Hypothalamic transcriptome response to simulated diel earthen pond hypoxia cycles in channel catfish (Ictalurus punctatus).

Commercial culture of channel catfish (Ictalurus punctatus) occurs in earthen ponds that are characterized by diel swings in dissolved oxygen concentration that can fall to severe levels of hypoxia, which can suppress appetite and lead to suboptimal growth. Given the significance of the hypothalamus in regulating these processes in other fishes, an investigation into the hypothalamus transcriptome was conducted to identify specific genes and expression patterns responding to hypoxia. Channel catfish in normoxic water were compared with catfish subjected to 12 h of hypoxia (20% oxygen saturation; 1.8 mg O2/L; 27°C) followed by 12 h of recovery in normoxia to mimic 24 h in a catfish aquaculture pond. Fish were sampled at 0-, 6-, 12-, 18-, and 24-h timepoints, with the 6- and 12-h samplings occurring during hypoxia. A total of 190 genes were differentially expressed during the experiment, with most occurring during hypoxia and returning to baseline values within 6 h of normoxia. Differentially expressed genes were sorted by function into Gene Ontology biological processes and revealed that most were categorized as "response to hypoxia," "sprouting angiogenesis," and "cellular response to xenobiotic stimulus." The patterns of gene expression reported here suggest that transcriptome responses to hypoxia are broad and quickly reversibly with the onset of normoxia. Although no genes commonly reported to modulate appetite were found to be differentially expressed in this experiment, several candidates were identified for future studies investigating the interplay between hypoxia and appetite in channel catfish, including adm, igfbp1a, igfbp7, and stc2b.NEW & NOTEWORTHY Channel catfish are an economically important species that experience diel episodic periods of hypoxia that can reduce appetite. This is the first study to investigate their transcriptome from the hypothalamus in a simulated 24-h span in a commercial catfish pond, with 12 h of hypoxia and 12 h of normoxia. The research revealed functional groups of genes relating to hypoxia, angiogenesis, and glycolysis as well as individual target genes possibly involved in appetite regulation.

Reference genomes of channel catfish and blue catfish reveal multiple pericentric chromosome inversions.

BACKGROUND: Channel catfish and blue catfish are the most important aquacultured species in the USA. The species do not readily intermate naturally but F1 hybrids can be produced through artificial spawning. F1 hybrids produced by mating channel catfish female with blue catfish male exhibit heterosis and provide an ideal system to study reproductive isolation and hybrid vigor. The purpose of the study was to generate high-quality chromosome level reference genome sequences and to determine their genomic similarities and differences. RESULTS: We present high-quality reference genome sequences for both channel catfish and blue catfish, containing only 67 and 139 total gaps, respectively. We also report three pericentric chromosome inversions between the two genomes, as evidenced by long reads across the inversion junctions from distinct individuals, genetic linkage mapping, and PCR amplicons across the inversion junctions. Recombination rates within the inversional segments, detected as double crossovers, are extremely low among backcross progenies (progenies of channel catfish female × F1 hybrid male), suggesting that the pericentric inversions interrupt postzygotic recombination or survival of recombinants. Identification of channel catfish- and blue catfish-specific genes, along with expansions of immunoglobulin genes and centromeric Xba elements, provides insights into genomic hallmarks of these species. CONCLUSIONS: We generated high-quality reference genome sequences for both blue catfish and channel catfish and identified major chromosomal inversions on chromosomes 6, 11, and 24. These perimetric inversions were validated by additional sequencing analysis, genetic linkage mapping, and PCR analysis across the inversion junctions. The reference genome sequences, as well as the contrasted chromosomal architecture should provide guidance for the interspecific breeding programs.

Pyrrocidine, a molecular off switch for fumonisin biosynthesis.

Sarocladium zeae is a fungal endophyte of maize and can be found co-inhabiting a single seed with Fusarium verticillioides, a major mycotoxigenic food safety threat. S. zeae produces pyrrocidines A and B that inhibit the growth of F. verticillioides and may limit its spread within the seed to locations lacking S. zeae. Although coinhabiting single seeds, the fungi are generally segregated in separate tissues. To understand F. verticillioides' protective physiological response to pyrrocidines we sequenced the F. verticillioides transcriptome upon exposure to purified pyrrocidine A or B at sub-inhibitory concentrations. Through this work we identified a F. verticillioides locus FvABC3 (FVEG_11089) encoding a transporter critical for resistance to pyrrocidine. We also identified FvZBD1 (FVEG_00314), a gene directly adjacent to the fumonisin biosynthetic gene cluster that was induced several thousand-fold in response to pyrrocidines. FvZBD1 is postulated to act as a genetic repressor of fumonisin production since deletion of the gene resulted in orders of magnitude increase in fumonisin. Further, pyrrocidine acts, likely through FvZBD1, to shut off fumonisin biosynthesis. This suggests that S. zeae is able to hack the secondary metabolic program of a competitor fungus, perhaps as preemptive self-protection, in this case impacting a mycotoxin of central concern for food safety.

CRISPR/Cas9-mediated genome editing of Schistosoma mansoni acetylcholinesterase.

CRISPR/Cas9-mediated genome editing shows cogent potential for the genetic modification of helminth parasites. We report successful gene knock-in (KI) into the genome of the egg of Schistosoma mansoni by combining CRISPR/Cas9 with single-stranded oligodeoxynucleotides (ssODNs). We edited the acetylcholinesterase (AChE) gene of S. mansoni targeting two guide RNAs (gRNAs), X5 and X7, located on exon 5 and exon 7 of Smp_154600, respectively. Eggs recovered from livers of experimentally infected mice were transfected by electroporation with a CRISPR/Cas9-vector encoding gRNA X5 or X7 combining with/ without a ssODN donor. Next generation sequencing analysis of reads of amplicon libraries spanning targeted regions revealed that the major modifications induced by CRISPR/Cas9 in the eggs were generated by homology directed repair (HDR). Furthermore, soluble egg antigen from AChE-edited eggs exhibited markedly reduced AChE activity, indicative that programed Cas9 cleavage mutated the AChE gene. Following injection of AChE-edited schistosome eggs into the tail veins of mice, an significantly enhanced Th2 response involving IL-4, -5, -10, and-13 was detected in lung cells and splenocytes in mice injected with X5-KI eggs in comparison to control mice injected with unmutated eggs. A Th2-predominant response, with increased levels of IL-4, -13, and GATA3, also was induced by X5 KI eggs in small intestine-draining mesenteric lymph node cells when the gene-edited eggs were introduced into the subserosa of the ileum of the mice. These findings confirmed the potential and the utility of CRISPR/Cas9-mediated genome editing for functional genomics in schistosomes.

Transcriptome and binding data indicate that citral inhibits single strand DNA-binding proteins.

The mechanism of phytotoxicity of citral was probed in Arabidopsis thaliana using RNA-Seq and in silico binding analyses. Inhibition of growth by 50% by citral downregulated transcription of 9156 and 5541 genes in roots and shoots, respectively, after 1 h. Only 56 and 62 genes in roots and shoots, respectively, were upregulated. In the shoots, the downregulation increased at 3 h (6239 genes downregulated, vs 66 upregulated). Of all genes affected in roots at 1 h (time of greatest effect), 7.69% of affected genes were for nucleic acid binding functions. Genes for single strand DNA binding proteins (SSBP) WHY1, WHY 2 and WHY3 were strongly downregulated in the shoot up until 12 h after citral exposure. Effects were strong in the root at just 1 h after the treatment and then at 12 and 24 h. Similar effects occurred with the transcription factors MYC-2, ANAC and SCR-SHR, which were also significantly downregulated for the first hour of treatment, and downregulation occurred again after 12 and 24 h treatment. Downregulation of ANAC in the first hour of treatment was significantly (P < 0.0001) decreased more than eight times compared to the control. In silico molecular docking analysis suggests binding of citral isomers to the SSBPs WHY1, WHY2, and WHY3, as well as with other transcription factors such as MYC-2, ANAC and SCR-SHR. Such effects could account for the profound and unusual effects of citral on downregulation of gene transcription.

Repeated polyploidization of Gossypium genomes and the evolution of spinnable cotton fibres.

Polyploidy often confers emergent properties, such as the higher fibre productivity and quality of tetraploid cottons than diploid cottons bred for the same environments. Here we show that an abrupt five- to sixfold ploidy increase approximately 60 million years (Myr) ago, and allopolyploidy reuniting divergent Gossypium genomes approximately 1-2 Myr ago, conferred about 30-36-fold duplication of ancestral angiosperm (flowering plant) genes in elite cottons (Gossypium hirsutum and Gossypium barbadense), genetic complexity equalled only by Brassica among sequenced angiosperms. Nascent fibre evolution, before allopolyploidy, is elucidated by comparison of spinnable-fibred Gossypium herbaceum A and non-spinnable Gossypium longicalyx F genomes to one another and the outgroup D genome of non-spinnable Gossypium raimondii. The sequence of a G. hirsutum A(t)D(t) (in which 't' indicates tetraploid) cultivar reveals many non-reciprocal DNA exchanges between subgenomes that may have contributed to phenotypic innovation and/or other emergent properties such as ecological adaptation by polyploids. Most DNA-level novelty in G. hirsutum recombines alleles from the D-genome progenitor native to its New World habitat and the Old World A-genome progenitor in which spinnable fibre evolved. Coordinated expression changes in proximal groups of functionally distinct genes, including a nuclear mitochondrial DNA block, may account for clusters of cotton-fibre quantitative trait loci affecting diverse traits. Opportunities abound for dissecting emergent properties of other polyploids, particularly angiosperms, by comparison to diploid progenitors and outgroups.

A novel duplex ddPCR assay for the diagnosis of schistosomiasis japonica: proof of concept in an experimental mouse model.

The current World Health Organization strategic plan targets the elimination of schistosomiasis as a public health problem by 2025 and accurate diagnostics will play a pivotal role in achieving this goal. DNA-based detection methods provide a viable alternative to some of the commonly used tests, notably microscopy and serology, for the diagnosis of schistosomiasis. The detection of parasite cell-free DNA in different clinical samples is a recent valuable advance, which provides significant benefits for accurate disease diagnosis. Here we validated a novel duplex droplet digital PCR assay for the diagnosis of Chinese (SjC) and Philippine (SjP) strains of Schistosoma japonicum infection in a mouse model. The assay proved applicable for both SjC and SjP infections and capable of detecting infection at a very early intra-mammalian stage in conveniently obtainable samples (urine and saliva) as well as in serum and feces. The target DNA copy numbers obtained in the assay showed a positive correlation with the infection burden assessed by direct traditional parasitology. The potential to detect parasite DNA in urine and saliva has important practical implications for large-scale epidemiological screening programmes in the future, particularly in terms of logistical convenience, and the assay has the potential to be a valuable additional tool for the diagnosis of schistosomiasis japonica.

Specific humoral response of hosts with variable schistosomiasis susceptibility.

The schistosome blood flukes are some of the largest global causes of parasitic morbidity. Further study of the specific antibody response during schistosomiasis may yield the vaccines and diagnostics needed to combat this disease. Therefore, for the purposes of antigen discovery, sera and antibody-secreting cell (ASC) probes from semi-permissive rats and sera from susceptible mice were used to screen a schistosome protein microarray. Following Schistosoma japonicum infection, rats had reduced pathology, increased antibody responses and broader antigen recognition profiles compared with mice. With successive infections, rat global serological reactivity and the number of recognized antigens increased. The local antibody response in rat skin and lung, measured with ASC probes, increased after parasite migration and contributed antigen-specific antibodies to the multivalent serological response. In addition, the temporal variation of anti-parasite serum antibodies after infection and reinfection followed patterns that appear related to the antigen driving the response. Among the 29 antigens differentially recognized by the infected hosts were numerous known vaccine candidates, drug targets and several S. japonicum homologs of human schistosomiasis resistance markers-the tegument allergen-like proteins. From this set, we prioritized eight proteins that may prove to be novel schistosome vaccine and diagnostic antigens.

Metabolomic and transcriptomic insights into how cotton fiber transitions to secondary wall synthesis, represses lignification, and prolongs elongation.

BACKGROUND: The morphogenesis of single-celled cotton fiber includes extreme elongation and staged cell wall differentiation. Designing strategies for improving cotton fiber for textiles and other uses relies on uncovering the related regulatory mechanisms. In this research we compared the transcriptomes and metabolomes of two Gossypium genotypes, Gossypium barbadense cv Phytogen 800 and G. hirsutum cv Deltapine 90. When grown in parallel, the two types of fiber developed similarly except for prolonged fiber elongation in the G. barbadense cultivar. The data were collected from isolated fibers between 10 to 28 days post anthesis (DPA) representing: primary wall synthesis to support elongation; transitional cell wall remodeling; and secondary wall cellulose synthesis, which was accompanied by continuing elongation only in G. barbadense fiber. RESULTS: Of 206 identified fiber metabolites, 205 were held in common between the two genotypes. Approximately 38,000 transcripts were expressed in the fiber of each genotype, and these were mapped to the reference set and interpreted by homology to known genes. The developmental changes in the transcriptomes and the metabolomes were compared within and across genotypes with several novel implications. Transitional cell wall remodeling is a distinct stable developmental stage lasting at least four days (18 to 21 DPA). Expression of selected cell wall related transcripts was similar between genotypes, but cellulose synthase gene expression patterns were more complex than expected. Lignification was transcriptionally repressed in both genotypes. Oxidative stress was lower in the fiber of G. barbadense cv Phytogen 800 as compared to G. hirsutum cv Deltapine 90. Correspondingly, the G. barbadense cultivar had enhanced capacity for management of reactive oxygen species during its prolonged elongation period, as indicated by a 138-fold increase in ascorbate concentration at 28 DPA. CONCLUSIONS: The parallel data on deep-sequencing transcriptomics and non-targeted metabolomics for two genotypes of single-celled cotton fiber showed that a discrete developmental stage of transitional cell wall remodeling occurs before secondary wall cellulose synthesis begins. The data showed how lignification can be transcriptionally repressed during secondary cell wall synthesis, and they implicated enhanced capacity to manage reactive oxygen species through the ascorbate-glutathione cycle as a positive contributor to fiber length.

Development of a Large Set of Microsatellite Markers in Zapote Mamey (Pouteria sapota (Jacq.) H.E. Moore & Stearn) and Their Potential Use in the Study of the Species.

Pouteria sapota is known for its edible fruits that contain unique carotenoids, as well as for its fungitoxic, anti-inflammatory and anti-oxidant activity. However, its genetics is mostly unknown, including aspects about its genetic diversity and domestication process. We did high-throughput sequencing of microsatellite-enriched libraries of P. sapota, generated 5223 contig DNA sequences, 1.8 Mbp, developed 368 microsatellites markers and tested them on 29 individuals from 10 populations (seven wild, three cultivated) from Mexico, its putative domestication center. Gene ontology BLAST analysis of the DNA sequences containing microsatellites showed potential association to physiological functions. Genetic diversity was slightly higher in cultivated than in the wild gene pool (HE = 0.41 and HE = 0.35, respectively), although modified Garza-Williamson Index and Bottleneck software showed evidence for a reduction in genetic diversity for the cultivated one. Neighbor Joining, 3D Principal Coordinates Analysis and assignment tests grouped most individuals according to their geographic origin but no clear separation was observed between wild or cultivated gene pools due to, perhaps, the existence of several admixed populations. The developed microsatellites have a great potential in genetic population and domestication studies of P. sapota but additional sampling will be necessary to better understand how the domestication process has impacted the genetic diversity of this fruit crop.

Anthelminthic activity of the cyclotides (kalata B1 and B2) against schistosome parasites.

The risk of reduced sensitivity of the human schistosomes to praziquantel has led to efforts to find new therapies. Here, the cyclotides kalata B1 (kB1), kalata B2 (kB2), MCoCC-1, and MCoTI-II, cyclic peptides extracted from plants and shown to be potent against nematodes and insects, were tested for antischistosome activity. In vitro assays showed that high concentrations (500-1000 µg/mL) of either kB1 or kB2 killed Schistosoma japonicum and Schistosoma mansoni adults within 5 min, whereas MCoTI-II and MCoCC-1 had no effect. Lethal concentrations to kill 50% of the population for kB2 was 15.5 ± 7.4 µg/mL at 1 h for male S. japonicum (Philippine strain). Males were more susceptible than females. kB2 showed higher antischistosome activity than kB1 and killing time was concentration-dependent. Mode of action studies revealed that kB1 and 2 lysed the tegument of adult worms. Lysis of myofibrils was not demonstrated, but longitudinal and radial muscle fibers were distorted, an observation consistent with strong coiling of the parasites after drug exposure. A single dose of kB2 administered either orally or intravenously, reduced worm burdens in S. japonicum-infected mice from 15% to 60%. However, treatment of S. mansoni-infected mice did not result in reduction in worm burdens. Our studies show that kB2 acts as a promising antischistosomal against Philippine S. japonicum, and it or other cyclotides may be developed further as general anthelminthics. With thousands of cyclotides predicted to occur in plants, and the amenability of these peptides to combinatorial variation, there is potential for their exploitation as wide-spectrum anthelminthics.

Tetraspanins on the surface of Schistosoma mansoni are protective antigens against schistosomiasis.

Schistosomes are blood-dwelling flukes that infect 200 million people worldwide and are responsible for hundreds of thousands of deaths annually. Using a signal sequence trap, we cloned from Schistosoma mansoni two cDNAs, Sm-tsp-1 and Sm-tsp-2, encoding the tetraspanin (TSP) integral membrane proteins TSP-1 and TSP-2. We raised antibodies to recombinant TSP fusion proteins and showed that both proteins are exposed on the surface of S. mansoni. Recombinant TSP-2, but not TSP-1, is strongly recognized by IgG1 and IgG3 (but not IgE) from naturally resistant individuals but is not recognized by IgG from chronically infected or unexposed individuals. Vaccination of mice with the recombinant proteins followed by challenge infection with S. mansoni resulted in reductions of 57% and 64% (TSP-2) and 34% and 52% (TSP-1) for mean adult worm burdens and liver egg burdens, respectively, over two independent trials. Fecal egg counts were reduced by 65-69% in both test groups. TSP-2 in particular provided protection in excess of the 40% benchmark set by the World Health Organization for progression of schistosome vaccine antigens into clinical trials. When coupled with its selective recognition by naturally resistant people, TSP-2 seems to be an effective vaccine antigen against S. mansoni.

Development of CRISPR/Cas13a-based assays for the diagnosis of Schistosomiasis.

BACKGROUND: Schistosomiasis is a disease that significantly impacts human health in the developing world. Effective diagnostics are urgently needed for improved control of this disease. CRISPR-based technology has rapidly accelerated the development of a revolutionary and powerful diagnostics platform, resulting in the advancement of a class of ultrasensitive, specific, cost-effective and portable diagnostics, typified by applications in COVID-19/cancer diagnosis. METHODS: We developed CRISPR-based diagnostic platform SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing) for the detection of Schistosoma japonicum and S. mansoni by combining recombinase polymerase amplification (RPA) with CRISPR-Cas13a detection, measured via fluorescent or colorimetric readouts. We evaluated SHERLOCK assays by using 150 faecal/serum samples collected from Schistosoma-infected ARC Swiss mice (female), and 189 human faecal/serum samples obtained from a S. japonicum-endemic area in the Philippines and a S. mansoni-endemic area in Uganda. FINDINGS: The S. japonicum SHERLOCK assay achieved 93-100% concordance with gold-standard qPCR detection across all the samples. The S. mansoni SHERLOCK assay demonstrated higher sensitivity than qPCR and was able to detect infection in mouse serum as early as 3 weeks post-infection. In human samples, S. mansoni SHERLOCK had 100% sensitivity when compared to qPCR of faecal and serum samples. INTERPRETATION: These schistosomiasis diagnostic assays demonstrate the potential of SHERLOCK/CRISPR-based diagnostics to provide highly accurate and field-friendly point-of-care tests that could provide the next generation of diagnostic and surveillance tools for parasitic neglected tropical diseases. FUNDING: Australian Infectious Diseases Research Centre seed grant (2022) and National Health and Medical Research Council (NHMRC) of Australia (APP1194462, APP2008433).

Immune-mediated mechanisms of parasite tissue sequestration during experimental cerebral malaria.

Cerebral malaria is a severe complication of malaria. Sequestration of parasitized RBCs in brain microvasculature is associated with disease pathogenesis, but our understanding of this process is incomplete. In this study, we examined parasite tissue sequestration in an experimental model of cerebral malaria (ECM). We show that a rapid increase in parasite biomass is strongly associated with the induction of ECM, mediated by IFN-gamma and lymphotoxin alpha, whereas TNF and IL-10 limit this process. Crucially, we discovered that host CD4(+) and CD8(+) T cells promote parasite accumulation in vital organs, including the brain. Modulation of CD4(+) T cell responses by helminth coinfection amplified CD4(+) T cell-mediated parasite sequestration, whereas vaccination could generate CD4(+) T cells that reduced parasite biomass and prevented ECM. These findings provide novel insights into immune-mediated mechanisms of ECM pathogenesis and highlight the potential of T cells to both prevent and promote infectious diseases.

Analysis of rhodopsin G protein-coupled receptor orthologs reveals semiochemical peptides for parasite (Schistosoma mansoni) and host (Biomphalaria glabrata) interplay.

Schistosomiasis is a medically significant disease caused by helminth parasites of the genus Schistosoma. The schistosome life cycle requires chemically mediated interactions with an intermediate (aquatic snail) and definitive (human) host. Blocking parasite development within the snail stage requires improved understanding of the interactions between the snail host and the Schistosoma water-borne free-living form (miracidium). Innovations in snail genomics and aquatic chemical communication provide an ideal opportunity to explore snail-parasite coevolution at the molecular level. Rhodopsin G protein-coupled receptors (GPCRs) are of particular interest in studying how trematode parasites navigate towards their snail hosts. The potential role of GPCRs in parasites makes them candidate targets for new antihelminthics that disrupt the intermediate host life-cycle stages, thus preventing subsequent human infections. A genomic-bioinformatic approach was used to identify GPCR orthologs between the snail Biomphalaria glabrata and miracidia of its obligate parasite Schistosoma mansoni. We show that 8 S. mansoni rhodopsin GPCRs expressed within the miracidial stage share overall amino acid similarity with 8 different B. glabrata rhodopsin GPCRs, particularly within transmembrane domains, suggesting conserved structural features. These GPCRs include an orphan peptide receptor as well as several with strong sequence homologies with rhabdomeric opsin receptors, a serotonin receptor, a sulfakinin (SK) receptor, an allatostatin-A (buccalin) receptor and an FMRFamide receptor. Buccalin and FMRFa peptides were identified in water conditioned by B. glabrata, and we show synthetic buccalin and FMRFa can stimulate significant rates of change of direction and turn-back responses in S. mansoni miracidia. Ortholog GPCRs were identified in S. mansoni miracidia and B. glabrata. These GPCRs may detect similar ligands, including snail-derived odorants that could facilitate miracidial host finding. These results lay the foundation for future research elucidating the mechanisms by which GPCRs mediate host finding which can lead to the potential development of novel anti-schistosome interventions.

Occupational Therapy Utilization in Veterans With Dementia: A Retrospective Review of Root Cause Analyses of Falls Leading to Adverse Events.

INTRODUCTION: Falls in persons with dementia are associated with increased mortality. Occupational therapy (OT) is a rehabilitation discipline, which has, among its goals, the promotion of safety and fall prevention in older adults and those with dementia. The purpose of this study was to evaluate root cause analysis (RCA) data to identify causes of falls with adverse events in patients with dementia who were referred to or receiving OT services within the Veterans Health Administration (VHA). METHODS: This study used retrospective review of RCAs within the National Center for Patient Safety database for the VHA. The RCA database was searched using these terms: falls with adverse events, dementia, and OT. Descriptive statistical analysis of demographic information, location, occurrence of orthopedic fracture, and mortality was used. All root causes were qualitatively categorized using thematic analysis of determined causes. RESULTS: Eighty RCAs were included in analysis. Mean age of veterans included was 80 years; 96% were male; 76% resulted in hip fracture; and 20% died as a result of the fall. Occupational therapy evaluations occurred within 7 days of admission to VHA and falls most frequently occurred within 4 days of OT evaluation. Most common causes included inappropriate or lack of equipment (21%), need for falls/rehabilitation assessment (20%), compliance/training to fall protocol of all staff (19%), and behavior/medical status (17%). CONCLUSIONS: Earlier identification for OT evaluation need may improve access to services, and use of proper equipment to decrease frequency of falls may improve patient safety for older adults with dementia.

Identification of Schistosoma mansoni miracidia attractant candidates in infected Biomphalaria glabrata using behaviour-guided comparative proteomics.

Schistosomiasis, caused by infection with Schistosoma digenetic trematodes, is one of the deadliest neglected tropical diseases in the world. The Schistosoma lifecycle involves the miracidial infection of an intermediate freshwater snail host, such as Biomphalaria glabrata. Dispersing snail host-derived Schistosoma miracidia attractants has been considered a method of minimising intermediate host infections and, by extension, human schistosomiasis. The attractiveness of B. glabrata to miracidia is known to be reduced following infection; however, the relationship between duration of infection and attractiveness is unclear. Excretory-secretory proteins (ESPs) most abundant in attractive snail conditioned water (SCW) are key candidates to function as miracidia attractants. This study analysed SCW from B. glabrata that were naïve (uninfected) and at different time-points post-miracidia exposure (PME; 16h, 1-week, 2-weeks and 3-weeks PME) to identify candidate ESPs mediating Schistosoma mansoni miracidia behaviour change, including aggregation and chemoklinokinesis behaviour (random motion, including slowdown and increased turning rate and magnitude). Miracidia behaviour change was only observed post-addition of naïve and 3W-PME SCW, with other treatments inducing significantly weaker behaviour changes. Therefore, ESPs were considered attractant candidates if they were shared between naïve and 3W-PME SCW (or exclusive to the former), contained a predicted N-terminal signal peptide and displayed low identity (<50%) to known proteins outside of the Biomphalaria genus. Using these criteria, a total of 6 ESP attractant candidates were identified, including acetylcholine binding protein-like proteins and uncharacterised proteins. Tissue-specific RNA-seq analysis of the genes encoding these 6 ESPs indicated relatively high gene expression within various B. glabrata tissues, including the foot, mantle and kidney. Acetylcholine binding protein-like proteins were highly promising due to their high abundance in naïve and 3W-PME SCW, high specificity to B. glabrata and high expression in the ovotestis, from which attractants have been previously identified. In summary, this study used proteomics, guided by behavioural assays, to identify miracidia attractant candidates that should be further investigated as potential biocontrols to disrupt miracidia infection and minimise schistosomiasis.

Identification of Putative Neuropeptides That Alter the Behaviour of Schistosoma mansoni Cercariae.

Elucidating the infectivity of Schistosoma mansoni, one of the main etiological agents of human schistosomiasis, requires an improved understanding of the behavioural mechanisms of cercariae, the non-feeding mammalian infective stage. This study investigated the presence and effect of cercariae-derived putative neuropeptides on cercarial behaviour when applied externally. Cercariae were peptidomically analysed and 11 neuropeptide precursor proteins, all of which were specific to the Schistosoma genus and most of which highly expressed in the cercarial stage, were identified in cercariae for the first time. Protein-protein interaction analysis predicted the interaction of various neuropeptide precursors (e.g., Sm-npp-30, Sm-npp-33, Sm-npp-35) with cercarial structural proteins (e.g., myosin heavy chain and titin). In total, nine putative neuropeptides, selected based on their high hydrophobicity and small size (~1 kilodalton), were tested on cercariae (3 mg/mL) in acute exposure (1 min) and prolonged exposure (360 min) behavioural bioassays. The peptides AAYMDLPW-NH2, NRKIDQSFYSYY-NH2, FLLALPSP-OH, and NYLWDTRL-NH2 stimulated acute increases in cercarial spinning, stopping, and directional change during active states. However, only NRKIDQSFYSYY-NH2 caused the same behavioural changes at a lower concentration (0.1 mg/mL). After prolonged exposure, AAYMDLPW-NH2 and NYLWDTRL-NH2 caused increasing passive behaviour and NRKIDQSFYSYY-NH2 caused increasing body-first and head-pulling movements. These findings characterise behaviour-altering novel putative neuropeptides, which may inform future biocontrol innovations to prevent human schistosomiasis.

Transcriptomic Responses of Fusarium verticillioides to Lactam and Lactone Xenobiotics.

The important cereal crops of maize, rye, and wheat constitutively produce precursors to 2-benzoxazolinone, a phytochemical having antifungal effects towards many Fusarium species. However, Fusarium verticillioides can tolerate 2-benzoxazolinone by converting it into non-toxic metabolites through the synergism of two previously identified gene clusters, FDB1 and FDB2. Inspired by the induction of these two clusters upon exposure to 2-benzoxazolinone, RNA sequencing experiments were carried out by challenging F. verticillioides individually with 2-benzoxazolinone and three related chemical compounds, 2-oxindole, 2-coumaranone, and chlorzoxazone. These compounds all contain lactam and/or lactone moieties, and transcriptional analysis provided inferences regarding the degradation of such lactams and lactones. Besides induction of FDB1 and FDB2 gene clusters, four additional clusters were identified as induced by 2-benzoxazolinone exposure, including a cluster thought to be responsible for biosynthesis of pyridoxine (vitamin B6), a known antioxidant providing tolerance to reactive oxygen species. Three putative gene clusters were identified as induced by challenging F. verticillioides with 2-oxindole, two with 2-coumaranone, and two with chlorzoxazone. Interestingly, 2-benzoxazolinone and 2-oxindole each induced two specific gene clusters with similar composition of enzymatic functions. Exposure to 2-coumranone elicited the expression of the fusaric acid biosynthetic gene cluster. Another gene cluster that may encode enzymes responsible for degrading intermediate catabolic metabolites with carboxylic ester bonds was induced by 2-benzoxazolinone, 2-oxindole, and chlorzoxazone. Also, the induction of a dehalogenase encoding gene during chlorzoxazone exposure suggested its role in the removal of the chlorine atom. Together, this work identifies genes and putative gene clusters responsive to the 2-benzoxazolinone-like compounds with metabolic inferences. Potential targets for future functional analyses are discussed.

Transcriptomic Response of Fusarium verticillioides to Variably Inhibitory Environmental Isolates of Streptomyces.

Fusarium verticillioides is a mycotoxigenic fungus that is a threat to food and feed safety due to its common infection of maize, a global staple crop. A proposed strategy to combat this threat is the use of biological control bacteria that can inhibit the fungus and reduce mycotoxin contamination. In this study, the effect of multiple environmental isolates of Streptomyces on F. verticillioides was examined via transcriptome analysis. The Streptomyces strains ranged from inducing no visible response to dramatic growth inhibition. Transcriptionally, F. verticillioides responded proportionally to strain inhibition with either little to no transcript changes to thousands of genes being differentially expressed. Expression changes in multiple F. verticillioides putative secondary metabolite gene clusters was observed. Interestingly, genes involved in the fusaric acid gene cluster were suppressed by inhibitory strains of Streptomyces. A F. verticillioides beta-lactamase encoding gene (FVEG_13172) was found to be highly induced by specific inhibitory Streptomyces strains and its deletion increased visible response to those strains. This study demonstrates that F. verticillioides does not have an all or nothing response to bacteria it encounters but rather a measured response that is strain specific and proportional to the strength of inhibition.