among lungworm species, but, in general, snake lungworms were longer lived than anuran worms. Anuran lungworms were poorly suited for transmission via transport hosts, whereas snake lungworms were consistently capable of establishing infections using transport hosts. Overall, these observations suggest that snake and anuran lungworms have discrepant life cycles and life history strategies.