Francisella tularensis subspecies holarctica isolated from 3 patients who had been bitten by arthropods. Next-generation sequencing showed substantial isolate similarity. Clinicians should consider bloodstream F. tularensis infections for patients with signs/symptoms of ulceroglandular tularemia, and surveillance of potential vectors should be intensified.Tularemia is increasing in Austria. We report  Francisella tularensis. Although F. tularensis subspecies tularensis  is associated with severe infections, F. tularensis subsp. holarctica  causes less severe illness  led to sign/symptom resolution.Patient 1 was a 64-year-old woman who had received a tick bite in the right inframammary region. After experiencing fever, chills, and a general feeling of discomfort, along with nausea and vomiting, she sought emergency care when symptoms persisted. Clinical examination detected a 20-mm skin lesion surrounded by a reddish hem and central necrotic ulcer. Results of serologic testing for Lyme borreliosis were negative. Stab incision of the skin lesion was performed, and no bacteria grew in culture of the sampled pus. Blood cultures grew F. tularensis. Antimicrobial treatment, changed to oral ciprofloxacin (250 mg 2×/d), led to substantial signs/symptom improvement.Patient 2 was a 5-year-old girl seen at a pediatric outpatient clinic for retroauricular pain after a tick bite 9 days earlier. Clinical examination showed a swollen and reddened pinna, a pus-covered 5-mm retroauricular skin lesion with a central ulcer  Figure, F. tularensis grew on anaerobic blood culture. Therapy with ciprofloxacin was initiated and later switched to oral doxycycline (100 mg 2×/d) because of potential allergic reaction to ciprofloxacin, followed by substantial sign/symptom improvement. The findings suggest local transmission of F. tularensis after an insect or tick bite.Patient 3 was a 76-year-old man who was hospitalized after reporting fever, generalized discomfort, nausea, and vertigo. A red, painless skin lesion with central crust on the left thigh, which probably developed after an arthropod bite, and inguinal lymphadenopathy were noted. Antimicrobial treatment with ampicillin/sulbactam was started because of suspected pneumonia. Eight days after hospitalization, https://www.analyticon.eu/de) to test samples, and all were positive for F. tularensis. Real-time PCR , performed for subspecies determination according to manufacturer instructions, yielded F. tularensis subsp. holarctica (https://www.biomerieux.fr) and interpreted results according to Clinical and Laboratory Standards Institute guidelines (F. tularensis subsp. holarctica biovar II (genotype B.12), which was later confirmed by whole-genome sequencing (WGS) to be subclade B.34/B.35.We used multiplex PCR , 150-bp paired-end, by using a QIAGEN MagAttract HMW DNA Kit (https://www.qiagen.com) for DNA isolation and a Nextera XT DNA Library Prep Kit  for library preparation. We assembled whole-genome sequences de novo by using SPAdes (https://www.ridom.de) with the published core-genome multilocus sequence typing scheme : biosample SAMN31677967 , accession no. JAPKFK000000000; biosample SAMN31677968 , accession no. JAPKFJ000000000; and biosample SAMN32382778 , accession no. JAPZIK000000000  is found mainly in western Europe and biovar II (genotype B.12) in northern and eastern Europe  and ticks as vectors for tularemia in Austria.Bloodstream infections with  genomes  Table, wF. tularensis transmission in Austria. Aside from other tickborne diseases endemic to Austria, clinicians should consider tularemia as a cause of signs/symptoms that follow tick bites, especially when combined with fever, enlarged and painful lymph nodes, and skin ulcers. Diagnosis can be achieved by molecular testing. Genomic data are essential for understanding dissemination and invasion of certain genotypes that may cause systemic infections. To confidently declare that certain genetic subpopulations are associated with systemic infections, a larger number of isolates and further research are needed. For that reason, Austria established large-scale monitoring of arthropod vectors for the presence of vectorborne pathogens, including F. tularensis, to provide public health authorities with knowledge about infection risk for the exposed population.Our findings show that ticks represent underestimated vectors for Francisella tularensis infection, Austria, 2022.Additional information for 3 cases of tickborne


Bloodstream infections (BSIs) produced by antibiotic-resistant bacteria (ARB) cause a substantial disease burden worldwide. However, most estimates come from high-income settings and thus are not globally representative. This study quantifies the excess mortality, length of hospital stay (LOS), intensive care unit (ICU) admission, and economic costs associated with ARB BSIs, compared to antibiotic-sensitive bacteria (ASB), among adult inpatients in low- and middle-income countries (LMICs).n = 13,012 from their inception to August 1, 2022). We only included quantitative studies. Our final sample consisted of n = 109 articles, excluding studies from high-income countries, without our outcomes of interest, or without a clear source of bloodstream infection. Crude mortality, ICU admission, and LOS were meta-analysed using the inverse variance heterogeneity model for the general and subgroup analyses including bacterial Gram type, family, and resistance type. For economic costs, direct medical costs per bed-day were sourced from WHO-CHOICE. Mortality costs were estimated based on productivity loss from years of potential life lost due to premature mortality. All costs were in 2020 USD. We assessed studies’ quality and risk of publication bias using the MASTER framework. Multivariable meta-regressions were employed for the mortality and ICU admission outcomes only. Most included studies showed a significant increase in crude mortality (odds ratio (OR) 1.58, 95% CI [1.35 to 1.80], p < 0.001), total LOS , and ICU admission  for ARB versus ASB BSIs. Studies analysing Enterobacteriaceae, Acinetobacter baumanii, and Staphylococcus aureus in upper-middle-income countries from the African and Western Pacific regions showed the highest excess mortality, LOS, and ICU admission for ARB versus ASB BSIs per patient. Multivariable meta-regressions indicated that patients with resistant Acinetobacter baumanii BSIs had higher mortality odds when comparing ARB versus ASB BSI patients . Excess direct medical costs were estimated at $12,442  for ARB versus ASB BSI per patient, with an average cost of $41,103  due to premature mortality. Limitations included the poor quality of some of the reviewed studies regarding the high risk of selective sampling or failure to adequately account for relevant confounders.We conducted a systematic review by searching 4 medical databases  caused by antibiotic-resistant bacteria (ARB) have multifaceted impacts, including higher admission to intensive care units (ICUs), prolonged hospitalisations, and high economic and societal costs worldwide.Despite the global burden, most evidence on the excess burden of ARB BSIs has been derived from high-income countries; comparatively, there are limited data from low- and middle-income countries (LMICs).We employed a systematic literature review and subsequent meta-analysis of 109 individual studies to quantify the impact of ARB BSIs in hospitalised patients from LMICs.Based mostly on crude data comparisons ignoring the possible influence of confounding factors, we found that ARB BSIs, compared to BSIs caused by antibiotic-sensitive bacteria (ASB), were associated with substantially longer stays in hospitals and ICUs, higher mortality, and increased direct medical and productivity costs.Our findings highlight the excess morbidity, mortality, and costs associated with ARB BSIs and the sparsity of data from LMICs.Targeted strategies to improve the prevention, detection, and treatment of resistant BSIs in LMICs are required to reduce the economic and disease burden. Escherichia coli bloodstream infections (BSIs) caused by third-generation cephalosporins resistant E. coli was more than triple in LMICs compared to high-income countries,  ), and high variation for ICU admission  and LOS  for the meta-analysis run by specific groups  compared to the average mortality estimates reported in When applying the leave-one-out method to our meta-analyses, we observed that after assessing the effect of every single study on the overall estimates, the numbers presented a relative variation with respect to overall estimates ranging between −2% and 4% for mortality (OR 95% CI [1.57 to 1.58]), −8% and 4% for ICU admission (OR 95% CI [1.95 to 1.97]), and −10% and 4% for LOS (SMD 95% CI [0.48 to 0.50]) . Others have also shown considerably increased LOS as a result of MRSA compared with sensitive S. aureus: Tsuzuki and colleagues  and high-scores [HS] obtained from the MASTER scale. Table Z. Checklist of information that should be included in new reports of global health estimates. Table AA. PRISMA Checklist. Fig A. Density of the studies over time. Fig B. Violin and kernel density estimate plots for the main outcomes and by ARB susceptibility. Fig C. Relationship between the main outcomes. Fig D. Meta-analysis using all the studies reporting mortality rates. Fig E. Subgroup meta-analysis using all the studies reporting mortality rates/odds for critical (N = 72) and high-priority (N = 22) pathogens according to the WHO criteria. Fig F. Subgroup meta-analysis using all the studies reporting mortality rates by bacterium’s family name. Fig G. Subgroup meta-analysis using all the studies reporting mortality rates by WHO Region. Fig H. Subgroup meta-analysis using all the studies reporting mortality rates by income level. Fig I. Meta-analysis results using all the studies reporting the mean and SD for the length of stay at the hospital. Fig J. Subgroup meta-analysis using all the studies reporting the mean and SD for the length of stay at the hospital for critical and high-priority pathogens according to the WHO. Fig K. Subgroup meta-analysis using all the studies reporting the mean and SD for the length of stay at the hospital for Enterococcus spp., Enterobacteriaceae, Moraxellaceae, Pseudomonadaceae, and Staphyloccocaceae. Fig L. Subgroup meta-analysis using all the studies reporting the mean and SD for the length of stay at the hospital by income level. Fig M. Subgroup meta-analysis using all the studies reporting the mean and SD for the length of stay at the hospital by WHO region. Fig N. Meta-analysis results using all the studies reporting ICU admission rates. Fig O. Subgroup meta-analysis using all the studies reporting ICU admission rates for critical pathogens according to the WHO criteria. Fig P. Subgroup meta-analysis using all the studies reporting ICU admission rates for high-priority pathogens according to the WHO criteria. Fig Q. Subgroup meta-analysis using all the studies reporting ICU admission rates for Enterobacteriaceae. Fig R. Subgroup meta-analysis using all the studies reporting ICU admission rates for Enterobacteriaceae. Fig S. Subgroup meta-analysis using all the studies reporting ICU admission rates for Moraxellaceae. Fig T. Subgroup meta-analysis using all the studies reporting ICU admission rates for Pseudomonadaceae. Fig U. Subgroup meta-analysis using all the studies reporting ICU admission rates for Staphylococcaceae. Fig V. Subgroup meta-analysis using all the studies reporting ICU admission rates by resistance type (ESBL+). Fig W. Subgroup meta-analysis using all the studies reporting ICU admission rates by WHO region: Americas. Fig X. Subgroup meta-analysis using all the studies reporting ICU admission rates by WHO region: Eastern Mediterranean. Fig Y. Subgroup meta-analysis using all the studies reporting ICU admission rates by WHO region: Europe. Fig Z. Subgroup meta-analysis using all the studies reporting ICU admission rates by WHO region: Southeast Asia. Fig AA. Subgroup meta-analysis using all the studies reporting ICU admission rates by WHO region: Western Pacific region. Fig AB. Subgroup meta-analysis using all the studies reporting ICU admission rates by income level: Low and lower-middle income countries. Fig AC. Subgroup meta-analysis using all the studies reporting ICU admission rates by income level: Upper-middle income countries. Fig AD. Subgroup analysis for studies reporting unadjusted ORs. Fig AE. Subgroup analysis for studies reporting unadjusted ORs, by bacteria’s gram type or WHO criticality category . Fig AF. Subgroup analysis for studies reporting unadjusted ORs, by specific bacterium. Fig AG. Subgroup analysis for studies reporting adjusted ORs. Fig AH. Subgroup analysis for studies reporting adjusted ORs, by bacteria’s gram type . Fig AI. Subgroup analysis for studies reporting adjusted ORs, by specific bacterium. Fig AJ. Subgroup analysis for studies reporting adjusted and unadjusted ORs simultaneously, general mortality estimates. Fig AK. Subgroup analysis for studies reporting adjusted and unadjusted ORs simultaneously, mortality rates by Gram type or WHO criticality list classification . Fig AL. Subgroup analysis for studies reporting adjusted and unadjusted ORs simultaneously, mortality rates by bacterium family. Fig AM. Doi plots for Model 1  and by outcome based on Tables Q, R, and S. Fig AN. Funnel plots for Model 1  and by outcome based on Tables Q, R, and S. Fig AO. Influence analysis for Model 1 using the mortality outcome compared to the general estimates and without subgroup analyses. Fig AP. Influence analysis for Model 1 using the ICU admission outcome compared to the general estimates and without subgroup analyses. Fig AQ. Influence analysis for Model 1 using the length of hospital stay outcome compared to the general estimates and without subgroup analyses. Fig AR. Meta-analysis results disaggregated by specific and prioritised antibiotic-bacterium pairs for mortality. Fig AS. Meta-analysis results disaggregated by carbapenem-resistant Enterobacteriaceae for mortality. Fig AT. Meta-analysis results disaggregated by specific and prioritised antibiotic-bacterium pairs for LOS. Fig AU. Meta-analysis results disaggregated by carbapenem-resistant Enterobacteriaceae for LOS. Fig AV. Meta-analysis results disaggregated by specific and prioritised antibiotic-bacterium pairs for ICU admission. Fig AW. Meta-analysis results disaggregated by carbapenem-resistant Enterobacteriaceae for ICU admission. Fig AX. Graphical results of Table V. Fig AY. Distribution of the Master scale scores by outcome. Fig AZ. Kernel density estimate of the Master scale scores by outcome. Fig BA. Percentage of full completion by MASTER scale main safeguard and outcome.(PDF)Click here for additional data file.S1 DataMasterData spreadsheet. Description and data extracted from each included study. MasterScale spreadsheet. Application of the MASTER scale by outcome and study. Summary MasterScale spreadsheet. Summary statistics per safeguard/item of the application of the MASTER scale.(XLSX)Click here for additional data file.


Biomphalaria pfeifferi is the world’s most widely distributed and commonly implicated vector snail species for the causative agent of human intestinal schistosomiasis, Schistosoma mansoni. In efforts to control S. mansoni transmission, chemotherapy alone has proven insufficient. New approaches to snail control offer a way forward, and possible genetic manipulations of snail vectors will require new tools. Towards this end, we here offer a diverse set of genomic resources for the important African schistosome vector, B. pfeifferi.B. pfeifferi (Kenya), smaller in size than known genomes of other planorbid schistosome vectors. In a total of 505 scaffolds (N50 = 3.2Mb), 430 were assigned to 18 large linkage groups inferred to represent the 18 known chromosomes, based on whole genome comparisons with Biomphalaria glabrata. The annotated B. pfeifferi genome reveals a divergence time of 3.01 million years with B. glabrata, a South American species believed to be similar to the progenitors of B. pfeifferi which undertook a trans-Atlantic colonization < five million years ago.Based largely on PacBio High-Fidelity long reads, we report a genome assembly size of 772 Mb for FReD gene family which is far more similar in its composition to B. glabrata than to Bulinus truncatus, a vector for Schistosoma haematobium. Provision of this annotated genome will help better understand the dependencies of trematodes on snails, enable broader comparative insights regarding factors contributing to susceptibility/ resistance of snails to schistosome infections, and provide an invaluable resource with respect to identifying and manipulating snail genes as potential targets for more specific snail control programs.The genome for this preferentially self-crossing species is less heterozygous than related species known to be preferential out-crossers; its smaller genome relative to congeners may similarly reflect its preference for selfing. Expansions of gene families with immune relevance are noted, including the  Biomphalaria pfeifferi is the world’s most widely distributed and commonly implicated vector snail for the causative agent of human intestinal schistosomiasis, Schistosoma mansoni. Understanding the basis of host-parasite compatibility relies on functional genomic resources, which will be helpful for developing genetic-based control of schistosomes and their vector snails. Here we report a high-quality, de novo–assembled whole genome for B. pfeifferi (PacBio High-Fidelity long reads), with in-depth gene model annotation for protein-coding and nonprotein-coding genes. Using these enriched molecular data, a divergence date of 3.01 Mya was calculated for B. pfeifferi and B. glabrata, consistent with the idea of Biomphalaria colonization of Africa from South America, and placing an upper boundary on the age of S. mansoni in Africa. The spread of S. mansoni was likely favored by its high degree of compatibility with B. pfeifferi-like snails. In addition, our evidence supports the idea that B. pfeifferi is a preferentially self-crossing species with a more homozygous and smaller genome than related species like B. glabrata known to be preferential out-crossers. Furthermore, several immune-related gene families have expanded differently between B. pfeifferi to B. glabrata. Resources provided here will deepen our general knowledge of molluscan biology, host-parasite co-evolution, vector biology, and genomics. Biomphalaria pfeifferi  is a freshwater planorbid gastropod widely distributed across sub-Saharan Africa and Madagascar, with populations also found in the Sahara and southwest Asia [Schistosoma mansoni, the world’s most common causative agent of intestinal schistosomiasis [ Krauss, 848 is a est Asia . The impsomiasis .Schistosomiasis remains entrenched as one of the world’s most prevalent and debilitating neglected tropical diseases (NTDs), infecting over 200 million people, most of whom live in sub-Saharan Africa . At a tiB. pfeifferi, and release multitudes of free-swimming cercariae into the water that are remarkably effective in finding and penetrating the skin of human hosts. Aquatic habitats and the vector snails living in them lie at the heart of schistosomiasis transmission and as the world’s demands for shrinking aquatic resources increase, there will be profound changes in store for snails and the schistosomes they host [Throughout much of sub-Saharan Africa, the underlying conditions that have always favored the transmission of schistosomiasis remain prevalent. Poverty, associated with poor sanitation, use of surface waters from natural habitats for basic needs, and limited access to health services, continue to enable the transmission of schistosomiasis . Althoughey host –10.Biomphalaria are known to be susceptible to S. mansoni infection [B. pfeifferi, the related species B. glabrata is particularly noteworthy, not only because it serves as an important vector of S. mansoni in the Neotropics, but also because genome resources are available for it [B. straminea, for which a genome sequence has also recently been produced [S. mansoni in South America, and has proved to be invasive, having colonized southeast Asia where its role as a potential host for S. mansoni has become a concern [Bulinus truncatus, a related planorbid snail that hosts Schistosoma haematobium, the causative agent of urinary schistosomiasis, has also recently been provided [Approximately 18 of the world’s 34 species of nfection . In addie for it . Additioproduced , plays a concern ,15. A geprovided .Biomphalaria pfeifferi is notable among Biomphalaria species for two reasons. Across its broad geographic range, individuals highly susceptible to S. mansoni infections are present, regardless of the geographic origin of the S. mansoni isolate being tested [Biomphalaria colonist, and its potential role as an ancestral host for S. mansoni as the latter arose in Africa [B. pfeifferi is distinctive because, although capable of out-crossing, it is a strong preferential self-fertilizer [B. pfeifferi of freshwater habitats on a large area of land reclaimed for agriculture as a result of construction of the Diama dam on the Senegal River contributed to an epidemic of S. mansoni occurring in the region thereafter [B. pfeifferi populations may also help explain the high susceptibility of at least some lineages of B. pfeifferi to potentially fast-evolving parasites like S. mansoni, but also to several additional trematode species, at least 19 of which are known to infect this snail [g tested –20. Thisn Africa ,21. Secortilizer ,23. Thisereafter . Thus, tis snail .B. pfeifferi might be exploited to achieve control.Schistosomiasis control efforts are still remarkably unidimensional and rely heavily on treatment of people with praziquantel both to improve individual health and to diminish transmission. The molluscan vectors of schistosomes have long been recognized as a much-needed alternative target for schistosomiasis control efforts, but efforts in this sphere have not proceeded past basic approaches such as the use of molluscicides, habitat modifications or introductions of potential snail predators or competitors . To makeB. pfeifferi exposed to S. mansoni and molluscicides have been undertaken [B. pfeifferi are surprisingly limited compared to some other schistosome snail vector species  the nature and consequences of its peculiar population structure, and how it differs from other Biomphalaria species in this regard, and how we might manipulate it to our advantage; 2) how B. pfeifferi responds rapidly to environmental change and how it might respond to looming long-term environmental changes like warming climates or increased pollution; 3) how we might devise specific ways to disrupt snail reproduction or selectively kill schistosome-infected snails; 4) the symbiont community supported by B. pfeifferi, including potential snail pathogens or essential metabolic partners; 5) how we might alter or enhance the ability of B. pfeifferi to overcome its susceptibility to schistosome infection, such as by diminishing its attractiveness for schistosome infectious stages or by enhancing immune responsiveness; and 6) add to the growing database of molluscan genomics that will provide increasing general insights into the many unique features of the Phylum Mollusca.Our hope is additional genomic information for B. pfeifferi, one which has been interrogated for its representation of several gene families believed to be relevant to its role in serving as an intermediate host for S. mansoni. Furthermore, we provide comparisons where possible with other schistosome snail hosts for which genome information is available, as part of an ongoing process to develop more extensive genome resources that will aid in answering the many daunting questions, we have posed above.Below we detail the results obtained from application of PacBio HiFi long-read sequencing technology to obtain a high quality and fully annotated genome for This project was undertaken with the approval of Kenya’s National Commission for Science, Technology and Innovation (permit number NACOSTI/P/15/9609/4270), National Environment Management Authority (NEMA/AGR/46/2014), and snails were exported with the approval of the Kenya Wildlife Service permit numbers 0004754 and 0001680.B. pfeifferi specimens were collected along the vegetated margins of Kasabong Stream  and Asao Stream, Kenya . Snails were brought to the Center for Global Health Research, Kisian, Kenya, where they were checked for trematode parasites, and uninfected snails were pooled and used to establish a colony. Survivors were subsequently transported to and maintained at the University of New Mexico in 20 L plastic containers filled with 5 L of artificial spring water with high aeration using airstones. A Weco Wonder Shell was placed in snail tanks monthly for additional calcium. Snails were fed twice weekly with veggie sticks and snail growth sticks, AquaticBlendedFoods Super Freshwater Snail Mix #2. Plastic plants were added to the snail containers to allow the snails more surface area for egg laying. Algae were allowed to grow on the plastic sides of the snail tanks, and the water was changed every 1–2 months. Snails were maintained on a 12:12 light-dark cycle (25–27°C).In 2017, B. pfeifferi (8 mm shell diameter) was crushed with shell in liquid nitrogen. The crushed snail was moved to a tube containing 5 ml of 30 mM Tris, 10 mM EDTA, and 1% SDS (pre-dissolved in 50 ml of water). Proteinase K  was added and the solution was placed in 55°C water bath for an hour. DNA was precipitated from the SDS solution with 2x ethanol. Precipitated DNA was centrifuged at 4500 x g and resuspended in 5 ml of QIAGEN G2 solution . Another 50 μl of Proteinase K was added. Once the precipitated DNA was fully dissolved in G2 solution, a Qubit assay  was used to measure DNA concentration and the sample was loaded onto a Qiagen genomic tip column and the Qiagen Genomic DNA Preparation protocol was followed. Once the DNA was eluted from the column it was precipitated with 2x ethanol. The precipitated DNA was resuspended and dissolved in 500 μl of Elution Buffer. The Qubit assay indicated a DNA yield of 33.8 ng/μl. Genomic DNA was sheared using the Diagenode Megaruptor 2  and checked on an Advanced Analytical Fragment Analyzer (Agilent) to determine the size of the sheared DNA. Once sheared, the sample was cleaned using 0.5x AMPure beads  and resuspended in 90 μl of PacBio EB buffer. A Qubit assay then measured 140 ng/μl of DNA. DNA (10 μg) was used in PacBio’s HiFi library preparation protocol , Version 02 (May 2019)), then the sample was 1x AMPure cleaned, and 4 μg of DNA was loaded on the SageElf (ACCELA). The top 4 elution fractions from the SageElf were run on the Fragment Analyzer. The best fraction of DNA was from well 3. A final clean-up was applied with a 0.5x AMPure cleanup and eluted in 12 μl of EB buffer before proceeding to the binding and annealing step.One The prepared DNA sample was loaded in a PacBio Sequel II–SMRT Cell instrument  for whole genome sequencing. HiFi reads centered at 15–20 kb were obtained after 30 hours of sequencing.www.lifeasible.com). B. pfeifferi, B. glabrata BB02 and iM line were investigated. In each species or strain, 3 individual snails were used separately for the measurement. The garden plant hosta (Hosta plantaginea) (Praying Hands’ 2-2-2) was used as control DNA. The hosta DNA was calibrated using tomato Solanum lycopersicum, in which the seeds were collected from Ohio State University and the DNA content (2C) was determined to be 2.02 Gb [www.wolflabs.co.uk). After extraction and staining of nuclei, the measurement was conducted in a BD Accuri C6 cytometer (BD Biosciences), using 20-mW laser illumination at 488 NM, FL-2 585/40-nm bandpass filter. Tested snail DNA content = hosta DNA content x fluorescence intensity of the tested snail / fluorescence intensity of control hosta.Measurement of nuclear DNA content (2C) was performed at Lifeasible, Shirley, NY  and Illumina paired end reads (BB02) downloaded from NCBI sequence Read Archive (SRA) PRJNA12879 [Genome size estimations by cope 2.0 , and K-m Merqury  with Pachttps://github.com/sanger-pathogens/assembly-stats), Benchmarking Universal Single-Copy Ortholog assessment tool BUSCO 4.1.4 [Initial genome assembly was performed on PacBio HiFi subreads using Hifiasm 0.16.1-r375  with defCO 4.1.4 , and QUACO 4.1.4 .The genome assembly was cleaned and filtered based on supportive read depth, GC content, and sequence similarity search results using BLAST  and DIAMA typical eukaryote genome annotation workflow contains three stages: repeat masking, gene model prediction, and functional annotation . We starab initio predictions, RNA sequencing (RNA-Seq) alignments, and sequence similarity-based searches against known transcript sequences.Gene models were predicted using EVidence Modeler 06/25/2012 , which iab initio prediction evidence is comprised of core single copy orthologs identified using BUSCO customized HMM models built from tBLASTn [de novo assembled B. pfeifferi transcripts [The  tBLASTn  and AUGU tBLASTn . Evidencnscripts  to the cnscripts .B. pfeifferi schistosome infection studies [B. pfeifferi genome. The mapping rates are 82.6% for BLAT  and 83.0% for GMAP . Raw RNA-Seq sequences were first downloaded from the NCBI Sequence Read Archive (SRA) using the prefetch program, then converted to FASTQ format using the fastq-dump program in SRA toolkit 2.9.6 (https://trace.ncbi.nlm.nih.gov/Traces/sra/sra.cgi?view=software). Low quality sequences in FASTQ reads were filtered and trimmed using Trimmomatic 0.36 [S. mansoni, schistosome sequences were filtered out by mapping cleaned RNA-Seq reads to Schistosoma mansoni Puerto Rico strain genome V7 PRJEA36577 obtained from WormBase Parasite . Clean reads were mapped to the cleaned, repeats-masked B. pfeifferi genome assembly using HISAT2. The alignments of RNA-Seq reads and assembled transcripts were used as input for the BRAKER 2.1.1 annotation pipeline [RNA sequence evidence was generated from genome mapping of RNA-Seq data (NCBI PRJNA383396) from  studies ,28,48 us studies . A total studies . Transcrtic 0.36 . A slidi BLAT 1308/158,095pipeline  was usedWith the evidence described above, EVidence Modeler was used to predict gene models. BRAKER2 evidence was given weight 10 and all other evidence weight 1 based on BUSCO reports of source evidence and results of different weights combinations. The predicted gene models were sorted, renamed based on contig order and gene start location. Transcript, coding and protein sequences were extracted using GffRead 0.9.12  with opt−5; 2) BLASTp to the NCBI non-redundant protein database (NR) with the same cut off as above; 3) BLASTn to the NCBI non-redundant nucleotide database (NT) with minimum identity of 60% and maximum E value 10−5; 4) conserved functional domain by InterProScan 5.45 [Biomphalaria specific IgSF domains were predicted by a B. glabrata specific HMM model [Functional annotation for predicted gene models was performed by sequence similarity searches. Extracted coding sequences and protein sequences were searched as queries against reference databases: 1) BLASTp to UniProt  databasecan 5.45 . BiomphaMM model  with minhttps://github.com/tseemann/barrnap) with options “—kingdom euk—threads 40”.Transfer RNAs (tRNAs) were predicted using tRNAscan-SE 2.0.9  with staB. pfeifferi genome were extracted. Additionally, a highly automated pipeline with classified miRNA CMs, named mirMachine pipeline 0.2.11.2, was used to find miRNAs with the node set to Mollusca. The miRNAs predicted from the two methods were merged and duplicated records with overlapping location were removed. miRNAs in B. pfeifferi and B. glabrata were grouped using cd-hit-est 4.8.1 [For microRNAs (miRNAs), conserved RNA sequence similarity sharing sequence and/or structural similarities within the genome were identified. Sequence profiles containing covariance models (CMs) of known non-coding RNAs were downloaded from the Rfam database, and used as queries to search within the assembled genome using Infernal (INFERence of RNA ALignment) software , with opst 4.8.1 .B. pfeifferi genome assembly. The genome-guided transcriptome assembly was based on the RNA-Seq read-to-genome alignment using Trinity 2.8.5 [The annotation of long non-coding RNAs (lncRNAs) was initiated by mapping known RNA-Seq reads to the clean ty 2.8.5 . Assemblty 2.8.5 . PossiblB. pfeifferi genome assembly were aligned to the B. glabrata iM line genome sequences using minimap2 program [To facilitate a whole genome alignment, the cleaned contig sequences of the  program  with opt program  with sug program  and Shin program  in the R program .B. pfeifferi contigs that best matched to B. glabrata iM line with the largest non-redundant scaffold’s coverage were identified. These best-matched B. pfeifferi contigs were sorted according to iM line and scaffolds per iM line’s 18 linkage groups. Inferred linkage groups (18) were assigned to B. pfeifferi contigs according to their best matched iM line linkage group. Data transformations were processed using in house Linux scripts and R tidyverse package [Using the whole genome alignment information generated above, the  package .B. glabrata iM line  genome ID JAKZJL000000000), Bu. truncatus (JAGDYQ000000000), and Elysia marginata (BMAT00000000). For out-group Radix auricularia (MUZC00000000), no annotated genes in NCBI were available, so the protein sequences for core Mollusca orthologs were predicted from the genome sequences using BUSCO.Protein sequences were obtained from NCBI for species with annotated genome assemblies: With all protein sequences, orthologs were identified using OrthoFinder 2.5.4 , and a sB. pfeifferi and B. glabrata was calculated based on one fixed calibration constraint on the time tree: the appearance of Bulinus in the fossil record at least 19–20 Mya [The timetree was inferred by applying the RelTime method ,70 to th9–20 Mya .B. pfeifferi genome, we further classified and analyzed a list of immune response-related gene families of interest, including fibrinogen-related domain-containing proteins (FReDs) [Aplysia californica/brasiliana pheromonal complex, attractin, seductin, enticin, and temptin [Based on the predicted gene models with InterProScan, UniProt, DeepTMHMM  in the B (FReDs) ,55,74–78 (FReDs) ,80, and  (FReDs) , AIG (av (FReDs) , biompha (FReDs) ,83, G-pr (FReDs)  and Toll (FReDs) ,84,85. W temptin ,87.2+ binding EGF-like domain, DSDXD in temptin [Basic gene family classification was based on sequence similarity search using the BLASTn or BLASTp program mentioned above against NCBI database, and Uniprot databases with a minimum of 85% identify and coverage. Higher level gene family classification was based on conservative domain structures, e.g., fibrinogen-related proteins (FREP) containing immunoglobulin superfamily (IgSF) and fibrinogen (FBG) domains ,55; AIGs temptin ,90.B. pfeifferi FReDs in this study were used to construct a maximum likelihood tree through IQ-TREE [Full-length protein sequences of all identified  IQ-TREE  with sta IQ-TREE  in IQ-TRB. pfeifferi fibrinogen-related domain-containing proteins (FReDs) family, one that complements and does not conflict with the nomenclature developed for the B. glabrata FReDs family. This is important because of the immunological role of FREPs (one of the core groups of FReDs) in host responses in B. glabrata to parasites [B. pfeifferi.A systematic nomenclature is presented in this study  for the arasites ,93,94, aB. pfeifferi FREPs were assigned based on IgSF and FBG domain verification and the presence of a signal peptide. FREP1 to FREP14 have been reported in B. glabrata studies [B. pfeifferi sequence similarity BpFREP1 to BpFREP14 were identified (≥ 85% sequence identity at nucleotide level) to known B. glabrata FREPs. Additionally, for some BpFREPs, an additional numeral was added to distinguish individual variants (sequence similarity between 85% to 100%), e.g., BpFREP12.1, 12.2, and so on. Several BpFREPs had no obvious similarity to the known FREPs in the B. glabrata genome. Such new BpFREPs were named starting with BpFREP15, and so on. studies ,55,95,96B. pfeifferi sFReDs (single Fibrinogen-Related Domain-containing proteins) and FReMs -like region) was also made in reference to sequence similarity to the fibrinogen domains in B. pfeifferi FREPs; sFReDs and FReMs were assigned names following the BpFREPs with the closest fibrinogen sequence. For instance, BpsFReD12.1 shows closest similarity to the fibrinogen domain in BpFREP12.1, and BpsFReD12.4 shows high sequence similarity to the fibrinogen domain in BpFREP12.4. For B. pfeifferi sFReDs that have no similarity to any known FReDs [B. pfeifferi FReMs, e.g. BpFReM1, BpFReM2, and so on.Naming of wn FReDs , they weB. glabrata , including strains varying in susceptibility to S. mansoni [B. pfeifferi in such comparisons. Therefore, BpFREP 2, 3, and 4 transcript sequences were aligned and blasted against B. glabrata sequence similarity. For each FREP gene of interest, protein sequences from the B. glabrata BB02 genome [B. glabrata BS90 and M line strain transcript sequences [B. pfeifferi transcript sequences (this study) were aligned together using the multiple sequence alignment program MAFFT [Several single nucleotide variants (SNV) have been identified among BgFREP 2,3, and 4 derived from different strains or isolates of  mansoni . It was 2 genome , similarequences ,97, and am MAFFT  and visuam MAFFT .B. pfeifferi FREPs were predicted based on the protein sequences using the Alphafold2 algorithm [B. glabrata genome was submitted and processed with the same Alphafold2 analysis.The three-dimensional (3D) structures of selected lgorithm  integratlgorithm , a platflgorithm . To provB. glabrata genome-wide association studies (GWAS) have been undertaken to identify candidate genes in particular chromosome regions associated with resistance (or susceptibility) to infection with S. mansoni [B. glabrata, can be found in the B. pfeifferi genome. The gene sequences from the previous GWAS studies were extracted [B. pfeifferi, with query sequence coverage and identity ≥85% as a cutoff value.Several  mansoni ,103–106.xtracted  and wereSignal peptide searches were performed using SignalP 4.0 . Venn diB. pfeifferi haploid genome size assembled from PacBio HiFi reads is 772 Mb, smaller than observed for other related schistosome vector snails, including two other species of Biomphalaria and Bu. truncatus  or B. glabrata iM line  using the same technique (An independent estimation of genome size based on fluorescence cell sorting intensity in comparison with known standards (Lifeasible) yielded a value of 913 Mb for echnique . The conB. pfeifferi are indicative of a homozygous genome [B. glabrata is indicative of a greater degree of heterozygosity  in s genome  Fig 3A)B. pfeiffzygosity .B. pfeifferi genome heterozygosity rate to be 1.01% (PacBio HiFi reads), which is lower than 1.52% for outbred B. glabrata BB02. For comparisons sake, the deliberately inbred iM line with a history of 70+ generations of forced selfing had a heterozygosity rate of 0.197% [Secondly, k-mer analysis using GenomeScope showed the f 0.197% .B. pfeifferi genome derives from RNA-Seq data  Unip methods .B. pfeifferi is 329 Mb (B. glabrata iM line (374 Mb) or B. glabrata iBS90 (391 Mb). Additionally, the proportion of the genome occupied by repetitive elements is slightly smaller in B. pfeifferi (42.73%) than in either B. glabrata strain .The overall portion of the genome occupied by repetitive elements for s 329 Mb , smallerB. pfeifferi are Gypsy/DIRS1 (6–17 Mb), simple repeats (5–6 Mb) and RTE/Bov-B iM line (17 Mb). In contrast, the hobo-Activator family, a type of DNA transposon, was increased in representation by 1.4–2 Mb in B. pfeifferi compared to the B. glabrata genome.As noted in B. pfeifferi were determined by comparison with the 18 LGs of B. glabrata iM line, which were genetically determined using F2 offspring bred from two lines of B. glabrata [B. pfeifferi genome assembly were assigned to 18 LGs  for glabrata . Overallo 18 LGs . The ordB. pfeifferi and B. glabrata iM line. These SVs occupied 8% of the B. pfeifferi genome  of the inversion in B. pfeifferi, and 87% (784/901 Kbp) of the comparable region in B. glabrata iM line. Further details can be found in A total of 12 types of structural variation (SV) of >1Kb were identified and summarized from a whole genome comparison between i genome . SVs hadi genome  and consB. pfeifferi genome. The duplicated regions may relate to the expansion of the FReD (fibrinogen-related domain-containing proteins) family, the size of which is increased in comparison to B. glabrata. The genes affected by the SVs would be good candidates to explore with respect to explaining the phenotypic differences between the two species.Duplicated region changes (DUP and INVDP) are the major SVs detected, accounting for 22.5 Mb (2.9%) of the B. glabrata iM line , B. pfeifferi , Bu. truncatus , Elysia marginata , and Radix auricularia , and the time tree , and two congeneric species, B. pfeifferi and B. glabrata, the latter represented by two isolates originating from Brazil, BB02 and iBS90, and one mixed ancestry isolate, iM line. The category of “shared among all five genomes” far outweighed the number of Biomphalaria-specific ortho-groups . The large number of ortho-groups shared by iM line and iBS90  and the relatively large number  shared by Biomphalaria representatives exclusive of B. glabrata BB02, may reflect differences in sequencing techniques. The number of ortho-groups unique to each of the taxa examined ranged from 164 to 485, not surprisingly with the value for Bu. truncatus being higher than for the more-closely related Biomphalaria representatives.A total of 12,421 ortho-groups were shared among the 5 genomes sampled in A total of 7,054 single copy orthologs were detected in common to all five genomes. These comprise the core set of genes among these extant species. These universal single copy genes serve as good bench markers for expected gene content in genomes or transcriptomes. As a reference, the Mollusca Benchmarking Universal Single-Copy Orthologue (BUSCO) single copy orthologs number is 5,295. The higher number of 7,054 we observed among our five genomes is no doubt influenced by their relatively high degree of relatedness as compared to relatedness among all molluscs as a whole.B. pfeifferi and were able to obtain 514 tRNAs , 757 rRNAs, 77 miRNAs, and 2,954 long non-coding RNAs, or lncRNAs  have a signal peptide. Of the 55, 8 had one IgSF loop and 47 had two IgSF domains. The interceding region (ICR) in FREPs refers to the sequence between the IgSF and FBG domains; it forms coiled coils and is usually <150 aa in length. However, we found 21 B. pfeifferi FREPs with a longer ICR (152~322 aa) and 34 BpFREPs with an ICR of more typical length of <150aa . A more detailed overview of B. pfeifferi FReDs is in Compared with the congener  genomes , more FRFReD genes in the B. glabrata BB02 and B. pfeifferi genomes, a Maximum Likelihood tree was generated  seem to represent an expansion from a shared ancestral sequence, 17_tig_2702_BGLB000021 being the counterpart in B. glabrata BB02. Conversely, note the FREP4 cluster in B. glabrata BB02 that is lacking in B. pfeifferi.To compare enerated . OverallB. glabrata BB02, BS90 and M line with representatives from the B. pfeifferi genome, looking for single nucleotide variants (SNV). Similar transcript sequences for FREP2, 3.1, 3.2 were identified , was relatively conserved between species with SNVs particularly noted in the B. pfeifferi FBG domain which was also shorter than the corresponding domain in B. glabrata. Both FREP3.1 and 3.2 contain two IgSF domains  and a C-terminal domain CLECT, characteristic of C-type lectins. Based on the gene structures, 20 of the 63 C-type lectin encoding genes contain one or two IgSF loops, thereby fitting the criteria of CREPs . Among tglabrata .The 43 remaining C-type lectin domain-encoding genes either contain the CLECT domain only or the CLECT domain in combination with some other conserved motif, such as an Apple-like domain, Rhodopsin 7-helix transmembrane protein, or Trimeric LpxA-like superfamily member .B. pfeifferi as compared to one known from B. glabrata [With respect to GREPs which are comprised of an N-terminal signal peptide, one or two IgSF domains, IgSF1 and IgSF2, and a C-terminal galectin domain GLECT, none were found in glabrata . HoweverB. pfeifferi genome (B. pfeifferi genome (in We explored the location within the whole genome of the VIgLs gene families in the i genome . The genenome in .B. pfeifferi were found clustered within hotspots on two scaffolds (BP014 and BP029), both of which reside tin linkage group LG13. These two scaffolds are 8.2 Mb apart, separated by scaffolds BP031 and BP062. No obvious clustered hot spots were found for other VIgLs like CREPs, or isolated IgSF or sFReDs.With the exception of a few scattered members, FREPs in B. glabrata because some were found to be constitutively highly expressed in snails resistant to S. mansoni as compared to schistosome-susceptible snails [B. glabrata genome [The AIG (avrRpt2-induced gene) family are characterized by the presence of an AIG1 domain. Members of the AIG gene family are frequently represented as GIMAPs (GTPase of the immunity associated protein family), characterized by presence of the AIG1 domain along with coiled-coil domains. AIG family members first attracted attention in e snails . An expaa genome .B. pfeifferi genome, 69 AIG genes were identified (B. glabrata BB02 genome (47 GIMAPs and 15 AIGs). The remaining 7 which are distinctive to B. pfeifferi include 2 GIMAPs and 5 AIGs. As is the case with B. glabrata, most of the B. pfeifferi AIGs contain only 1 AIG1 domain, but a few contain two AIG1 domains, or a special domain, such as a Death domain, or a Hedgehog/Intein domain. More details can be found in In the entified , of whicB. glabrata GWAS studies published prior to 2022 [S. mansoni. Using the list of potential candidate resistance genes summarized from these studies by Lu et al., (2022) [B. pfeifferi genome with the query sequence coverage and identity ≥85% as cutoff values. We also searched the recently published GWAS study of B. glabrata iM line and iBS90 genomes by Bu et al., 2022 [Several  to 2022 –106 pred, (2022)  and showl., 2022 . ResultsB. glabrata GWAS resistance regions can be confirmed for B. pfeifferi. For instance, three B. pfeifferi fructose-bisphosphate aldolase genes similar in sequence to genes  of the Guadeloupe Resistance Complex [B. pfeifferi genome , an aerolysin domain consisting of 5 β-strands with an insertion loop corresponding to the pore-forming transmembrane domain (TMD) . Twenty- defense ,83.B. glabrata biomphalysins, the transmembrane domains were searched using the PRED-TMBB server (PREDiction of TransMembrane Beta Barrels proteins) [B. pfeifferi genome  [B. glabrata biomphalysin 10  with 97.8% of identity and 100% coverage. It’s possible either a downstream aerolysin domain was missed during genome assembly, or a deletion occurred within this gene. Therefore, BP08351 is included in the B. pfeifferi genome as a partial biomphalysin (Using criteria provided for roteins) ,89, and i genome , 8 of wh. (2021) . Interesphalysin .B. glabrata (11–21 HSPs) [B. glabrata defense reactions [B. straminea genome [Heat shock proteins (HSPs) are known to play a vital role in the synthesis, transport and folding of proteins. HSP70s are unique in the HSP family due to their high degree of conservation . They ha21 HSPs) –118, andeactions ,120. Seva genome .B. pfeifferi genome  were identified in the i genome , four ofB. glabrata BB02 genome [B. glabrata snails susceptible or resistant to S. mansoni infection [B. pfeifferi genome to NCBI databases, combined with InterProScan and Uniport for GPCR domains prediction, and identified 219 putative GPCRs (B. pfeifferi GPCRs. For instance: 96 GPCRs contained the domain of “Rhodopsin 7-helix transmembrane proteins” (G3DSA:1.20.1070.10); 39 GPCRs had a “Rhodopsin-like GPCR superfamily signature” (PR00237); 31 GPCRs contained “G_PROTEIN_RECEP_F2_4 DOMAIN-CONTAINING PROTEIN” (PTHR45902), and 10 GPCRs contained “MYOSUPPRESSIN RECEPTOR 1, ISOFORM B-RELATED” (PTHR46273). Considering the number and complexity of GPCRs revealed in B. glabrata and B. pfeifferi, and their responsiveness to schistosome infection, the GPCRs would seem to comprise a fruitful subject for future investigation.G-protein coupled receptors (GPCR) play an important role in environmental sensing ,121 and nfection . In thisve GPCRs . There wB. glabrata BB02 genome, 27 complete, 9 partial, and 20 TLR pseudogenes were identified [B. glabrata to S. mansoni infections [B. pfeifferi, by BLAST analysis, 43 TLRs were identified. Of these 43, 17 are considered as complete TLRs with an N-terminal signal peptide, a series of leucine-rich repeats (LRRs), a transmembrane domain, and an intracellular Toll/Interleukin-1 receptor (TIR) domain. The remainder are categorized as TLR partials because they lack one or more of the conserved domains (Toll-like receptors (TLRs) are known to be important pathogen recognition receptors (PRRs) responding to pathogen-associated molecular pattern molecules or PAMPs, triggering activation of immune signaling pathways ,122–124.fections ,125. In  domains .Aplysia californica/brasiliana pheromonal complex of attractin, seductin, enticin, and temptin. Similar to previous observations for B. glabrata [B. pfeifferi [B. glabrata BB02 and iBS90 genomes, we found 12 predicted partial temptin-like genes in B. pfeifferi , whereas B. glabrata iM line had 11 . All 13 predicted temptin-like  genes also grouped closely with B. glabrata temptin-like gene, including Bp30710RA with XP_013086708.1 from B. glabrata, a recombinant form of which was shown to attract B. glabrata in T-maze experiments  to B. pfeifferi genome sequence greatly increases our knowledge base for this understudied species, one that plays a pivotal role in maintaining schistosome transmission across much of the Afrotropical region and in southwest Asia. B. pfeifferi is well-known for its widespread involvement in S. mansoni transmission, and in broad terms should be considered a highly relevant vector species representing remarkable compatibility with schistosome infection.The The genome sequence was obtained using PacBio high-fidelity (HiFi) sequencing technology which generates accurate long reads  . HiFi seB. pfeifferi genome is smaller (772 Mb) than known genomes for other Biomphalaria or Bulinus species, and it contains proportionately about the same amount of repetitive DNA as genomes of close relatives  returned consistent results that the B. pfeifferi genome size is smaller than the B. glabrata genome. Additionally, the PacBio HiFi reads in this study have a mean length of 18Kbp and 37x depth, which leaves less opportunity for collapsing repeats. Therefore, despite the caveat that the B. pfeifferi genome might have a reduced genome size due to repeats collapsing during the assembly process, we have reached the conclusion that the B. pfeifferi genome size is just naturally smaller than the B. glabrata genome.Surprisingly, the elatives . The numB. pfeifferi as compared to other planorbid genomes. This is also supported by heterozygosity of SNP genotypes from RNA-Seq data: for B. pfeifferi, this ranged from 7–28%, whereas for outbred B. glabrata M line the value was 39%. The values for B. pfeifferi were closer to the 6% value noted for B. glabrata iM line which had been deliberately inbred for 81 generations. The low degree of heterozygosity for B. pfeifferi is consistent with the strong, but not exclusive, predilections for self-crossing as opposed to out-crossing for this snail species [B. glabrata which is naturally a preferential out-crosser [B. pfeifferi, even though it is not an exclusive selfer, both diminished heterozygosity and smaller genome sizes may be consequences of its relative reliance on selfing. Sequencing of additional individuals of B. pfeifferi, and of different schistosome vectors also known to be selfers  [Genome-based k-mer analysis  suggeste species , a trait-crosser . In nema-crosser . So, forB. glabrata BB02 [B. glabrata M line and BS90 lab strains [B. pfeifferi, matching the known haploid number of 18 chromosomes for this and other species of Biomphalaria. A total of 53.5% of the B. pfeifferi genome exhibits synteny with the B. glabrata iM line genome assembly [B. pfeifferi and the remaining 11 species of African Biomphalaria from the trans-Atlantic colonization of a B. glabrata-like ancestral snail [B. glabrata and B. pfeifferi of 3.01 million years, a rate that falls into the range of 2.62–3.47 million years proposed earlier as the separation time for these two species [Bulinus in the fossil record which does not necessarily reflect the earliest appearance of this genus in evolutionary history. We return to a discussion of the implications of this study with respect to relationships between Biomphalaria species and schistosomes and other trematodes below.Building on the pioneering work of the genome project for field-collected ata BB02  and of s strains , 18 wellassembly , as mighB. glabrata BB02 genome, two lncRNAs were predicted [B. pfeifferi. As our knowledge of genome regulation improves, the significance of the many lncRNAs identified will become more apparent: they will provide insights into regulation of gene expression at multiple levels .As compared to other available schistosome vector snail genomes, we provide a relatively comprehensive list of the non-coding RNA genes found, including rRNAs, tRNAs, miRNAs, and lncRNAs. In the redicted  as compaBiomphalaria genomes [B. glabrata and B. pfeifferi had similar overall FReD profiles, with comparable numbers of sFReDs, FReMs and FREPs. By contrast, Bu. truncatus was found to have only a single FREP and relatively more sFReDs. The number of FREPs found in B. pfeifferi (55) exceeded that found in B. glabrata (39). The sole FREP from Bu. truncatus possesses two IgSF domains which are relatively dissimilar from the IgSF domains found in B. pfeifferi or B. glabrata BB02/iM line/iBS90. In fact, IgSF domains in Bu. truncatus FReDs were only retrieved from predicted 3D protein structures showing the typical 8-beta sheets featured in IgSF domains. It will be of particular interest to chart the expansion or contraction of FReDs across the Planorbidae as more data become available. Bulinus is a relatively early diverging genus in the family and is even placed by some in a separate family, the Bulinidae. In contrast, Biomphalaria represents a more derived lineage [Among the many gene families identifiable in  genomes , we chos lineage ,136. ProB. pfeifferi FREPs were striking, notable both for their differences from Alphafold2 predictions for Bu. truncatus FREPs, and for how impactful the interceding region is in dictating their overall structure. With respect to four FREPs noteworthy in B. glabrata for their responsiveness to S. mansoni infection [B. pfeifferi. Generally, the versions from B. pfeifferi were more divergent in base composition than were the various B. glabrata representatives. It was somewhat surprising that the fibrinogen domains appeared more prone to variation than the IgSF domains. Further analysis of the functional roles of FREP IgSF and fibrinogen domains will be facilitated by use of 3-dimensional structures such as those shown in S. mansoni [The three-dimensional structures predicted by Alphafold2 for nfection , FREPs 2 mansoni  to be mo mansoni .B. pfeifferi. This gene was found to be down-regulated early during the course of S. mansoni infection in schistosome-susceptible B. glabrata M line snails, but was persistently overexpressed in S. mansoni-resistant BS90 snails following exposure to infection [S. mansoni, if linked to FREP4, might result from two different processes: down-regulation in susceptible strains of B. glabrata, or loss of the gene in B. pfeifferi. In contrast, FREP2 and 3 have the same copy numbers in the two related Biomphalaria species, suggesting their stable maintenance is significant. Furthermore, not only has FREP4 disappeared in B. pfeifferi but a novel FREP, FREP12, is present, indicative of the dynamic nature of the FReD gene family, exhibiting both loss and expansion.It was noteworthy that no sequence similar to FREP4 was found in nfection . This maB. pfeifferi (20) than in B. glabrata (4). Their role in schistosome interactions requires more study, but proteins with sequence similarity to CREP IgSF sequences have been found adherent to S. mansoni sporocysts [B. glabrata susceptible or resistant to S. mansoni have also found distinctive response profiles for C-type lectins which normally lack an IgSF domain [B. glabrata although 111 lectins representing multiple categories are known to be present [B. pfeifferi has 43 C-type lectins and Bu. truncatus has 101 mostly C-type lectins [With respect to other molecules with IgSF domains, CREPs have both an IgSF domain and a C-terminal C-type lectin domain . More CRorocysts . PreviouF domain . The num present . B. pfei lectins . Further lectins .Biomphalaria is the AIG gene family, which is expanded in B. glabrata with 91 members, with some members known to be expressed at constitutively high levels in snails resistant to S. mansoni [B. pfeifferi also has an expanded AIG family containing 69 members. The presence and size of this gene family in other planorbid snails is as yet unknown, as is the role they might play when up-regulated in schistosome-resistant snails.Another gene family known from  mansoni . B. pfeiB. glabrata has 23 pore-forming-encoding biomphalysin genes [B. pfeifferi was found to have 14 representatives, strongly suggesting these horizontally acquired genes of bacterial origin have survival value for Biomphalaria. Biomphalysins have been shown to adhere to and damage schistosome larvae [S. mansoni sporocysts has been noted by [B. pfeifferi, 43 complete, partial or pseudogene TLRs were recovered, whereas B. glabrata was found to have 56, and Bu. truncatus 123.in genes , and B. e larvae ,140. Tolnoted by . In B. pBiomphalaria defense from schistosomes are HSP70 genes [B. straminea has an enriched complement of HSP70s (41) relative to B. glabrata (11–21) and B. pfeifferi with 11. In molluscs, particularly bivalves, HSP70s are among the most frequently investigated molecules and are believed to play an important role in protection from thermal stress [B. straminea or other invasive gastropods. The role of several of these gene families in snail-schistosome interactions remains to be more fully investigated, and the perspective offered by comparative genomics will prove to be an insightful way forward.Another prominent group of molecules implicated in 70 genes ,120. Thel stress . It willB. pfeifferi is its strong preference for self-fertilization, often viewed as an advantageous way to quickly re-populate fluctuating habitats [Aplysia: temptin, attractin, seductin, and enticin [B. glabrata [B. pfeifferi, comparable in number to the outcrossing B. glabrata with 12 temptin genes.One of the most distinctive features of the biology of habitats . One pos enticin ,128,143.glabrata . We founB. glabrata transcriptomic study of Lu et al. [S. mansoni cercariae, four temptin genes were up-regulated while one was down-regulated relative to unexposed controls. Why snails effectively castrated by S. mansoni would be producing more temptins is somewhat baffling if their role is only in attraction of conspecifics. In B. pfeifferi exposed to S. mansoni [B. pfeifferi temptin-like genes was unaffected by S. mansoni infection, suggesting they might have other functions unrelated to reproduction, a supposition supported by other studies [B. pfeifferi temptin genes and how they differ from other Biomphalaria species. Differences in mating behavior between species might be influenced by expression of distinctive suites of temptins, or by sequence differences among the encoded proteins, hypotheses deserving additional study.In the u et al. , five te mansoni , some te studies ,145,146, studies . FurtherB. pfeifferi (219), though fewer than reported from B. glabrata (241) or Bu. truncatus (709). Further specific analysis of the complements of GPCRs present in the different schistosome vector snails is clearly a topic deserving additional study, not only with respect to how they respond to the presence of schistosome parasites per se, but also how it might relate to differences in their ability to perceive a huge range of environmental cues. Transcriptomic results [Another group of molecules with a largely unappreciated impact on the biology of freshwater gastropods are the membrane-associated signal-receiving G-protein coupled receptors (GPCRs). Not surprisingly, several GPCRs are present in  results  highlighB. glabrata [B. pfeifferi. Not surprisingly, comparable regions with similar gene composition were found in the B. pfeifferi genome. Nonetheless, both B. glabrata and B. pfeifferi are comprised of individuals that largely, but not necessarily exclusively, exhibit a natural state of susceptibility to S. mansoni across their respective geographic ranges [S. mansoni is just one of several species of castrating digenetic trematodes known to infect either snail species: both support diverse communities of trematodes [B. pfeifferi that occupy habitats subject to rapid change, the threat imposed by parasites and the presumed benefits offered by regular out-crossing may be offset by the advantages of rapid colonization achieved by selfing, especially if the risk of trematode infection is small.The schistosome-resistance gene complexes identified by genome-wide association studies in glabrata ,103–106 c ranges . So, undematodes ,148–150.B. glabrata-like ancestor from South America to Africa in an interval, as again suggested by this study, probably less than five million years ago. Some caution is required in interpreting this date because, although it is agreement with other estimates of the divergence’s times of the two species [Bulinus, and this may underestimate the actual origin time of demonstrable Bulinus snails. Similar issues have been reported by others in attempts to estimate divergence times [B. pfeifferi being the first-diverging African Biomphalaria species among them [Schistosoma, which were Afro-Asian in origin and distribution. A snail species likely to have encountered the progenitor of S. mansoni or the related S. rodhaini in Africa is B. pfeifferi. The susceptibility shown by B. pfeifferi to S. mansoni today may reflect this early period of intimate association. Interestingly, any specific accommodation B. pfeifferi and related African snails may have made to S. mansoni and its relatives would be independent from whatever specific accommodation B. glabrata may have made with S. mansoni . This would be because S. mansoni did not encounter B. glabrata until 400–500 years ago, when S. mansoni-infected people were first transported from Africa to the New World. It would prove interesting to learn if the accommodations made (if any) by B. pfeifferi to S. mansoni prove to have any resemblance to those made independently by B. glabrata to S. mansoni at a later time. In other words, how prominent might schistosome resistance genes be in either species in nature, and to what extent might selection have converged on the same genes in each species as they independently encountered S. mansoni? Also, resistance may be manifested very differently between the two species, being present in distinct largely self-propagating lineages in B. pfeifferi, whereas resistance traits might segregate more freely among out-breeding B. glabrata. Once again, comparative genomics as offered by this and other forthcoming genome studies, as for species like the African out-crossing species Biomphalaria sudanica, will help us delve into these and related subjects in depth.Lastly, consider the peculiar colonization history of a  species , our prece times ,70. It wong them ,11, woulS1 Table(XLSX)Click here for additional data file.S2 TableNotes: 1: * Most repeats fragmented by insertions or deletions have been counted as one element. 2: Repeats were identified using RepeatMasker 4.0, with customized repeat libraries, which was generated using RepeatModeler 2.0.1 followed by filtering out protein like sequences using BLAST and InterProScan domain prediction (details in Method).(XLSX)Click here for additional data file.S3 Table(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 TableMultiple sheets in the Excel table recorded the details of the inversion between the two genomes: provided are inversion region locations; annotations and sequence similarities of 24 reciprocal best hits; annotations of all genes within the inversion region in the two genomes; ordered gene locations and MCscan scores for genes within the inversion region.(XLSX)Click here for additional data file.S6 Table(XLSX)Click here for additional data file.S7 Table(XLSX)Click here for additional data file.S8 Table(XLSX)Click here for additional data file.S9 Table(XLSX)Click here for additional data file.S10 Table(XLSX)Click here for additional data file.S11 Table(XLSX)Click here for additional data file.S12 Table(XLSX)Click here for additional data file.S13 Table(XLSX)Click here for additional data file.S14 Table(XLSX)Click here for additional data file.S15 Table(XLSX)Click here for additional data file.S16 Table2+ predictive binding site, DSDXD. Only one of the temptin-like genes contained a signal peptide.Note: Predicted genes were run in BLAST and then searched for signal peptides and the conserved Ca(XLSX)Click here for additional data file.S1 FigB. glabrata [B. pfeifferi [B. glabrata; Bp, B. pfeifferi; Sm, S. mansoni; dpe, days post-exposure; Moll, molluscicide treated; R#, replicate number. BpSmMollR3 means B. pfeifferi infected with S. mansoni and treated with molluscicide, the 3rd replicate.The bar shows the proportion of homozygous (blue), or heterozygous (orange) SNPs found in the individual snails used in RNA-Seq studies of glabrata  and B. pfeifferi ,28,48. A(TIF)Click here for additional data file.S2 FigB. glabrata; Bp, B. pfeifferiAbbreviations: Bg, (TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S5 FigThe free-standing IgSF domain-containing genes (free-standing IgSF in purple) and variable immunoglobulin and lectin domain-containing molecules (VIgLs) gene families including FREPs (blue), CREPs (yellow), and sFReDs (green) were marked on scaffolds according to predicted genomic locations.(TIF)Click here for additional data file.S6 FigB. pfeifferi temptin-like  genes are shown in bold and each clade is color coded. All the B. pfeifferi temptin-like genes group with B. glabrata temptin-like genes, either from B. glabrata BB02, iM line, or iBS90 genomes. One temptin-like gene grouped with BB02, two grouped with iM line, one grouped with iBS90, and the rest of the 8 temptin-like genes all grouped together. The clade with the star is the BgTempin clade from Pila et al. (2017) [B. glabrata is attracted to this protein. The tree with the highest log likelihood (-1627.03) is shown. A discrete Gamma distribution was used to model evolutionary rate differences among sites ). The rate variation model allowed for some sites to be evolutionarily invariable . The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. This analysis involved 52 amino acid sequences. All positions containing gaps and missing data were eliminated (complete deletion option). There was a total of 46 positions in the final dataset. Evolutionary analyses were conducted in MEGA X by Kumar et al., (2018) [The evolutionary history was inferred by using the Maximum Likelihood method and JTT matrix-based model from Jones et al. (1992) . An (*) . (2017) , a study, (2018) .(TIF)Click here for additional data file.


However, further exploration is necessary to elucidate the mechanisms underlying the association between oral microbiota and tumors. This review systematically summarizes the reported correlations between oral microbiota and common cancers while also outlining potential mechanisms that may guide biological tumor treatment.Cancer remains a significant global challenge, with an estimated 47% increase in cancer patients from 2020 to 2040. Increasing research has identified microorganism as a risk factor for cancer development. The oral cavity, second only to the colon, harbors more than 700 bacterial species and serves as a crucial microbial habitat. Although numerous epidemiological studies have reported associations between oral microorganisms and major systemic tumors, the relationship between oral microorganisms and cancers remains largely unclear. Current research primarily focuses on respiratory and digestive system tumors due to their anatomical proximity to the oral cavity. The relevant mechanism research mainly involves 47% dominant oral microbial population that can be cultured  AccordiFusobacterium nucleatum (F. nucleatum) and Porphyromonas gingivalis , can promote the infiltration of immunosuppressive cells and interfere with the function of immune killer cells, thereby protecting tumor cells from immune system surveillance and clearance  Induce and aggravate chronic inflammation and promote the occurrence and development of tumors by the components of themselves . (ii) Relearance . In summ2.2.1.P. gingivalis, F. nucleatum, Treponema dentinosum and Streptococcus , the bacterium responsible for stomach cancer, can also be detected in the oral cavity  dominated by myeloid-derived immunosuppressive cells (MDSC) and tumor-associated macrophages, thereby promoting CRC progression  ranks third in terms of mortality rate, with 830,180deaths (4.7%) reported . Well-esgression . Regrett disease . P. ginghe liver . Additioatocytes . Hence, 2.4.H. pylori infection is a well-established risk factor for gastric cancer, which ranks as the fourth most common cancer globally, with the new incidence of gastric cancer in China was 44% in 2020 (H. pylori, followed by oral microorganisms---Proteobacteria and Firmicutes (H. pylori infected patients will develop into gastric cancer, so it is reasonable to speculate that other biological factors may be involved in the occurrence and development of gastric cancer (Peptostreptococcus stomatis, Streptococcus anginosus, Parvimonas micra, Slackia exigua and Dialister pneumosintes in gastric cancer (Fusobacterium, Clostridium and Lactobacillus, suggesting their potential as biomarkers for early diagnosis of gastric cancer (P. gingivalis has also been reported to be associated with an increased risk of gastric cancer in Asians ( in 2020 . An analrmicutes . Howeverc cancer . Study hc cancer . A recenc cancer . P. gingn Asians . However2.5.Proteobacteria and Firmicutes are the predominant microbial groups found in the breast tissue, which exhibit tolerence toward fatty acids infiltration. Therefore, it can be considered that there are unique and diverse microbial groups in the breast (Breast cancer is the fifth most commonly diagnosed tumor worldwide . Its rise breast .P. gingivalis and F. nucleatum, in breast cancer patients (F. nucleatum could spread through blood and colonize breast cancer sites in a FAP2-dependent manner, thereby impairing anti-tumor immunity and promoting the progression of breast cancer, while the corresponding metronidazole antibacterial treatment could delay the progression of tumor. In addition, F. nucleatum colonized in breast cancer can also activate NF-κB through TLR4/MyD88 pathway, creating an immunosuppressive TME by recruiting immunosuppressive cells, and promoting the immune escape of breast cancer cells by MYC-dependent up-regulation of PD-L1 and CD47 in breast cancer (For a considerable duration, it has been postulated that alterations in the intestinal microbiota within breast tissue can exert influence on hormone levels and contribute to the pathogenesis of breast cancer . Meanwhipatients . Howevert cancer . In conc2.6.Due to the anatomical relationship between oral cavity and digestive tract, there is a higher likelihood of cross-talk between oral microorganisms and tumors in digestive system . In addiEnterococcus faecalis, F. nucleatum and P. gingivalis (Enterococcus faecalis can promote the proliferation of oral cancer cells through the H2O2-mediated EGFR signaling pathway (F. nucleatum promotes the progression of oral cancer by regulating cell cycle, inducing oral inflammation, regulating anti-tumor immune response and promoting epithelial-mesenchymal transition of oral cancer cells (P. gingivalis and F. nucleatum share similarities (in vitro transcriptomic study involving three oral squamous cell carcinoma cell lines, CAL27, SCC4, and SCC25, and three oral commensals, Streptococcus, Neisseria aureus, and Haemophilus parainfluis, with P. gingivalis as a positive control, found that only Streptococcus exhibited significant antitumor properties alone. The other two commensal bacteria showed the coexistence of pro-tumor and anti-tumor effects, while P. gingivalis only had pro-tumor effects on SCC4 cell line (P. gingivalis can mediate autophagy to induce G1 phase arrest in oral squamous cell carcinoma cells (Oral microorganisms that have been clearly associated with the progression of oral cancer include but are not limited to ngivalis . Enteroc pathway . F. nucler cells . The carlarities . Interesell line . In addima cells . TherefoP. gingivalis and F. nucleatum are the primary oral microorganisms reported to be associated with esophageal cancer (P. gingivalis promotes esophageal cancer involves the activation of NF-κB (F. nucleatum can also activate NOD1/RIPK2/NF-κB signaling pathway (Six studies used 16S rRNA sequencing to analyze saliva and oral swab samples collected from patients with esophageal cancer, revealing significant alterations of oral microorganisms. Although the dominant groups were different, they all suggested that oral microorganisms may be potential screening markers for esophageal cancer . Currentl cancer . The defof NF-κB , PTEN/Akof NF-κB , EMT (Chof NF-κB  and apopof NF-κB , which c pathway , AHR/CYP pathway , NLRP3 i pathway  and auto pathway . It also pathway .P. gingivalis and F. nucleatum as important biomarkers for distinguishing pancreatic cancer patients (F. nucleatum has been found to stimulate the secretion of cytokines, including GM-CSF and CXCL1, thus promoting tumor cell proliferation and migration (Furthermore, recent studies have highlighted the potential of patients . Notablyigration . However3.P. gingivalis and F. nucleatum. This review provides a systematic overview of the association between oral microbiota and the world’s most common malignancies, and summarizes the possible mechanisms reported in the literature. At present, it is believed that oral microorganisms are widely associated with the malignant transformation and progression of tumors. Changes in the abundance of oral microorganisms may be used as potential biomarkers for predicting tumorigenesis, and targeted elimination of related harmful oral microorganisms is expected to become a new strategy for cancer treatment. However, it has also been reported that oral microorganisms play a dual role in tumor biology, which either promotes or inhibits tumor depends on the interaction between microorganisms, host and TME. Oral microorganisms have the ability to colonize extra-oral organs, such as the lungs, colorectum, and stomach (F. nucleatum, interacts with human inhibitory receptors TIGIT to facilitate the evasion of tumor cells from NK cells and T cells (F. nucleatum can activate the STING signaling pathway and enhance the expression of PD-L1, thereby exhibiting heightened susceptibility toward PD-L1 immune blockade therapy (With the development of technology and interdisciplinary science, the research on microorganisms and cancer has become a hot topic. Due to the anatomical connection, the current reports mainly focus on digestive system tumors, and mainly investigate the common dominant bacteria in the oral microbiome, such as  stomach . They em stomach . The ana stomach . Intratu stomach . Moreove stomach . Studies T cells . Additio therapy . TherefoZL, W-JL, Y-YZ, and G-TY: conceptualization. ZL and W-JL: formal analysis and writing the original draft. ZL, WJ-L, HCu, K-LZ, HCh, Y-YZ, and G-TY: writing review and editing. Y-YZ and G-TY: supervision and funding acquisition. All authors contributed to the article and approved the submitted version.


Several insects are known as vectors of a wide range of animal and human pathogens causing various diseases. However, they are also a source of different substances, such as the Antimicrobial Peptides (AMPs), which can be employed in the development of natural bioactive compounds for medical, veterinary and agricultural applications. It is well known that AMP activity, in contrast to most classical antibiotics, does not lead to the development of natural bacterial resistance, or at least the frequency of resistance is considered to be low. Therefore, there is a strong interest in assessing the efficacy of the various peptides known to date, identifying new compounds and evaluating possible solutions in order to increase their production. Moreover, implementing AMP modulation in insect rearing could preserve insect health in large-scale production. This review describes the current knowledge on insect AMPs, presenting the validated ones for the different insect orders. A brief description of their mechanism of action is reported with focus on proposed applications. The possible effects of insect diet on AMP translation and synthesis have been discussed. AMPs are found in phylogenetically distant organisms, varying from eukaryotes to prokaryotes . Even ifostraca)  4]. . https:/Harmonia axyridis  (Coleoptera: Coccinellidae) with more than 50 putative AMPs [Neoponera goeldii (Forel) (Hymenoptera: Formicidae) capable of producing more than 15 different Ponericins [Hermetia illucens (L.) (Diptera: Stratiomyidae) with 57 predicted genes [With nearly one million species described, insects are an important source of bioactive compounds ,6. Despiive AMPs , the antnericins  or Hermeed genes .Oxya chinensis subsp. sinuosa Mistshenko (Orthoptera: Acrididae), 26 novel AMP sequences were identified [Periplaneta americana L. (Blattodea: Blattidae) allowed the identification of 86 putative peptides [Since NGS and third generation sequencing are spreading, bioinformatic approaches to identify putative AMPs have been proposed in several papers. From the transcriptome of the grasshopper entified . Comparientified , helpingentified   (Coleoptera: Tenebrionidae) with the reducing agent dithiothreitol, the peptide loses its activity against multidrug resistant Staphylococcus aureus [Most of the AMPs are amphipathic molecules, positively charged in the hydrophilic portion. This is necessary for the recognition of the primary target, the microorganism’s membrane ,37. By ts aureus . Moreoves aureus . Anothers aureus . AMPs ins aureus , thus mas aureus . To describe the activity against membranes, four different models have been proposed so far. Barrel-stave model: after reaching the threshold concentration, AMPs rotate and insert into the membrane bilayer, forming pores. The pore surface is made by the amount of α-helical peptides  Figure A. An exaEscherichia coli, inhibiting DNA, RNA and protein synthesis [E. coli but not the human homologue Hsp70, impeding the correct protein folding [E. coli FtsZ, a bacterial tubulin homolog part of the divisome complex, impeding the correct cell division and leading to cell death [The pore-forming effect is not the only known mechanism of action. Some AMPs can impede the uptake of thymidine, uridine and histidine in ynthesis . Moreove folding . Apidaec folding . Temporill death . C18G inll death .E. coli, preventing ribosome release once reaching the stop codon, causing a general shutdown of the translation process [Furthermore, some AMPs can impede translation. Api137, a derivative of Apidaecin with improved serum stability, binds to the release factors RF1 and RF2 of  process . Other p process ,55,56.Coprinopsis cinerea [AMPs can also impair cell wall synthesis. The defensins Lucifensin, Gallicin and Plectasin, the latter of fungal origin, directly binds to the precursor Lipid II of Gram-positive bacteria . Plectas cinerea ,59. Anti cinerea .C. albicans death by impairing cell ion balance; Ca2+ enters the cytosol while K+ exits. This ion homeostasis imbalance induces apoptosis and, subsequentially, cell death [C. albicans death by pore formation and by activating the apoptotic process via metacaspase activation and ROS generation [Fusarium graminearum depends on two mechanisms. Metchnikowin impairs cell wall synthesis by targeting the enzyme βglucanosyltransferase Gel1, as well as inhibiting succinate dehydrogenase, limiting mitochondrial energetic production [Some insect AMPs proved to induce cell apoptosis besides having a pore-forming activity against fungal pathogens. Cecropin A leads to ll death . Similarneration . Metchnioduction . Periplaoduction . Buda Deoduction  reportedThere are also AMPs with antiviral activity. The human cathelicidin-derived GF-17 can elicit type-I interferon signaling and directly inactivate Zika virus . MelittiStaphylococcus strains against some AMPs, such as Magainin 2 and Colistin [E. coli susceptibility to different AMPs [E. coli [To express antimicrobial activity, AMPs must also overcome the microorganismal cell wall. Peptidoglycan is not dense enough to impede AMPs from reaching the membrane . MoreoveColistin . This inColistin . Similarent AMPs . Loss-of[E. coli . The del[E. coli .Fusarium oxysporum f. sp. vasinfectum hyphae [Candida albicans needs an intact cell wall. RsAFP2 interacts with cell wall glucosylceramides, and loss-of-function mutation of genes involved in cell wall synthesis increases C. albicans tolerance to this AMP [The fungal cell wall harbors receptors essential for the activity of some AMPs C. Plant m hyphae . The toxm hyphae . The Defthis AMP . Moreovethis AMP . Several applications have been proposed for insect AMPs so far, either in the healthcare system, agriculture or food production chain. T. castaneum can inhibit MDR Streptococcus pneumoniae growth in vitro, decreasing cytokines production in infected macrophages [S. aureus strains in vitro, increases infected Caenorabditis elegans adult survivors, and shows no cytotoxicity against human erythrocytes [T. castaneum Defensin 3 with higher cationicity and decreased hydrophobicity, can counteract S. aureus growth through membrane permeation, but there is evidence of a possible block of cell division [Oxysternon conspicillatum are active against MDR bacteria, such as Klebsiella pneumoniae and Pseudomonas aeruginosa, but generally not against Gram-positive bacteria with limited hemolytic activity [P. aeruginosa, E. coli and Staphylococcus, with high stability to pH, salinity and temperature variations, showing no significant cytotoxic or hemolytic activity [Many insect AMPs, such as some Defensins, Thanatins and Coleoptericins, present low to no lithic activity against human erythrocytes ,79,80. Mrophages . The samhrocytes . The Defdivision . Oxysteractivity . Furtheractivity .Drosophila melanogaster (Meigen) (Diptera: Drosophilidae), larvae affected by hyperplasia of lymph gland have a higher expression of AMP coding genes [Bombyx mori L. (Lepidoptera: Bombycidae) selectively inhibits gastric cancer cell growth in vitro without harming healthy cell development and reduces cancer growth in mice in vivo [In addition to exerting a defensive function in immune challenges, an antitumoral activity of insect AMP was assessed. In ng genes . An ectong genes . Focusin in vivo . The sam in vivo . Cecropi in vivo . Further in vivo ,94. CA-MTriatoma pallidipennis (Stål) (Hemiptera: Reduviidae), actively increases Trypanosoma cruzi, Trypanosoma rangeli and Leishmania major death and altered morphology [Leishmania panamensis [Glossina morsitans morsitans Westwood (Diptera: Glossinidae) inhibits Trypanosoma brucei growth in vitro [Another property of insect AMPs is the ability to contrast tripanosomatids. The recombinant insect Defensin rDef1.3, derived from a Defensin of rphology . Cecropinamensis , while Ain vitro . Moreovein vitro .Cercospora nicotianae [Fusarium graminearum but has a low effect against the basidiomycetes Piriformospora indica and Rhizoctonia solani, a mycorrhizal and a pathogenic fungus, respectively [Citrus sinensis have been transformed with insect AMP coding construct. The expression of attacin A in the apoplast results in significantly lower susceptibility to Xanthomonas axonopodis pv. citri [Brassica napus for the expression of cecropin P1 confers resistance to the bacterial pathogen Erwinia carotovora and the fungus Fusarium sporotrichioides [The world’s population is continuously growing and, therefore, the food and feed demand. To ensure adequate production, it is mandatory to protect plants from pathogens . A recencotianae . Transgeectively . Not onlv. citri . The trachioides . Intereschioides . Xanthomonas oryzae pv. oryzae, mainly by forming pores in the pathogen membrane but also by binding to DNA and RNA intracellularly [Erwinia carotovora subsp. atrosepica and Erwinia chrysanthemi, prevent damage formation [Few studies have been conducted on applications different from genetic engineering. One example is given by Melittin injected in and sprayed on rice plants. It effectively protects plants from llularly . Differellularly . Treatmeormation .Scophthalmus maximus survival to inoculation with Edwardsiella tarda, whilst a high supplementation does not change fish survival and deeply affects turbot gut microbiota composition [Aeromonas veronii [Insect AMPs also showed positive health effects in animal farming, resulting in a possible alternative to the wide antibiotics usage . A Cecroposition . The mod veronii . Moreove veronii . The sup veronii . Whilst  veronii .With a constantly growing world population and the increase of pathogen strains resistant to commonly used food preservatives, there is a need for identifying novel food preservatives and developing active packages to increase the shelf life of products. For this purpose, AMPs have been widely studied ,114,115.Musca domestica L. (Diptera: Muscidae) larvae, showed bactericidal activity against E. coli, P. aeruginosa, Salmonella typhimurium, Shigella dysenteriae, S. aureus and Bacillus subtilis. The antibacterial activity was almost equivalent to sodium benzoate in orange juice [Listeria monocytogenes, with both a pore-forming activity and deleterious interaction with pathogen DNA. Furthermore, biofilm formation is strongly impaired by Jelleine-1 [Hf-1, a peptide purified from ge juice . Jelleinlleine-1 . M. domestica pupae showed promising preservation on chilled pork meat [T. molitor larvae challenged with edible Lactiplantibacillus plantarum rich in AMPs displayed high antimicrobial activity against food-borne bacteria and aflatoxin-producing fungal strains. This extract shows high stability in a wide range of temperature, salinity and pH, resulting as an optimal soy sauce preservative [Since AMPs act synergistically in natural conditions, the use of insect extracts containing AMPs has also been proposed. A mix of nineteen AMPs belonging to seven different peptide families extracted from ork meat . An extrervative . Apart fervative ,115.D. melanogaster, the fat body metabolism switches from an anabolic to a catabolic response, with energy redirected to immune response [In mass rearing of insects intended for AMP extraction or to produce peptide-rich insect products, it is necessary to identify strategies to increase their content. To date, this is achieved by infecting insects with microorganisms. Immune challenge to increase AMP production presents important drawbacks. It has been shown that, in immune challenged response . Furtherresponse . The eneresponse ,31,32,33response .Tenebrio molitor, Spodoptera littoralis  (Lepidoptera: Noctuidae), Spodoptera exempta  (Lepidoptera: Noctuidae), as well as other insects facing an immune threat prefer to ingest high protein feed [Apis mellifera carnica Pollmann (Hymenoptera: Apidae) fed protein diet enriched with Sinapis spp., Asparagus spp. and Castanea spp. monofloral pollen showed higher apidaecin 1 and abaecin coding gene expression level [H. illucens and T. molitor rearing [Since diet modulation is leading to promising positive results for both immune response regulation and insect growth, diet optimization is also becoming a topical issue to counteract disease outbreaks in large-scale production . The dieein feed ,134,135.on level . Brewers rearing ,33.H. illucens [Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae) larvae. The uptake and incorporation of phytosterols into insect cell membranes lead to membrane alterations and leaky cells. This, in turn, could lead to a recognition of self-damage or induction of general stress responses resulting in the upregulation of AMP coding genes after feeding [Different diets showed to deeply affect AMP transcription in illucens ,31,32. Hillucens ,138. Theillucens ,31. The illucens . The stiillucens . It is d feeding . Moreove feeding  probably feeding ,141.Galleria mellonella L.  larvae deprived of food for seven days demonstrated increased susceptibility to infection by C. albicans due to cellular and humoral response reduction. Starving leads to a reduction in the expression of a variety of AMPs and immune proteins. Lipocalin coding gene expression was reduced by approximately 14% in starved larvae [Not only composition but also the abundance of feeding substrate is crucial for the survival of immune-challenged insects ,143. Gald larvae ,141.D. melanogaster. The infection with Gram-positive bacteria activates the Toll-pathway, which enhances AMP transcription but also inhibits insulin receptors [step mutants, an essential component of insulin/insulin-like growth factor signaling (ILS), and chico mutants . In both cases, increased production of AMPs was obtained in non-infected insects [A deep correlation between immune and metabolic responses has been shown in eceptors . Intereseceptors . Recentleceptors ,47. A fu insects .Although the rearing diet could positively influence AMP transcription, it does not always correspond to a higher translation. Biological assays or analyses of specific AMPs are needed to confirm it . Indeed,The possibility to modulate AMP production with the diet opens new perspectives in the industrial rearing of insects as food and feed. On the one hand, it can be an important tool to preserve the health of the insects, avoiding the use of antibiotics and preventing mass death with important economic losses. On the other hand, it launches the use of insects as food or feed not only in terms of nutritional value but also as a novel ingredient that can improve the health of humans and livestock with immunity-enhancing effects. In particular, a diet-dependent expression of AMPs translated into diet-dependent profiles of inhibitory activities against a spectrum of bacteria opens a new and important chance to transfer these AMPs in the final consumers.