End resection and telomere healing of DNA double-strand breaks during nematode programmed DNA elimination.

Most DNA double-strand breaks (DSBs) are harmful to genome integrity. However, some forms of DSBs are essential to biological processes, such as meiotic recombination and V(D)J recombination. DSBs are also required for programmed DNA elimination (PDE) in ciliates and nematodes. In nematodes, the DSBs are healed with telomere addition. While telomere addition sites have been well-characterized, little is known regarding the DSBs that fragment nematode chromosomes. Here, we used embryos from the nematode Ascaris to study the timing of PDE breaks and examine the DSBs and their end processing. Using END-seq, we characterize the DSB ends and demonstrate that DNA breaks are introduced before mitosis, followed by extensive end resection. The resection profile is unique for each break site, and the resection generates 3' overhangs before the addition of telomeres. Interestingly, telomere healing occurs much more frequently on retained DSB ends than on eliminated ends. This biased repair of the DSB ends in Ascaris may be due to the sequestration of the eliminated DNA into micronuclei, preventing their ends from telomere healing. Additional DNA breaks occur within the eliminated DNA in both Ascaris and Parascaris, ensuring chromosomal breakage and providing a fail-safe mechanism for nematode PDE.

The activity of leaf extracts, fractions, and isolated compounds from Ptaeroxylon obliquum against nine phytopathogenic fungi and the nematode Meloidogyne incognita.

Phytopathogenic fungi and nematodes cause great losses in economically important crops and food production especially in developing countries. To minimize the use of fungicides and nematicides, researchers have concentrated on the use of natural products for crop disease prevention or control. The aim of the study was to investigate the antifungal activity of Ptaeroxylon obliquum leaf extracts, fractions, and isolated compounds (obliquumol and a mixture of lupeol and ß-amyrin) and nematocidal activity of fractions (hexane, chloroform and 30% water in methanol and the isolated compounds) on Meloidogyne incognita. Nine phytopathogenic fungi (Aspergillus niger, A. parasiticus, Colletotrichum gloeosporioides, Fusarium oxysporum, Penicillium digitatum, P. expansum, P. italicum, P. janthinellum, and Rhizoctonia solani) were used for testing and nematocidal activity was determined on motility of plant parasitic nematode Meloidogyne incognita race 2 juveniles. Serial microdilution test was utilized to determine the minimum inhibitory concentration (MIC) of each sample against the fungus. Motility tests was done on the second-stage juveniles (J2s) of M. incognita. The most susceptible phytopathogenic fungal species to the acetone crude leaf extracts were A. niger, C. gloeosporioides and P. digitatum with MIC of 80 µg/ml which is considered pharmacological significant. Rhizoctonia solani was the most susceptible fungus against obliquumol and, lupeol and ß-amyrin mixture with MIC values of 8 µg/ml and 16 µg/ml respectively. Lupeol & ß-amyrin mixture had good activity on juvenile motility at high concentrations used which was significantly high (p ≤ 0.05) after 24 h, further incubation resulted in temporary paralysis at lower concentrations. Fractions and obliquumol showed good activity after 48 h, stable paralysis was observed up to 72 h. The extracts and isolated compounds may be useful as fungicides if the in vitro results can be confirmed under field conditions at levels not toxic to beneficial soil organisms.

Heterocheilus floridensis sp. n. (Nematoda: Heterocheilidae) from the West Indian manatee Trichechus manatus (Trichechidae, Sirenia) in Florida, USA.

Morphological data are used to describe a new nematode species, Heterocheilus floridensis sp. n. (Heterocheilidae) from the digestive tract of the Florida manatee Trichechus manatus latirostris (Harlan) (Trichechidae, Sirenia) from Florida, USA. Examination by light and scanning electron microscopy revealed that the new species differs from the related Heterocheilus tunicatus Diesing, 1839 mainly by having dentigerous ridges on the inner surface of the lips, a median unpaired papilla located anterior to the cloaca, and a considerably larger body size. Sequence data for subunits I and II of mitochondrial cytochrome oxidase gene, 18S small subunit and 28S ribosomal RNA genes were provided for molecular characterisation of the new species. However, the current unavailability of homologous sequence data for congeneric specimens precluded a molecular assessment of the morphological species hypothesis, and ascaridoid phylogenetic hypotheses could not be advanced. Specimens of Heterocheilus sp. collected from the Antillean manatee Trichechus manatus manatus Linnaeus in Puerto Rico, on loan from the US National Museum of Natural History, were morphologically consistent with the new species, so apparently all congeneric nematodes reported from both subspecies of the West Indian manatee Trichechus manatus Linnaeus and previously identified as H. tunicatus belong rather to H. floridensis sp. n. Heterocheilus hagenbecki (Khalil et Vogelsang, 1932) Sprent 1980 is here considered to be a species inquirenda. A key to valid species of Heterocheilus Diesing, 1839 is provided.

Carbon dioxide shapes parasite-host interactions in a human-infective nematode.

Skin-penetrating nematodes infect nearly one billion people worldwide. The developmentally arrested infective larvae (iL3s) seek out hosts, invade hosts via skin penetration, and resume development inside the host in a process called activation. Activated infective larvae (iL3as) traverse the host body, ending up as parasitic adults in the small intestine. Skin-penetrating nematodes respond to many chemosensory cues, but how chemosensation contributes to host seeking, intra-host development, and intra-host navigation - three crucial steps of the parasite-host interaction - remains poorly understood. Here, we investigate the role of carbon dioxide (CO2) in promoting parasite-host interactions in the human-infective threadworm Strongyloides stercoralis. We show that S. stercoralis exhibits life-stage-specific preferences for CO2: iL3s are repelled, non-infective larvae and adults are neutral, and iL3as are attracted. CO2 repulsion in iL3s may prime them for host seeking by stimulating dispersal from host feces, while CO2 attraction in iL3as may direct worms toward high-CO2 areas of the body such as the lungs and intestine. We also identify sensory neurons that detect CO2; these neurons are depolarized by CO2 in iL3s and iL3as. In addition, we demonstrate that the receptor guanylate cyclase Ss-GCY-9 is expressed specifically in CO2-sensing neurons and is required for CO2-evoked behavior. Ss-GCY-9 also promotes activation, indicating that a single receptor can mediate both behavioral and physiological responses to CO2. Our results illuminate chemosensory mechanisms that shape the interaction between parasitic nematodes and their human hosts and may aid in the design of novel anthelmintics that target the CO2-sensing pathway.

Nematode Uptake Preference toward Different Nanoplastics through Avoidance Behavior Regulation.

Expounding bioaccumulation pathways of nanoplastics in organisms is a prerequisite for assessing their ecological risks in the context of global plastic pollution. Invertebrate uptake preference toward nanoplastics is a key initial step of nanoplastic food chain transport that controls their global biosafety, while the biological regulatory mechanism remains unclear. Here, we reveal a preferential uptake mechanism involving active avoidance of nanoplastics by Caenorhabditis elegans and demonstrate the relationship between the uptake preference and nanoplastic characteristics. Nanoplastics with 100 nm in size or positive surface charges induce stronger avoidance due to higher toxicity, causing lower accumulation in nematodes, compared to the 500 nm-sized or negatively charged nanoplastics, respectively. Further evidence showed that nematodes did not actively ingest any types of nanoplastics, while different nanoplastics induced defense responses in a toxicity-dependent manner and distinctly stimulated the avoidance behavior of nematodes (ranged from 15.8 to 68.7%). Transcriptomics and validations using mutants confirmed that the insulin/IGF signaling (IIS) pathway is essential for the selective avoidance of nanoplastics. Specifically, the activation of DAF-16 promoted the IIS pathway-mediated defense against nanoplastics and stimulated the avoidance behavior, increasing the survival chances of nematodes. Considering the genetical universality of this defense response among invertebrates, such an uptake preference toward certain nanoplastics could lead to cascaded risks in the ecosystem.

First report of the barley root-knot nematode, Meloidogyne naasi Franklin, 1965 from Pennsylvania, with new host report.

Meloidogyne naasi Franklin, 1965, the barley root-knot nematode, was originally found in field crops such as cereals, grasses, and sugar beet (Beta vulgaris L.) in England and Wales, (Franklin,1965). This nematode is one of the most significant root-knot nematodes impacting grains in European countries (Santos et al. 2020). Among root-knot nematode species, M. naasi, exhibits a distinct preference for grasses, with documented impacts on turfgrasses leading to reduced growth and vigor (Skantar et al., 2023; Cook and Yeates, 1993). In September 2022, root-knot nematode females and second-stage juveniles (J2) were recovered from roots of fowl manna grass, Glyceria striata (Lam.) Hitchc., during a nematode survey on natural vegetation at the Allegheny National Forest (41°30'13.8"N 79°09'46.2"W). Second-stage juvenile specimens were recovered from soil using sugar centrifugal flotation (Jenkins, 1964). Small galls with egg masses were dissected from fowl manna grass roots originally collected from the surveyed areas. In parallel, five plants of non-infected fowl manna grass were placed in a pot in the greenhouse using naturally nematode-infested soil collected from the same forested area. Small galls and female specimens recovered from these plants were dissected and processed for further analyses. Female and J2 were fixed in 3% formaldehyde solution and processed to glycerin (Golden, 1990; Hooper, 1970). The specimens were examined by light microscopy, morphometric measurements, and molecular markers, which included the D2-D3 region of the large ribosomal subunit 28S, and the rDNA internal transcribed spacer region (ITS). The perennial pattern of five females analyzed morphologically were consistent to the patterns observed for M. naasi. The perennial patterns had coarse ridges on the cuticle in dorsal region forming broken irregular lines around anal and phasmid area. We also noted a prominent fold that covered some of the anus and showed a curved line between vulval slit and phasmids, typical of M. naasi. The area around the vulval area had a few or no striae except for a few lines radiating from the vulval slit as in the original description. Measurements of ten J2 had a body length ranged between 380 and 410 µm, stylet 11-13 µm, tail 50-70 µm long with a hyaline tail terminus between 12-22 µm in length, 4 lines in the lateral field, a and c ratio between 29.23-35.91 and 5.79-7.9 fitting the original description by Franklin, 1965 and others populations found in the USA (Skantar et al., 2023). The matrix codes for the female specimes are A32, B324, C3, D3 and for J2's A2, B21, C123, D1, E3, F12 (Subbotin et al., 2021). The amplified DNA fragments were sequenced, resulting in an 726 bp fragment flanked by the D2-D3 primers (PP097762), while for the ITS primers an 634 bp fragment was obtained (PP092043). Both generated sequences for the specimens collected in Pennsylvania revealed >99% similarity to M. naasi sequences deposited at GenBank, and therefore, validating the morphological analyses. Based on both morphological and molecular analyses the specimens collected in the state of Pennsylvania were identified as M.naasi. To our knowledge, this is the first report of this species from this state and being associated with naturally infected fowl manna grass.

The Impact of Peach Rootstocks and Winter Cover Crops on Reproduction of Ring Nematode.

Two peach rootstocks ('Guardian' and 'MP-29') and ten winter cover crops (rye, wheat, barley, triticale, oat, Austrian winter pea, crimson clover, balansa clover, hairy vetch, and daikon radish) were evaluated in a greenhouse environment to determine their suitability to host ring nematode, Mesocriconema xenoplax. Each crop was inoculated with 500 ring nematodes, and the experiments were terminated 60 days after inoculation. The reproduction factor (ratio of final and initial nematode population) ranged from 0 to 13.8, indicating the crops greatly varied in their host suitability to ring nematode. 'Guardian' has been known to tolerate ring nematode; however, results from the current study suggest the tolerance statement is anecdotal. Another peach rootstock, 'MP-29', was also a good host for ring nematode, suggesting an urgency to develop ring nematode-resistant peach rootstocks. Wheat supported the least to no nematode reproduction while pea supported the greatest reproduction. The rest of the cover crops were poor to good hosts to ring nematodes. Although planting cover crops in peach orchards is not common, employing non or poor host crops can help suppress nematodes in addition to having soil health benefits. Furthermore, peach breeding programs should focus on finding and introgressing ring nematode resistance in commercial rootstocks.

Integrated approaches for Solenopsis invicta (Hymenoptera: Formicidae) management: Insights from laboratory studies with entomopathogenic nematodes and insecticides.

BACKGROUND: In agricultural pest management, especially in combatting the invasive red imported fire ant (RIFA, Solenopsis invicta), significant challenges emerge due to the constraints of solely depending on chemical insecticides or entomopathogenic nematodes (EPNs). The utilization of chemical insecticides carries environmental and ecological hazards, whereas EPNs, when applied independently, might not offer the immediate effectiveness necessary for adequate RIFA suppression. Acknowledging these hurdles, our study investigates a synergistic method that integrates EPNs with chemical insecticides, aiming to fulfill the urgent demand for more efficient and environmentally friendly pest control solutions. RESULTS: Our evaluation focused on the interaction between the highly pathogenic Steinernema riobrave 7-12 EPN strain and prevalent insecticides, specifically beta-cypermethrin and a mixture of bifenthrin and clothianidin, applied at highly diluted recommended concentrations. The findings revealed a notable increase in RIFA mortality rates when EPNs and these insecticides were used together, outperforming the results achieved with each method individually. Remarkably, this enhanced efficacy was especially evident at lower concentrations of the bifenthrin-clothianidin mixture, indicating a valuable approach to minimizing reliance on chemical insecticides in agriculture. Furthermore, the high survival rates of EPNs alongside the tested insecticides indicate their compatibility and potential for sustained use in integrated pest management programs. CONCLUSION: Our research underscores the effectiveness of merging EPNs with chemical insecticides as a powerful and sustainable strategy for RIFA management. This combined approach not only meets the immediate challenges of pest control in agricultural settings but also supports wider environmental objectives by reducing the dependency on chemical insecticides. This article is protected by copyright. All rights reserved.

Evaluation of Weed Species for Host Status to the Root-Knot Nematodes Meloidogyne enterolobii and M. incognita Race 4.

Weeds that compete with valuable crops can also host plant-parasitic nematodes, acting as a source of nematode inoculum in a field and further damaging crops. The host status of 10 weed species commonly found in North Carolina, USA, was determined for the root-knot nematodes Meloidogyne enterolobii and M. incognita race 4 in the greenhouse. Each weed species was challenged with 5,000 eggs/plant of either M. enterolobii or M. incognita race 4, with five replicate plants per treatment in two separate greenhouse trials. Root galling severity and total number of nematode eggs per root system were recorded 60 days after inoculation. Reproduction factor (Rf = final nematode population/initial nematode population) was calculated to determine the host status of each weed species to M. enterolobii and M. incognita race 4. Four weed species (Datura stramonium, Digitaria sanguinalis, Senna obtusifolia, and Cyperus esculentus) were poor hosts (Rf < 1) to both nematode species, and roots of these weed plants did not display galling. Four weed species (Ipomoea hederacea, Amaranthus palmeri, Portulaca pilosa, and Ipomoea lacunosa) were hosts (Rf > 1) to both nematode species, and all had observable root gall formation. Sida rhombifolia and Cyperus rotundus were poor hosts to M. enterolobii but susceptible hosts to M. incognita. This study documents a differential host status of some common weeds to M. enterolobii and M. incognita race 4, and these results highlight the necessity of managing root-knot nematodes through controlling weeds in order to protect valuable crops.

Cotton host resistance as a tool for managing Rotylenchulus reniformis in Louisiana.

The reniform nematode, Rotylenchulus reniformis, is a major yield-limiting pest of upland cotton (Gossypium hirsutum) in the United States that has been steadily increasing in incidence in many states. Reniform nematode-resistant cotton cultivars have recently become commercially available for cotton producers; however, few field trials have evaluated their efficacy as a nematode management tool. The aim of this study was to evaluate reniform nematode population development, plant growth, and seed cotton yield of reniform nematode-resistant cotton cultivars in two nematode-infested fields in Louisiana. Replicated small-plot field trials were conducted in St. Joseph, LA (NERS field) and Winnsboro, LA (MRRS field) during the 2022 and 2023 growing seasons. In 2022, cultivars evaluated included: (1) DP 1646 B2XF (susceptible/tolerant), (2) DP 2141NR B3XF (resistant), (3) PHY 332 W3FE (resistant), (4) PHY 411 W3FE (resistant), and (5) PHY 443 W3FE (resistant). In 2023, an additional susceptible cotton cultivar, PHY 340 W3FE, was also included. All nematode-resistant cotton cultivars evaluated provided suppression of reniform nematode population development relative to that of the susceptible cotton cultivars, with suppression of nematode soil population densities at harvest ranging from 49 - 81% relative to DP 1646 B2XF. The resistant cultivar PHY 411 W3FE provided the most consistent suppression of reniform nematode population development, reducing reniform nematode soil population densities at harvest in both field locations and both trial years. In contrast, DP 2141NR B3XF only reduced soil population densities at harvest in the NERS field in 2023. Despite relatively consistent nematode suppression and improvements in plant vigor ratings and canopy coverage associated with the resistant cotton cultivars, a yield increase was only observed with PHY 332 W3FE and PHY 411 W3FE planted at the NERS field in 2023. Despite strong resistance to reniform nematode in the evaluated cotton cultivars, nematode soil population densities still increased during the growing season in plots planted with resistant cotton cultivars, emphasizing the need for additional management tactics to use alongside host resistance. This study indicates that new reniform nematode-resistant cotton cultivars show promising potential to reduce nematode population development during the growing season in Louisiana.

Fortify the defense frontline: MAPKs phosphorylate receptor-like cytoplasmic kinase to maintain plant resistance in soybean.

Mitogen-activated protein kinase (MAPK) activation is one of the significant immune events that respond to pathogens in plants. A MAPK cascade often contains a MAPK kinase kinase (MAPKKK), a MAPK kinase (MAPKK/MKK), and a MAPK. The well-characterized MAPK cascade, to date, is the MAPKKK3/4/5-MKK4/5-MPK3/6 module. Soybean cyst nematodes (SCN) is one of the most devastating soybean pathogens. However, the early immune components contributing to soybean resistance to SCN and the role of the MAPK cascade in the soybean-SCN interaction remain unclear. A recent study published in Plant Cell discovered that GmMPK3/6 phosphorylates a receptor-like cytoplasmic kinase (RLCK), CDG1-LIKE1 (GmCDL1), and maintains the stability of GmCDL1 in soybean. Remarkably, GmCDL1 enhances GmMPK3/6 activation and resistance to SCN by phosphorylating GmMAPKKK5 and activating the GmMAPKKK5-GmMKK4-GmMPK3/6 cascade. In addition, two L-type lectin receptor kinases (LecRKs), GmLecRK02g and GmLecRK08g, are involved in the GmCDL1 function after the perception of SCN. taken together, this study not only discovers a complete early immune pathway that responds to SCN infection in soybean, but also reveals a molecular mechanism by which plants maintain the activation of the MAPK cascade and resistance.

Reducing fecal egg count through selective breeding alters dorper lamb response to Haemonchus contortus in an artificial challenge trial.

Infection by gastrointestinal nematodes (GIN), particularly Haemonchus contortus, can be detrimental to sheep health and performance. Genetic susceptibility to GIN varies between breeds, with those lacking high levels of natural resistance often requiring frequent anthelmintic treatment when facing parasitic challenge. Genetic technology can serve as a tool to decrease GIN susceptibility via selection for sheep with reduced fecal egg count (FEC) estimated breeding values (EBVs). However, the physiological changes that result from implementation of this strategy are not well described. Additionally, there is a need for comparison of animals from recent selective breeding against breeds with inherent GIN resistance. In this study we administered a challenge of H. contortus to Dorper x White Dorper (DWD; n = 92) lambs that have been genetically selected for either low (DWD-) or high (DWD+) FEC EBVs and Barbados Blackbelly x Mouflon (BBM; n = 19) lambs from a genetically resistant breed backgrounds. Lamb FEC, packed-cell volume (PCV) and serum IgG were measured at intermittent levels over 5 weeks. At day 21 and day 35, the selectively bred DWD- had a lower mean FEC compared to DWD+, but were higher than BBM. Reductions in both PCV and serum IgG from initial day 0 levels were observed in DWD lambs, but not in BBM. Furthermore, from a subset of lambs (n = 24) harvested at day 21, DWD- only tended (p = 0.056) to have lower mean worm counts than DWD+, with BBM having the lowest mean worm count. Differentially expressed genes (DEGs) identified via RNA-sequencing of abomasal tissue at day 21 indicate a more pronounced Th2 immune response and more rapid worm expulsion occurred in iBBM than iDWD- and iDWD+ lambs. However, gene expression in DWD- suggests an association between reduced FEC EBV and gastric acid secretion and the ability to limit worm fecundity. Ultimately, selection of Dorper sheep for low FEC EBV can reduce susceptibility to GIN, but it will likely require multiple generations with this trait as a breeding priority before presenting a similar resistance level to Caribbean breeds.

Intraocular Parafilaria bovicola infection and surgical removal in a mixed breed heifer.

An approximately 1.5-year-old mixed breed heifer was presented for evaluation and treatment due to ocular pain affecting the right eye secondary to a live nematode within the anterior chamber. Ophthalmic examination revealed marked blepharospasm, evidence of chronic keratitis, uveitis, and a single, white, approximately 2.5 cm long, 0.5 mm thick, living parasite. The heifer underwent general anesthesia, and the parasite was removed using passive aqueous humor outflow following a stab incision into the anterior chamber. Twenty-four hours after removal of the intraocular parasite, the heifer displayed a significant improvement in ocular comfort, corneal edema, and uveitis. The nematode was identified via genetic analyses as Parafilaria bovicola, a filarial parasite of cattle. The heifer was discharged 5 days following nematode removal with a marked improvement in all ocular signs and apparently normal vision. To the authors' knowledge, there are no previous reports of confirmed intraocular filariosis caused by P. bovicola in the veterinary literature, nor are there reports detailing surgical removal of intraocular parasites in bovids.

Characterization of data observing Meloidogyne incognita, Neofusicoccum parvum, and Xylella fastidiosa infection effects on development of grapevine phenolic compound levels and resistance to subsequent Neofusicoccum parvum infections.

Grapevines encounter many different pathogens throughout their lifespans, including the bacterial pathogen Xylella fastidiosa, which causes Pierce's disease that results in vascular occlusion and eventual plant host death, the fungal pathogen Neofusicoccum parvum, which causes stem cankers that kill individual vines and reduce fruit yields, and the root knot nematode Meloidogyne incognita, which destroys root tissues that impacts host vigour. To date, little research has been conducted to examine how one infection could impact subsequent infections by the same or different pathogens despite this is important to ensure healthy vineyards. Therefore, grapevines initially infected with either X. fastidiosa, N. parvum, or M. incognita were subsequently infected with N. parvum eight weeks later to observe developing lesion lengths, which were assessed to determine grapevine resistance to infections. Collected data shows that when prior infections were present, the N. parvum lesions lengths were smaller. This suggests grapevines had induced resistance to combat infections. Further, defence-associated phenolics were measured by high-performance liquid chromatography to determine roles in observed resistance to the secondary N. parvum infections. Data shows that of the different phenolics examined, only stilbenoids were different due to infections, with lowered levels observed in plants that were infected compared with non-infected controls. These data provide insight into how infections by different pathogens could impact grapevine host resistance to new, subsequent pathogen infections.

First report of Meloidogyne hapla on hemp (Cannabis sativa) in Oregon.

Hemp is a crop that has gained interest in Washington and Oregon. As with other crops, hemp production faces challenges due to biotic factors, including plant-parasitic nematodes. During a survey for plant-parasitic nematodes associated with hemp, Meloidogyne sp. was found in a composite root sample collected in Oregon. Morphological characterization of second-stage juveniles identified the nematode as Meloidogyne hapla. Molecular identification confirmed the population as M. hapla. To our knowledge, this is the first report of M. hapla on hemp in the Pacific Northwest of the United States.

Anthraquinones against Cryptococcus neoformans sensu stricto: antifungal interaction, biofilm inhibition and pathogenicity in the Caenorhabditis elegans model.

Introduction. Cryptococcal biofilms have been associated with persistent infections and antifungal resistance. Therefore, strategies, such as the association of natural compounds and antifungal drugs, have been applied for the prevention of biofilm growth. Moreover, the Caenorhabditis elegans pathogenicity model has been used to investigate the capacity to inhibit the pathogenicity of Cryptococcus neoformans sensu stricto.Hypothesis. Anthraquinones and antifungals are associated with preventing C. neoformans sensu stricto biofilm formation and disrupting these communities. Antraquinones reduced the C. neoformans sensu stricto pathogenicity in the C. elegans model.Aim. This study aimed to evaluate the in vitro interaction between aloe emodin, barbaloin or chrysophanol and itraconazole or amphotericin B against growing and mature biofilms of C. neoformans sensu stricto.Methodology. Compounds and antifungal drugs were added during biofilm formation or after 72 h of growth. Then, the metabolic activity was evaluated by the MTT reduction assay, the biomass by crystal-violet staining and the biofilm morphology by confocal laser scanning microscopy. C. neoformans sensu stricto's pathogenicity was investigated using the nematode C. elegans. Finally, pathogenicity inhibition by aloe emodin, barbarloin and chrysophanol was investigated using this model.Results. Anthraquinone-antifungal combinations affected the development of biofilms with a reduction of over 60 % in metabolic activity and above 50 % in biomass. Aloe emodin and barbaloin increased the anti-biofilm activity of antifungal drugs. Chrysophanol potentiated the effect of itraconazole against C. neoformans sensu stricto biofilms. The C. elegans mortality rate reached 76.7 % after the worms were exposed to C. neoformans sensu stricto for 96 h. Aloe emodin, barbaloin and chrysophanol reduced the C. elegans pathogenicity with mortality rates of 61.12 %, 65 % and 53.34 %, respectively, after the worms were exposed for 96 h to C. neoformans sensu stricto and these compounds at same time.Conclusion. These results highlight the potential activity of anthraquinones to increase the effectiveness of antifungal drugs against cryptococcal biofilms.

First molecular identification of Haemonchus contortus (Nematoda: Trichostrongylidae), a blood-sucking gastric nematode of artiodactyles, in the ground beetle Carabus granulatus (Coleoptera: Carabidae).

Among gastrointestinal nematodes, Haemonchus contortus (Rudolphi) Cobb (order Strongylidae; family Trichostrongylidae) is one of pathogenic and economic importance in domestic and wild ruminants, including the European bison, Bison bonasus Linnaeus (order Cetartiodactyla; family Bovidae); a species on the International Union for Conservation of Nature's Red List of Threatened Species. Carabus granulatus Linnaeus (order Coleoptera; family Carabidae) is one of the most prevalent species of ground beetle, inhabiting a wide range of terrestrial ecosystems in Poland. Twenty-six ground beetles of this species inhabiting the Bialowieza Primeval Forest in eastern Poland were screened for the presence of DNA of pathogenic gastrointestinal nematodes of ruminants. Extracted DNA was sequenced and compared to reference sequences. In six insects, the presence of H. contortus DNA was detected. The obtained nucleotide sequences were homologous to each other and to the majority of the published DNA sequences of H. contortus isolates. The sequences were also identical to a sequence of H. contortus isolated from European bison in Poland. The study provides the first molecular evidence of the presence of H. contortus DNA in C. granulatus. The finding suggests that ground beetles may play a role in the transmission dynamics of this parasite.

Starvation resistance in the nematode Pristionchus pacificus requires a conserved supplementary nuclear receptor.

Nuclear hormone receptors (NHRs) are a deeply-conserved superfamily of metazoan transcription factors, which fine-tune the expression of their regulatory target genes in response to a plethora of sensory inputs. In nematodes, NHRs underwent an explosive expansion and many species have hundreds of nhr genes, most of which remain functionally uncharacterized. However, recent studies have reported that two sister receptors, Ppa-NHR-1 and Ppa-NHR-40, are crucial regulators of feeding-structure morphogenesis in the diplogastrid model nematode Pristionchus pacificus. In the present study, we functionally characterize Ppa-NHR-10, the sister paralog of Ppa-NHR-1 and Ppa-NHR-40, aiming to reveal whether it too regulates aspects of feeding-structure development. We used CRISPR/CAS9-mediated mutagenesis to create small frameshift mutations of this nuclear receptor gene and applied a combination of geometric morphometrics and unsupervised clustering to characterize potential mutant phenotypes. However, we found that Ppa-nhr-10 mutants do not show aberrant feeding-structure morphologies. Instead, multiple RNA-seq experiments revealed that many of the target genes of this receptor are involved in lipid catabolic processes. We hypothesized that their mis-regulation could affect the survival of mutant worms during starvation, where lipid catabolism is often essential. Indeed, using novel survival assays, we found that mutant worms show drastically decreased starvation resistance, both as young adults and as dauer larvae. We also characterized genome-wide changes to the transcriptional landscape in P. pacificus when exposed to 24 h of acute starvation, and found that Ppa-NHR-10 partially regulates some of these responses. Taken together, these results demonstrate that Ppa-NHR-10 is broadly required for starvation resistance and regulates different biological processes than its closest paralogs Ppa-NHR-1 and Ppa-NHR-40.

Characterisation of protective vaccine antigens from the thiol-containing components of excretory/secretory material of Ostertagia ostertagi.

Previous vaccination trials have demonstrated that thiol proteins affinity purified from Ostertagia ostertagi excretory-secretory products (O. ostertagi ES-thiol) are protective against homologous challenge. Here we have shown that protection induced by this vaccine was consistent across four independent vaccine-challenge experiments. Protection is associated with reduced cumulative faecal egg counts across the duration of the trials, relative to control animals. To better understand the diversity of antigens in O. ostertagi ES-thiol we used high-resolution shotgun proteomics to identify 490 unique proteins in the vaccine preparation. The most numerous ES-thiol proteins, with 91 proteins identified, belong to the sperm-coating protein/Tpx/antigen 5/pathogenesis-related protein 1 (SCP/TAPS) family. This family includes previously identified O. ostertagi vaccine antigens O. ostertagi ASP-1 and ASP-2. The ES-thiol fraction also has numerous proteinases, representing three distinct classes, including: metallo-; aspartyl- and cysteine proteinases. In terms of number of family members, the M12 astacin-like metalloproteinases, with 33 proteins, are the most abundant proteinase family in O. ostertagi ES-thiol. The O. ostertagi ES-thiol proteome provides a comprehensive database of proteins present in this vaccine preparation and will guide future vaccine antigen discovery projects.

Response of the symbiotic microbial community of Dioscorea opposita Thunb. cv. Tiegun to root-knot nematode infection.

Yam is an important medicinal and edible dual-purpose plant with high economic value. However, nematode damage severely affects its yield and quality. One of the major effects of nematode infestations is the secondary infection of pathogenic bacteria or fungi through entry wounds made by the nematodes. Understanding the response of the symbiotic microbial community of yam plants to nematodes is crucial for controlling such a disease. In this study, we investigated the rhizosphere and endophytic microbiomes shift after nematode infection during the tuber expansion stage in the Dioscorea opposita Thunb. cv. Tiegun yam. Our results revealed that soil depth affected the abundance of nematodes, and the relative number of Meloidogyne incognita was higher in the diseased soil at a depth of 16-40 cm than those at a depth of 0-15 cm and 41-70 cm. The abundance of and interactions among soil microbiota members were significantly correlated with root-knot nematode (RKN) parasitism at various soil depths. However, the comparison of the microbial alpha diversity and composition between healthy and diseased rhizosphere soil showed no difference. Compared with healthy soils, the co-occurrence networks of M. incognita-infested soils included a higher ratio of positive correlations linked to plant health. In addition, we detected a higher abundance of certain taxonomic groups belonging to Chitinophagaceae and Xanthobacteraceae in the rhizosphere of RKN-infested plants. The nematodes, besides causing direct damage to plants, also possess the ability to act synergistically with other pathogens, especially Ramicandelaber and Fusarium, leading to the development of disease complexes. In contrast to soil samples, RKN parasitism specifically had a significant effect on the composition and assembly of the root endophytic microbiota. The RKN colonization impacted a wide variety of endophytic microbiomes, including Pseudomonas, Sphingomonas, Rhizobium, Neocosmospora, and Fusarium. This study revealed the relationship between RKN disease and changes in the rhizosphere and endophytic microbial community, which may provide novel insights that help improve biological management of yam RKNs.