Diabetes mellitus (DM) is a group of metabolic disorders characterized by hyperglycemia, insulin insufficiency or insulin resistance, and many issues, including vascular complications, glycative stress and lipid metabolism dysregulation. Natural products from plants with antihyperglycemic, hypolipidemic, pancreatic protective, antioxidative, and insulin-like properties complement conventional treatments. Throughout this review, we summarize the current status of knowledge of plants from the Caribbean basin traditionally used to manage DM and treat its sequelae. Seven plants were chosen due to their use in Caribbean folk medicine. We summarize the antidiabetic properties of each species, exploring the pharmacological mechanisms related to their antidiabetic effect reported in vitro and in vivo. We propose the Caribbean flora as a source of innovative bioactive phytocompounds to treat and prevent DM and DM-associated complications. Diabetes mellitus (DM) is a chronic disturbance that occurs when the hypoglycemic hormone insulin is not released in adequate amounts or when its activity is ineffective due to metabolic resistance ,2. Type DM affects a large number of people, especially in low-and middle-income countries. According to the World Health Organization (WHO), about 422 million people worldwide have DM, and 1.6 million deaths are directly linked to DM or DM-related complications . In partDM is a disease of metabolic dysregulation that results in the buildup of aberrant sugar levels in the bloodstream. At the molecular level, chronic hyperglycemia leads to the accumulation of toxic advanced glycation end products (AGEs). AGEs are a diverse array of compounds formed through a non-enzymatic reaction known as glycation in which sugars or sugar metabolites attach to different biomolecules, such as proteins, impairing their function. Given that glycation is a sugar concentration-dependent reaction, high levels of glycative stress are a key pathophysiological feature in DM, being the monitorization of glycated hemoglobin A1c (HbA1c) and the most common blood test to diagnose DM ,9. AGE aConventional anti-DM therapies include lifestyle modifications , exercise, and weight loss), oral pharmacological agents, and subcutaneous pharmaceutical insulin . Oral anAnacardium occidentale L. (Anacardiaceae), Hyptis suaveolens (L.) Poit. (Lamiaceae), Persea americana Mill. (Lauraceae), Psidium guajava L. (Myrtaceae), Momordica charantia L. (Cucurbitaceae), Tecoma stans (L.) Juss. ex Kunth (Bignoniaceae) and Phyllanthus niruri L. (Phyllanthaceae). We classified these plants regarding their pharmacological activity and the mechanism of action of their key active phytocomponents.Herbal remedies have been traditionally used to maintain general health and well-being. Given the high prevalence of DM in the population of the Caribbean basin, there is popular knowledge on medicinal plants to prevent or treat DM ,22,23. TMany natural products isolated from plants, including polypeptides, carotenoids, flavonoids, alkaloids, terpenoids, saponins, tannins and glycosides, have previously shown antidiabetic properties ,73. The The first group contains phytochemicals that modulate metabolic pathways in which glucose acts as a substrate or as a product. Therefore, they affect gluconeogenesis, glycogenolysis, pentose phosphate pathways and glycogenesis. This group is also comprised of phytochemicals that affect glucose uptake and compounds that show α-glucosidase and α-amylase inhibitory activity. Group II harbors phytochemicals that lower triglyceride levels and cholesterol content, impacting hyperlipidemia, a pathophysiological feature of DM . Group Iwww.tramil.net) (accessed on 28 September 2021) [The Caribbean flora constitutes an untapped reservoir of natural biodiversity and a vast source of natural products. It has evolved under conditions of extreme environmental stress. It is exposed to multiple stressors, including high levels of UV radiation, high temperatures, anaerobic soils, strong winds, high salt and sulfide concentrations, oxidative stress, nutrient deficiency, extreme seasonality, phytopathogens and herbivory. The ability of Caribbean species to grow under these conditions is mainly due to specialized physiological processes that affect the chemical responses of the plants, triggering the biosynthesis of unique metabolites that include hydrocarbons, terpenoids, polyphenols, alkaloids, flavonoids, and quinones . Severaler 2021) . This muHere we summarize the available literature about the antidiabetic potential of these Caribbean species. We compile in vitro and in vivo experimental evidence supporting a beneficial impact of extracts or isolated fractions that follow the above-described antidiabetic action mechanisms .A. occidentale L.   and gallic acid [A. occidentale is also being explored using computational approaches. According to a ligand-based prediction model, 8 different hit compounds from the plant  catechin, protocatechuic acid, epigallocatechin, naringenin and (−) epicatechin) have potential action against glutamine-fructose-6-phosphate aminotransferase 1 (GFAT1) and dipeptidyl peptidase-4 (DPP-4), two promising therapeutic targets for DM management [ntale L.  is a trolic acid . The antnagement .A. occidentale [A. occidentale leaves had major beneficial effects in lowering blood glucose levels in streptozotocin-induced diabetic rats. The impact of A. occidentale extracts was similar to pioglitazone, a standard medicine used for DM management. After administration of 100 mg/kg of plant extract, the rats’ blood glucose levels showed an 8.01% and 19.25% decrease in their fasting blood glucose levels after 15 and 30 days, respectively [A. occidentale leaves showed protection against the hyperglycemic effects of streptozotocin in rats [Several in vivo studies strengthen the antidiabetic potential of plant extracts of identale ,41,81,82identale . The metectively . AnotherA. occidentale seeds and their active ingredient, AA, induced glucose absorption into C2C12 muscle cells in a dose-dependent manner. Nonetheless, the extracts from other plant sections  were inactive [The role of AA as a therapeutic agent on metabolic disorders, including fatty liver disease and DM, has been deeply investigated. AA in vitro administration reduced lipid accumulation in 3T3-L1 cells without observed cytotoxicity. Adipocyte differentiation was inhibited by reducing the expression of the fatty acid synthase (FAS) and peroxisome proliferator-activated receptor-gamma (PPAR-γ), two adipogenesis-related markers . Hydroetinactive . In addiinactive ,43.A. occidentale might affect different antidiabetic acting mechanisms. For example, the oral administration of the ethanolic extract of A. occidentale flowers revealed anti-inflammatory behavior in diabetic mice with sepsis and extended mice lifespan. The flower extract of the plant stimulated the recruitment and proliferation of macrophages and neutrophils and decreased IL-6, MCP-1, and TNF-α, three immunomodulatory cytokines [A. occidentale showed anti-inflammatory and antioxidant properties, reducing neutrophil infiltration and colon damage and lowering malondialdehyde (MDA) and pro-inflammatory cytokine levels. Additionally, cashew nut administration enhanced manganese superoxide dismutase (MnSOD) antioxidant activity and inhibited NF-kβ nuclear factor, a key player during inflammation [A. occidentale bark extract has also been reported to stimulate pancreatic β cell islet cells. The plant extract acted on β-cells similarly to sulfonylurea drugs, stimulating insulin discharge in a glucose-dependent manner [Compelling literature supports that, in addition to its hypoglycemic and hypolipidemic impact, ytokines . In a moammation . A. occit manner .A. occidentale extracts, the information about its toxicity and safety is scant. Regarding the acute toxicity of the hexane leaf extract on mice, the LD50 was reported to be 16 g/kg. Additionally, the oral subchronic treatment during 56 days at doses of 10 and 14 g/kg suggests liver and kidney toxicity since urea, creatinine and transaminase levels are impaired [Despite many in vitro and in vivo studies using impaired . Anacardiun genus can also be used in DM management. For instance, the ethanolic extract of Anacardium humile St. Hil leaves showed important in vitro antiglycation and antioxidant activity. It inhibited α-amylase activity in RAW264.7 cells, a mouse macrophage cell line [Furthermore, recent findings suggest that other plants from the ell line . H. suaveolens (L.) Poit.  Poit.  is an aractivity .H. suaveolens’ phytochemical profile contains alkaloids, carbohydrates, flavonoids, tannins, steroids, and terpenes [H. suaveolens leaves are hentriacontane, hentriacontanone, lupeol, diterpenoids, and triterpenoids. There is scant information about the pharmacological potential of these phytocompounds of H. suaveolens. The major therapeutical activity of the plant might reside in ursolic acid (UA), a pentacyclic triterpenoid. UA is a potent hypoglycemic agent acting as an insulin secretagogue and insulinomimetic. It enhances insulin secretion, transportation and uptake by the glucose transporter protein (GLU4) by stimulating the intracellular accumulation of calcium [terpenes . Other p calcium ,87.H. suaveolens extracts for DM management. The oral administration of the ethanolic extract of the leaves had a hypoglycemic impact in diabetic rats, also reducing cholesterol and triglyceride levels [H. suaveolens had antioxidant potential in different diabetic rat models and inhibitory potential on salivary α-amylase [H. suaveolens as an antihyperglycemic agent and for promoting the improvement of oxidative stress present this Caribbean species as a promising source of phytocompounds to fight against DM. Unfortunately, there is no information regarding H. suaveolens’ side effects and toxicity. Further studies are needed to address its suitability for pharmaceutical purposes.Growing evidence informs the promising potential of e levels . Additioe levels . The chl-amylase . These fHyptis genus has also been tested. In streptozotocin-induced diabetic rats, the administration of the ethanolic extract of Hyptis verticillata jacq. leaves decreased HbA1c and fasting blood glucose levels [H. verticillata ameliorated dyslipidemia (increasing the cardioprotective index and lowering the atherogenic coefficient and the atherogenic and coronary risk indices) [H. verticillata extract diminished oxidative stress ) and also ameliorated hepato-renal damage and lowered blood glucose levels of streptozotocin-induced rats [The antidiabetic potential of other plant species belonging to the e levels . Analogoindices) . The H. ced rats .P. americana, including phenol compounds, organic acids, alkaloids, diterpenoids and amino acids [Persea americana Mill.  is an arno acids . Avocadono acids . The nutno acids . Among tno acids ,95,96.P. americana leaves have been proposed as an interesting phytopharmaceutical source. In a comparative study, the effects of aqueous, ethanolic and methanolic leaf extracts of the plant were assessed in nicotinamide and streptozotocin-induced diabetic rats. The methanolic extract displayed the highest antidiabetic potential, but all the extracts significantly reduced glucose blood levels and ameliorated the hyperlipidemic state of the diabetic rats [P. americana leaves reduced blood glucose content in diabetic rats, restoring the intracellular energetic balance through the phosphorylation and the activation of protein kinase B (PKB), a central player in the AKT/PKB signaling pathway [P. americana leaves can modify normal renal function [tic rats . The aut pathway . This si pathway . The act pathway . Neverthfunction , furtherP. americana seeds have also demonstrated antidiabetic effects. The oral administration of an aqueous extract of P. americana seeds to alloxan-induced diabetic rats had significant tissue-protective effects on the pancreas, liver, and kidney and lowered blood glucose levels. The hypoglycemic effect was similar to glibenclamide, a drug used for T2D management [nagement . P. americana extracts have been linked to the inhibition of T2D key enzymes. The phenolic extracts of P. americana leaves and fruit inhibit both α-amylase and α-glucosidase in a dose-dependent manner [50 value , the peel had the highest inhibitory activity for α-amylase, while the leaves had the most inhibitory action for α-glucosidase. Moreover, the phenolic extracts inhibited induced-lipid peroxidation in the pancreas in a dose-dependent manner [Additionally, t manner . AccordiP. americana, other plants from the Persea genus have shown promising antiglycation and antidiabetic properties. In alloxan-induced diabetic rabbits, 24 days of treatment with the crude extract of Persea duthieion reduced the animals’ body weight and had significant antihyperglycemic effects. In particular, the ethyl acetate fraction of the extract had the highest antidiabetic activity [Persea indica inhibited BSA glycation in vitro and inhibited α-amylase, α-glucosidase, and aldose reductase activity. The antiglycative action was higher than that observed for aminoguanidine, a glycation protector compound [Apart from activity . Moreovecompound .Psidium guajava L. , a negative regulator of the leptin and insulin signaling pathways [P. guajava arises as a valuable phytopharmaceutical agent. In addition, the isolated polysaccharide fraction of P. guajava leaves lowered the total triglyceride and cholesterol contents, decreased fasting glucose levels and enhanced the superoxide dismutase activity in streptozotocin-induced diabetic rats [ipidemia . The oraced rats . The auta levels . Interespathways . Given tpathways , P. guajtic rats . P. guajava leaf aqueous extract also shows a strong inhibitory effect on Amadori products (fructosamines) that are intermediates in the production of AGEs. Gallic acid, catechin and quercetin are phenolic compounds from the leaves believed to possess antiglycation properties [P. guajava leaf extract, shows an important antiglycative and anticoagulant activity restoring the antithrombin III function previously inhibited by methylglyoxal, a potent precursor of glycation [operties . Furtherlycation . P. guajava has also been proposed as a therapeutic agent against DM-associated complications. The plant leaf extracts’ ethyl acetate fraction significantly reduced serum fructosamine and HbA1c levels. They lowered the expression of transforming growth factor β (TGF-β1), connective tissue growth factor (CTGF) and B-type natriuretic peptide (BNP) in the myocardium of diabetic rats, suggesting a cardioprotective role for the P. guajava extract [P. guajava triterpenoids resulted in increased serum insulin levels and insulin sensitivity index and ameliorated the renal damage of streptozotocin-induced diabetic rats [P. guajava extracts since there is no information available in the literature. extract . The inttic rats . These rTecoma stans (L.) Juss. ex Kunth  Juss. ex Kunth, including terpenoids, flavonoids, glycosides, unsaturated fatty acids and carotenoids [T. stans produce elevated levels of phytochemical substances with pancreatic lipase inhibitory activity and, thus, antidiabetic potential. Chrysoeriol, apigenin, luteolin, and verbascoside from T. stans leaves showed lipase inhibitory activity, with chrysoeriol and apigenin being the most bioactive compounds [T. stans thought to have a hypoglycemic effect. Chlorogenic acid reduced postprandial peaks of glucose and decreased liver triacylglycerol levels and fasting cholesterol plasma content in obese, hyperlipidemic and insulin-resistant rats [Approximately 120 different compounds have been isolated from otenoids . T. stanompounds . Interesompounds ,60,111. ant rats . Tecominant rats . Tecostaant rats , althougT. stans extracts in a diabetic context [T. stans stimulated the glucose uptake in insulin-sensitive and insulin-resistant murine or human adipocytes [In vivo evidence supports a beneficial effect of  context . These eipocytes . T. stans is being examined in clinical trials. The effectiveness of an herbal mixture made of Guazuma ulmifolia and T. stans was assessed in a randomized, double-blind, placebo-controlled trial. The group of T2D patients that were administered with the mixture showed a significant decrease in waist circumference, fasting glucose levels and HbA1c content [T. stans is a relevant issue that needs to be evaluated. The subchronic treatment with the plant extract showed no mortality and no adverse effects in rats [Recent studies report that the antidiabetic potential of  content . Further in rats . Momordica charantia L. : (I) afM. charantia has been tested in numerous in vivo and in vitro studies. The fruit extract showed significant antihyperglycemic activity by reducing the percentage of HbA1c and blood glucose levels and by inhibiting glycogenolysis in vitro [The hypoglycemic therapeutical effect of in vitro . Similarin vitro . Additioin vitro ,109,110.M. charantia has been proposed as a promising complementary agent for diabetes with antioxidant properties. M. charantia juice was fermented with Lactobacillus fermentum LLB3, a lactic acid bacterium, increasing its antioxidant properties [M. charantia juice, non-fermented M. charantia juice, or administered acarbose, a typical anti-diabetic drug. The fermented juice showed more anti-diabetic properties than the non-fermented one. Rats fed with fermented juice and with non-fermented juice reported a significant reduction in fasting blood glucose levels  in postprandial blood glucose levels  mg/mL and an increase in SOD levels  when compared with diabetic rats that were given only water. However, the antidiabetic effects were lower than those observed using acarbose. Rats within this group reported the lowest fasting and postprandial blood glucose levels  and the highest SOD levels (84.3 ± 3.95). In another study, the aqueous extract of the bitter melon significantly decreased blood glucose levels and HbA1c content and increased tissue glycogen serum insulin and glucagon-like peptide 1 (GLP-1) in streptozotocin-induced diabetic rats [M. charantia extract to streptozotocin-induced diabetic rats resulted in lowered serum glucose and in a reduction of the suppressor of cytokine signaling-3 (SOCS-3), c-Jun N-terminal kinase (JNK) and GLUT-4 content [The probiotic-rich fermented extract of operties . In thistic rats . Since G content , two key content ,123. DesM. charantia can also help to prevent complications from kidney failure. A recent study showed that M. charantia extracts improved blood glucose levels, defended against diabetic nephropathy, decreased body weight loss, and preserved hyperglycemia in rats with streptozotocin-induced T2D [uced T2D .Phyllanthus niruri L. (P. niruri with multiple pharmacological properties. Phytochemical research shows that the leaf extract contains alkaloids, flavonoids, saponins, tannins, lignins, terpenoids and coumarins but not hormones, glycosides or resins [iruri L.  is a perr resins . Due to P. niruri [Several studies highlight the anticholesterolemic, anti-inflammatory, anti-hyperuricaemic and potent anticancer and antimicrobial properties of . niruri ,126,127.P. niruri protects against diabetes-related renal disorder and pointed out that this herb may be used to treat diabetic nephropathy. Another study reported that oral administration of P. niruri extract from its aerial parts to alloxan-induced diabetic rats lowered HbA1c, decreased blood glucose levels and increased liver glycogen amount [P. niruri has been tested as a hypoglycemic agent regarding its antidiabetic potential. The administration of the plant extract improved the lipid profile and lowered the serum glucose levels of T2D rats . The autn amount .P. niruri [P. niruri extract inhibited superoxide and hydroxyl radical formation and possessed hydrogen peroxide scavenging activity by ameliorating glutathione peroxidase, catalase and superoxide dismutase function and lowering MDA and lipid peroxidation products. Unfortunately, there is no available information about P. niruri’s possible harmful effects. Further studies should be implemented to address the plant toxicity and dosage.Bioactive phytocompounds of P. niruri also enhance other antidiabetic acting mechanisms. For example, the α-glucosidase inhibitors corilagin and repandusinic acid A also have been discovered in the water extracts of . niruri . Additio. niruri . The P. Phyllanthus genus, several other plant species have shown antidiabetic potential. An in silico study revealed that ellagic acid, phytoestrogens, sesamine, kaempferol, zeatin, quercetin, and leucodelphinidin, natural products that can be found in Phyllanthus emblica L. (Phyllanthus amarus has reported diuretic, hypoglycemic and hypotensive effects in humans [Phyllanthus acidus. Skeels has also shown antioxidant, hypoglycemic and α-glucosidase inhibitory activity [In the blica L. , possessn humans . Finallyactivity . DM is a recurrent condition that calls for ongoing and long-term treatment and possesses a challenge for worldwide healthcare. In this scenario, natural products from plants represent an abundant pharmacological source to treat or prevent DM and its associated complications. A significant proportion of diabetic patients are regular users of these traditional medicines. Given that the Caribbean Basin represents a unique ethnopharmacological relevant area due to its floral diversity, the Caribbean flora represents an opportunity to discover novel bioactive phytocompounds to combat DM. This review describes the antidiabetic potential of seven widely used plants in the Caribbean region in folk medicine. However, the pharmacological information for each plant is scant. Further investigation is required to identify novel phytochemicals and determine which molecular and cellular pathways are involved in the protective anti-diabetic effect of these traditional herbal medicines. Additionally, a more intense effort in research is imperative to define the effective dosage and toxicity of the various plant extracts that are commonly used.


Crematogaster matsumurai (Forel 1901) is an important arboreal ant species commonly found on Phyllostachys heterocycla (Carr.) in Lishui, Zhejiang, China. This study analyzed the mitochondrial genome sequence of C. matsumurai and discussed its phylogenetic relationship in Hymenoptera. The circular mitochondrial genome was 16,028 bp long, including a standard set of 22 transfer RNAs (tRNAs), two ribosomal RNAs (rRNAs), and 13 protein-coding genes (PCGs), which showed the typical insect mitochondrial genome arrangement. The AT and GC contents of the mitochondrial genome sequence were 76.92% and 23.08%, respectively. The maximum-likelihood (ML) phylogenetic analysis based on whole mitochondrial genome sequences showed that C. matsumurai is closest to Crematogaster teranishii. Crematogaster (Hymenoptera: Formicidae: Myrmicinae) ants are diverse, widespread, and abundant in tropical, subtropical, and warm-temperate climates throughout the world  is an ant species commonly found in East Asia. It has been reported as a predator of some pests, such as Halyomorpha halys  in Lishui, Zhejiang, China. It builds insect nets nests on the leaves, stems, and branches of P. heterocycle (Carr.) to catch insects, such as aphids. Moreover, C. matsumurai develops mutualistic associations with other organisms. Despite its local abundance and important ecological interactions, the phylogenetic relationship of C. matsumurai within genus Crematogaster is controversial and in great need of modern revisionary studies  forest in Longquan City , Lishui City, Zhejiang Province, China, and preserved in pure ethanol. The collected samples were identified and stored at −40 °C in the Institute of Integrated Plant Protection Center, Lishui Academy of Agricultural and Forestry Sciences, Lishui, China  under the voucher number 20210805LQCM. Total genomic DNA was extracted by using E.Z.N.A.® Insect DNA kit and applied to 300 bp paired-end library construction using the Truseq SBS Kit (300 cycles) for Illumina sequencing. Sequencing was carried out on the Illumina NovaSeq 6000 platform . A total of 5449.1 Mb of raw reads were generated, and by employing the tool Trimmomatic v0.39, the reads having adapter contamination and the small pieces less than 75 bp in length after quality trimming were removed to obtain clean reads. De novo genome assembly were conducted by SPAdes v3.14.1. The mitochondrion genes were annotated using the online MITOS tool  protein database, Swiss-Prot, Clusters of Orthologous Groups (COGs), and Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) terms (Yin et al. C. matsumurai was closely related to C. teranishii (Crematogaster had a close relationship with Ochetellus in Hymenoptera. The genome sequence of C. matsumurai in this study might provide useful information for Hymenoptera researches.To reveal the phylogenetic relationship of ranishii . Meanwhi


Borrelia burgdorferi sensu stricto (s.s.), Anaplasma phagocytophilum, and Babesia microti within a fragmented landscape in Essex County, Massachusetts, USA. We studied communities at multiple spatial scales, including vegetation, edge type, and landscape  scales. A total of 16 study sites were chosen to represent four edge types: interior forest, pasture edge, natural edge, and residential edge. At each site, we trapped small mammals and conducted vegetation surveys and GIS analysis. Upon capture, a tissue sample was collected to analyze for presence of pathogens. Northern short-tailed shrew (Blarina brevicauda) abundance did not differ based on edge type, whereas abundance of the white-footed mouse (Peromyscus leucopus) was greatest at pasture edges, although the relationship was relatively weak. White-footed mouse abundance was negatively associated with amount of forested area within a 500-m radius, whereas northern short-tailed shrew abundance demonstrated a positive relationship with fragmentation indices at the 200-m radius. White-footed mice captured at interior-forest habitat were more likely be infected with B. burgdorferi (s.s.) than individuals from edge habitat. Greater prevalence of B. burgdorferi infection of white-footed mice in forest interiors compared to edge habitats counters previous studies. Reasons for this and implications are discussed.Habitat fragmentation and heterogeneity transform otherwise contiguous tracks of forest into smaller patches in the northeastern U.S. and likely impact abundances, movement patterns, and disease transmission pathways for small-mammal communities at multiple scales. We sought to determine the structure of a small-mammal community in terms of mammal abundance and infection prevalence of  Habitat fragmentation and loss are pervasive challenges to biological populations in New England, USA. Increases in agricultural, residential, and commercial development over the past four decades have led to consistent reductions in forested area in northern Massachusetts, with approximately 63% of the state remaining forested . Forest Peromyscus leucopus), whereas Glennon and Porter (2007) [Sorex genus prefer contiguous landscapes with minimal edge habitat. White-footed mice also may be more common in smaller than larger forest patches [The effects of habitat fragmentation on small mammals have been widely studied, but no unidirectional trends in abundance have been well-established . Howeverr 2007)  found sh  found s patches , 8.Borrelia burgdorferi sensu stricto (hereafter B. burgdorferi) (Lyme disease), Babesia microti (human babesiosis), and Anaplasma phagocytophilum (human granulocytic anaplasmosis) are the most prevalent tick-borne pathogens and are transmitted by their common vector, the black-legged tick (Ixodes scapularis) [Blarina brevicauda) are common members of the New England small-mammal community and serve as reservoirs for all three pathogens [B. burgdorferi and B. microti are more prevalent within forest interiors (50 m from an edge) than forest edges in Ixodes scapularis [Ixodes ricinus [B. burgdorferi infection prevalence, indicating that the core habitat (interiors) of patchy landscapes may represent high-risk zones for Lyme disease transmission [B. burgdorferi nor A. phagocytophilum prevalence appear to respond to changes in fragmentation along an urbanization gradient [A. phagocytophilum reservoirs remains unclear [Members of small-mammal communities serve as significant reservoirs in pathogen transmission. In New England, pularis) –12. Althathogens , 12–15. apularis  and at l ricinus . Furthersmission , 19. Neigradient , and the unclear .Peromyscus species and the northern short-tailed shrew as reservoirs of B. burgdorferi, A. phagocytophilum, and B. microti within a fragmented landscape in Essex County, Massachusetts, USA. Specifically, we considered how differences in fragmentation and habitat characteristics were related to abundance and infection prevalence of small mammals. We analyzed study sites in terms of vegetation composition, edge type, and landscape variation at 200 m, 500 m, and 1000 m. Based on the literature, we predicted that white-footed mice would be more abundant in natural, pasture, and residential edges and also negatively influenced by patch size, whereas northern short-tailed shrews would be more abundant in interior forest and negatively affected by habitat fragmentation. We also hypothesized that infection prevalence of small mammals would be positively related to fragmentation at multiple spatial scales and with greater understory vegetation.In this study, we sought to determine the abundance of a small-mammal community and determine the potential of This study is part of a larger research effort on differential habitat use by terrestrial vertebrates affected by edge type, landscape composition, and vegetation structure in northeastern Massachusetts . Essex CPinus strobus), eastern hemlock (Tsuga canadensis), oak (Quercus spp.), red maple (Acer rubrum), American beech (Fagus grandifolia), birch (Betula spp.), shagbark hickory (Carya ovata), horse chestnut (Aesculus hippocastanum), and cherry (Prunus spp.). Understory was dominated by seedlings and saplings of overstory trees, in addition to Japanese barberry (Berberis thunbergii), Japanese honeysuckle (Lonicera japonica), multiflora rose (Rosa multiflora), flowering dogwood (Cornus florida), poison ivy (Toxicodendron radicans), sassafras , and viburnam (Viburnum spp.). We selected sites that appeared to be similar in vegetation structure and tree species composition, and site characteristics did not appear to change between years. Sites were placed in mature forest (greater than 40 years old) at areas free from active disturbance  in forest patches at least 10 ha in size.Common overstory trees included white pine , weighed, tagged with aluminum tags, sampled by taking a small ear clipping or tail clipping (shrews), and released. Tissue samples were stored in 70% ethanol and collected for genetic species discrimination between Peromyscus spp. and for assessment of tick-borne pathogen infection among all small mammals.Upon capture, all small mammals were identified to the species level  of deciduous and coniferous trees separately as a measure of biomass. Within the 12-m radius, we also counted total number of logs  and snags (standing dead overstory trees).We conducted vegetation surveys at sites from June to July 2009 and during early September 2013. We adapted our vegetation survey protocol from James and Shugart (1970) , followihttp://maps.massgis.state.ma.us/map_ol/oliver.php). We measured four landscape metrics at each of the 16 study sites, including patch size , forested area, linear human edge (length of edge occurring at the interface between human-developed land and forest habitat), and linear wetland edge (length of edge occurring at the interface between a body of water and forest habitat). Other than forest patch size, these landscape variables were measured within three radii, including 200, 500 and 1000 m, representing distances above the home-range size and incorporating maximum long-distance dispersal movements for species we captured [Using ArcMap 9.3 (ESRI 2009) , we prodcaptured , 28. Thecaptured . Within ® Blood and Tissue kit  following the manufacturer’s recommended protocol and eluting in 50 ul H2O. Gel electrophoresis and concentration by NanoDrop (ThermoFisher) were used to assess DNA purity and quantity. Samples containing sufficient amounts of DNA for analysis were subjected to multiplex qPCR for detection of B. burgdorferi (gB31), A. phagocytophilum (msp4), and B. microti (18S) in a 96-well format using the methods developed by Hojgaard et al. (2014) [2O. Negative and positive controls were used in all tests and performed in triplicate. For the latter, a plasmid containing PCR-amplified target genetic loci was generously provided by Andrias Hojgaard and Joseph Piesman. Pipetting to mix samples was performed in a PCR hood; positive control samples were set up in a separate lab to ensure no contamination of experimental sample DNA. PCR was carried out on a CFX 96 Touch Real-Time PCR Detection System (BioRad) using the following parameters: initial denaturation for 3 min at 95°C was followed by 40 cycles of 95°C for 10 sec and 60°C for 1 min.DNA was extracted from small-mammal tissue samples using the Qiagen DNeasy. (2014) . BrieflyPeromyscus spp., 63 DNA samples were selected for genetic analysis, including 12, 9, 24, and 18 samples corresponding to Peromyscus individuals captured at interior forest, natural edge, pasture edge, and residential edge habitat, respectively. The protocol for identification was adopted from Tessier et al. (2004) [Peromyscus maniculatus, F9197) and the other a forward primer specific to white-footed mice . The reverse primer, H9375, was common among all PCR mixtures. Mixtures included 5 ul of 2X SSO Advanced SYBR Green Supermix, 3 ul H2O, 1ul template DNA, and 0.5 ul each of reverse and forward primers . PCR was performed on a CFX 96 Touch Real-Time PCR Detection System (BioRad) using the following parameters: 98°C for 3 min, 30 cycles of 10 sec at 98°C, 30 seconds at 56°C and 20 sec at 72°C, and a final 2 min extension at 72°C. DNA from a known deer mouse museum specimen was used as the positive deer mouse control. Analyses confirmed that all Peromyscus captures were white-footed mice.To corroborate field identifications of . (2004) . Two 10 a priori to test the hypotheses that white-footed mice would be more abundant in edge habitats with smaller patch sizes, while northern short-tailed shrews would respond positively to forest interiors and patch size. Abundances of masked shrew (Sorex cinereus), eastern chipmunk (Tamius striatus), and red squirrel (Tamiasciurus hudsonicus) were too low for analysis.To determine the effects of vegetation, edge type, and landscape characteristics on small mammal abundances, we used generalized linear models (GLM) with a Poisson error distribution. Dependent variables included total small-mammal abundance, white-footed mouse abundance, and northern short-tailed shrew abundance, with abundance being the total number of unique individuals captured at each site. Models were constructed B. burgdorferi, A. phagocytophilum, and B. microti infection prevalence in all small mammals captured as a function of vegetation, edge type, and landscape characteristics using generalized linear models (GLM) with a binomial error distribution. We used a binary indicator of infection as a dependent variable. Models were developed a priori to test the hypotheses that infection would be greater in more fragmented areas with greater understory vegetation.We modeled For all models, pairwise correlation coefficients were derived for independent variables to ensure that highly correlated variables (|r| > 0.7) were not included within the same model . All indThe 2012 season yielded 103 unique individuals with a trapping success rate of 4.3%, whereas the 2013 season yielded 92 unique individuals with a trapping success rate of 3.8%. These 195 total unique captures represented 5 small-mammal species, including white-footed mouse , northern short-tailed shrew (18), eastern chipmunk (5), red squirrel (2) and masked shrew (1). No individual was recaptured at a different site. Only white-footed mice and northern short-tailed shrews were captured in large enough abundances for statistical analysis. White-footed mouse abundance was greatest at pasture edges ; howeverGeneralized linear models of small mammal abundance suggest species correlated to both vegetation and landscape characteristics. Abundance of white-footed mice was greater at study sites with less forested area in a 500-m radius . NortherB. burgdorferi, A. phagocytophilum, and B. microti. White-footed mouse infection prevalence for B. burgdorferi  was greatest at interior-forest study sites compared to sites associated with a habitat edge  at 16 study sites across two years (A. phagocytophilum (mean infection = 5.4%) and B. microti (mean infection = 17.7%) did not differ significantly among edge types. Infection of northern short-tailed shrews with B. burgdorferi (mean infection = 6.3%), A. phagocytophilum (mean infection = 12.5%), and B. microti (mean infection = 25.0%) did not differ significantly among edge types. Most models for B. burgdorferi infection in white-footed mice included vegetation variables; we found a positive relationship between infection prevalence and presence of leaf litter and a negative relationship between infection prevalence and presence of vegetation ground cover   documentB. burgdorferi in white-footed mice was greater at forest-interior sites than at any edge-habitat site, while A. phagocytophilum and B. microti were not associated with any vegetation or edge characteristics. Greater B. burgdorferi infection prevalence in forest interiors may be a function of increased reservoir abundance [Turdus migratorius), or small mammals that are difficult to capture in Sherman traps, such as eastern chipmunks [Borrelia prevalence. Further analysis and sampling is required to establish the exact mechanism of this effect.Infection prevalence of bundance  althoughhipmunks . AdditioThe presence of induced edge and reduced forested habitat are closely associated with fragmentation processes, indicating that our results support an implicit positive relationship between small-mammal abundance and habitat fragmentation . AlthougB. burgdorferi prevalence and habitat fragmentation indices. General trends in the literature suggest that B. burgdorferi infection probabilities are correlated with white-footed mouse habitat preferences, where infection prevalence increases in fragmented landscapes. This pattern has been shown in black-legged ticks, where tick density and B. burgdorferi infection prevalence tend to increase with decreasing patch size and increasing patch isolation [B. burgdorferi infection in white-footed mouse was significantly higher in large forest patches compared to forest edges. They attributed this finding to the dilution effect, where edge habitats had greater encounter probabilities with alternative hosts. We did not directly test for the dilution effect as we did not estimate the abundances of alternative reservoirs to small mammals and we did not measure tick infestations on small mammals. However, the dilution effect is one possible explanation for our observations.We found a negative relationship between solation , 19. Oursolation  found thB. burgdorferi, significant models were not identified for infection prevalence for A. phagocytophilum and B. microti based on edge type or landscape characteristics. Other researchers have shown that B. microti prevalence in humans responds to amount of landscape-level edge habitat [A. phagocytophilum prevalence, based on black-legged tick infection, is not related to landscape-level composition [B. microti is typically detected through blood collection rather than tissue collection [P. leucopus and B. brevicauda combined) in our study suggest that collection of an ear or tail sample is a viable alternative to detect this pathogen, given the vascularization of this tissue. However, this comparison requires more direct analysis; lack of significant differences based on vegetation, edge type, or landscape characteristics may indicate an underestimate of this pathogen in our study.In contrast to findings for  habitat , but thaposition . Howeverposition  of thesellection , 52, relB. burgdorferi prevalence, emphasizing the importance of scale considerations in the ecology of small-mammal communities and disease transmission. In addition, this relationship between small-mammal infection prevalence and patch interiors is rare in the body of relevant literature and offers novel insight into the ecology of Lyme disease in the northeastern U.S. However, given small sample sizes in this study, additional research is required to corroborate these results and to help identify patterns for other disease pathogens for which small mammals are the common reservoir, including A. phagocytophilum and B. microti [Overall, we found multiscale correlations of vegetation, edge type, and landscape variables on small-mammal abundance and  microti .S1 File(XLSX)Click here for additional data file.


A suitable plant species was determined according to tolerance to WL during wetland vegetation recovery activities. Thus, two tree species  that showed a certain waterlogging tolerance were chosen to study their behaviors in different planting model and environmental conditions. Given that interspecific relationship should be considered during plant community construction, the eco-physiological adaptable mechanisms between these woody plants under well-watered condition and waterlogging stress were explored. Results showed that both species were waterlogging-tolerant species due to their ability to adapt to submergence conditions for 120 days. Moreover, C. operculatus possessed stronger tolerance to waterlogging stress because of a significant adventitious roots biomass accumulation. A competitive relationship was found between C. operculatus and S. jambos under well-watered condition, and C. operculatus showed better growth performance benefited from morphological responses  and endogenous hormone variations. In comparison, S. jambos suffered some negative effects when the well-watered mixed planting was used. However, the competitive relationship under well-watered condition was transformed into mutualistic relationship under waterlogging stress. The mixed planting under waterlogging condition significantly improved the tolerance of C. operculatus and S. jambos to waterlogging stress, compared with the monoculture., Especially, S. jambos showed improvements in root length, root surface area, and redox equilibration between lower levels of relative conductivity, malondialdehyde, and C. operculatus and S. jambos was altered by waterlogging stress, and both showed improved tolerance to waterlogging stress. This study can provide a glimmer of light on suitable plant species selection and plant community construction during the revegetation activities in wetland zones. C. operculatus and S. jambos represent potential candidates in wetland restoration in a mixed planting model.Competition and abiotic stress such as waterlogging (WL) represent main factors limiting plant growth and determining plant resistance and distribution patterns in wetland ecosystems. One of the basic steps for wetland restoration is to plant trees to ensure a quicker recovery and prevent erosion. Plant survival and adaptation are considered criteria of principal priority for the screening of plant species for wetland ecosystem restoration. WL influences plant species in wetlands  Cleistocalyx operculatus with better growth performance benefits from water-watered mixed planting pattern.– – C. operculatus possessed stronger tolerance to waterlogging stress than Syzygium jambos.S. jambos which has a low tolerance to waterlogging stress seems to gain more benefits.– Plant competitive interactions under well-watered conditions become mutualistic under waterlogged conditions; C. operculatus and S. jambos in the restoration of areas that are subject constantly to submergence conditions.– The results of this study showed a promising perspective to use 3) is involved in the regulation of waterlogging tolerance  is an important biotic factor affecting plant species distribution, population dynamics, community construction, and ecosystem services . It has Cleistocalyx operculatus and Syzygium jambos are often used in restoration and reconstruction of riparian and wetland areas. Indeed, Cleistocalyx operculatus and S. jambos belong to the Myrtaceae family. They are evergreen medicinal woody plants with waterlogging tolerance and often used in the restoration and reconstruction of riparian zones of tropical and subtropical regions (C. operculatus and S. jambos under well-watered condition and waterlogging stress were designed and used in answering the following questions: (1) Can these two species benefit from the mixed planting pattern under well-watered conditions? (2) Whether the woody plant with strong waterlogging tolerance gain more benefits than a plant species which possesses a less waterlogging tolerance when they are subjected to waterlogging in a mixed planting? (3) Whether waterlogging environments alter neighboring relationships between two species and whether competition or facilitation is preferred? Thus, changes in interspecific relationships between C. operculatus and S. jambos under well-watered and waterlogging conditions were explored through physiological analysis based on phenotypic traits, photosynthetic performance, lipid peroxidation, non-enzymatic antioxidant components, antioxidant enzymatic activities, and endogenous hormones.Although there are many studies that focused on the physiological and morphological adaptations of plant species commonly used in restoration practices for individuals grown in monoculture, there is still a poor understanding of how species interactions may induce competitive or facultative interactions that ultimately modify the environmental conditions that individual plants are growing in despite the anaerobic conditions associated with waterlogging in wetlands. This is of particular importance in different regions threatened by submergence conditions, where two species such  regions . In the C. operculatus and S. jambos from Zengcheng, Guangdong Province were planted in pairs in a 43 cm × 19 cm × 14 cm (L × W × H) plastic bar-shaped basin, which was filled with red soil, sand, and coconut coir in 4:2:1 (v:v:v) ratio. The chemical and physical properties of the red soil were: pH = 6.23, 33.65 g.kg−1 of organic carbon, 58.01 g.kg−1 of organic matter, 1.77 g.kg−1 of total nitrogen, 0.64 g.kg−1 of total phosphorus. Before treatment, fertilizer solution was applied two times at 300 ml in each pot . The experiment was carried out in a greenhouse at Hainan University . Healthy plants were selected for a randomized design with treatments consisting of combinations of two irrigation levels (well-watered and waterlogging conditions), and three different planting systems  were arranged. Each treatment had at least 24 replicates, which implied at least 144 pots for the whole experiment. The waterlogging depth was 10 cm above the soil surface. For each species, the well-watered and waterlogging treatments were marked as CK and W, respectively, in the monoculture, and marked as CK-M and W-M, respectively, in the mixed planting. For the well-watered treatment, pots were watered daily (100% field capacity). All the experiments were arranged in a complete randomized design. The morphological and physiological assays were determined after 120 days.Two 6-month-old tree species seedlings of 120-Height1, and IBN was calculated as N120-N1. The leaf area (LA) was determined before the end of the experiment with a portable area meter  and blade number (IBN), the plant height and number of blades were measured at the beginning (day 1) and the end (day 120) of treatments. The IPH was calculated as HeightThe young fresh leaves were collected at the end of the treatment. Chlorophyll a (Chl a), chlorophyll b (Chl b), total chlorophyll, and carotenoids (Caro) were quantified after extraction in 95% (v/v) ethanol with a spectrophotometer  at 470, 640, and 665 nm. The net photosynthetic rate (Pn), intercellular carbon dioxide (Ci), stomatal conductance (Gs), and transpiration rate (Tr) were determined once at the end of the treatment from 08:30 to 11:30 h using a 3051D photosynthetic device  according to the methods of Relative conductivity was measured according to the method described by Malondialdehyde (MDA) was determined according to the method of 2 was used as the standard curve for the calculation of the The production rate of The concentration of proline was measured with the method of Soluble proteins were determined according to the method described by Reduced GSH (reduced glutathione) and ASA (ascorbic acid) levels were measured according to the manufacturer’s instructions with the method of Fresh leaf samples (0.2 g) were ground and homogenized with tissue grinder instrument scientific laboratory homogenizer equipment . The mixture contained liquid nitrogen, 5 ml of 50 mM sodium phosphate buffer (pH 7.8), and 1% polyvinylpolypyrrolidone. The mixture was centrifuged at 10,000 rpm, 4°C for 15 min, and the supernatant was used to perform for the antioxidant enzymatic activities determination.−1·FW·min−1, POD (peroxidase) activity was determined with the method described Expressed as unit· g3, and JA-Me were extracted and purified with the methods described by 3, and JA-Me horseradish peroxidase used in ELISA were produced at the Phytohormones Research Institute, and fresh samples were sent for determination in China Agricultural University.Endogenous IAA, ABA, GAC. operculatus and S. jambos under waterlogging conditions.Experimental data processing statistics, analysis, and mapping were performed using SPSS 23.0, Canoco 5.0 and GraphPad Prism 9. All data were tested for normal distribution and homogeneity of variance before analysis. One-way analysis of variance followed by Tukey’s multiple comparison tests was used in establishing significant differences between pure planting and mixed planting under waterlogging stress at 1 and 5% levels of significance. Correlation analysis was performed with R software  under well-watered conditions compared to S. jambos. All waterlogging-treated seedlings from C. operculatus and S. jambos survived after 120 days of waterlogging treatment, but their growth rates significantly varied from each other. In C. operculatus, compared with CK treatment, CK-M treatment significantly increased the IPH and LA (p < 0.05), whereas non-significant differences in the IBN were found between the CK and CK-M treatments . In S. jambos, compared with CK treatment, W treatment significantly decreased the IPH, IBN, and LA in the group subjected to the monoculture. W treatment also significantly decreased IBN and LA in the mixed planting, but the W-M treatment significantly increased the IPH relative to the CK-M treatment  under the W-M treatment. Interestingly, the W treatment significantly increased the aboveground biomass and underground biomass of C. operculatus in the monoculture group, whereas S. jambos showed the opposite trends (p < 0.05). Compared with the monoculture group, the mixed planting under waterlogging conditions increased significantly the aboveground and underground biomass of S. jambos but only increased the underground biomass of C. operculatus . Further, S. jambos showed a significant increase of its surface areas of roots compared to C. operculatus. The results showed no significant differences in the root lengths and root surface areas of C. operculatus and S. jambos between the CK and CK-M treatments  but obviously decreased after the W treatment. The W-M treatment did not offset the decreases. In S. jambos, the Chl a, Chl b, total chlorophyll, and Caro levels were significantly lower in the CK-M and W treatments than in the CK treatment. The W-M treatment significantly improved these parameters, compared with the W treatment.As shown in p < 0.05; C. operculatus were significantly higher than those of S. jambos in the same treatment (p < 0.01), and the values of Gs and Tr in C. operculatus were significantly higher than those of S. jambos (p < 0.01) under mixed planting patterns (CK-M and W-M).The W treatment significantly decreased the levels of Pn, Gs, and Ci in both species in the monoculture group, and the mixed planting under waterlogging conditions (W-M) increased Pn and Ci in both species (S. jambos but had no significant effects on C. operculatus. Compared with the monoculture group (W), the mixed planting (W-M) significantly improved Fv/Fm in C. operculatus and decreased Fv/Fm in S. jambos under waterlogging conditions (p < 0.01). In addition, significant difference in Fv/Fm was found between C. operculatus and S. jambos in the CK and W-M treatments (p < 0.01).As shown in C. operculatus and significantly increased relative conductivity and S. jambos, compared with the monoculture (CK). Compared with the monoculture (W), the mixed planting under waterlogging conditions (W-M) significantly inhibited the accumulation of S. jambos (p < 0.05) and had insignificant effects on relative conductivity and MDA in C. operculatus (p ≥ 0.05).Compared with the CK treatment, the W treatment significantly increased relative conductivity , MDA con content  in both S. jambos (C. operculatus. The mixed planting under well water conditions (CK-M) significantly decreased proline content in both species and GSH content in C. operculatus and significantly increased the content of soluble protein and GSH in S. jambos, compared with the monoculture (CK). Compared with the monoculture (W), the mixed planting under waterlogging conditions (W-M) significantly decreased proline content in both species, GSH content in C. operculatus, and ASA in S. jambos. The W-M treatment significantly increased the content of soluble protein and GSH in S. jambos. In addition, compared with the W treatment, the W-M treatment significantly reduced relative conductivity in S. jambos (p < 0.05).Compared with the CK treatment, the W treatment significantly increased proline, soluble protein, GSH, and ASA content in . jambos –D and siS. jambos, and had effects on CAT activities in both species, compared with the monoculture . The mixed planting resulted in a slightly higher increase in SOD activity in C. operculatus than the monoculture under well-watered or waterlogging conditions and significantly decreased SOD activity in S. jambos (p < 0.05).The mixed planting under well water conditions (CK-M) significantly increased POD activities in both species, significantly decreased SOD activities in ture CK; . Compareture CK;  in both ture CK;  in S. jativities  in both 3 , the mixed planting under waterlogging conditions (W-M) significantly increased the content of GA3 and JA-Me in C. operculatus, significantly decreased their content in S. jambos, significantly decreased IAA content in C. operculatus, had no effect on S. jambos, and had no effects on ABA content in both species (Compared with the monoculture (CK), the mixed planting under well water conditions (CK-M) significantly increased the levels of IAA , ABA Fi, GA3 , Pn, fresh weight of original root biomass, root length, root surface area, MDA, GSH, ASA, 3, IAA, and JA-Me). The differences among treatment groups were significant and can be easily differentiated and identified  and the enhancement of biomass accumulation in C. operculatus in the CK-M treatment , and its growth and development were negatively affected by in contrast to C. operculatus in the pure planting (CK). Nevertheless, the mixed planting under waterlogging conditions significantly improved IPH, IBN, LA, pigment content, fresh weight of adventitious roots, accumulations of aboveground biomass and underground biomass, root length, and root surface area in S. jambos  and C. operculatus and S. jambos under waterlogging stress suggested that waterlogging caused some oxidative stress and damaged the integrity of the cell membrane. However, their levels in the leaves of both plants decreased when the mixed planting was used, suggesting that the patter efficiently alleviated the damage due to waterlogging stress, especially in S. jambos. In addition, the mixed planting under waterlogging condition induced significant increases in the levels of GSH and soluble proteins in S. jambos and slightly increased SOD and CAT activities in C. operculatus, suggesting that C. operculatus and S. jambos employed different antioxidant systems to maintain the redox equilibrium and improve their waterlogging tolerance under waterlogging stress in the mixed planting.Waterlogging can trigger oxidative stress on plants at the sub-cellular level by overproducing ROSs and promoting their accumulation, such as hydrogen peroxide ,Hainan Provincial Natural Science Foundation of China , and National Science Foundation of China (Grant Nos. 32060240 and 31660165).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.https://www.frontiersin.org/articles/10.3389/fpls.2022.869418/full#supplementary-materialThe Supplementary Material for this article can be found online at: Click here for additional data file.Click here for additional data file.


Spodoptera frugiperda (J. E. Smith), an emerging invasive pest worldwide, has posed a serious agricultural threat to the newly invaded areas. Although somatic sex differentiation is fundamentally conserved among insects, the sex determination cascade in S. frugiperda is largely unknown. In this study, we cloned and functionally characterized Doublesex (dsx), a “molecular switch” modulating sexual dimorphism in S. frugiperda using male- and female-specific isoforms. Given that Lepidoptera is recalcitrant to RNAi, CRISPR/Cas9-mediated mutagenesis was employed to construct S. frugiperda mutants. Specifically, we designed target sites on exons 2, 4, and 5 to eliminate the common, female-specific, and male-specific regions of S. frugiperda dsx (Sfdsx), respectively. As expected, abnormal development of both the external and internal genitalia was observed during the pupal and adult stages. Interestingly, knocking out sex-specific dsx variants in S. frugiperda led to significantly reduced fecundity and fertility in adults of corresponding sex. Our combined results not only confirm the conserved function of dsx in S. frugiperda sex differentiation but also provide empirical evidence for dsx as a potential target for the Sterile Insect Technique (SIT) to combat this globally invasive pest in a sustainable and environmentally friendly way. Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae), commonly known as fall armyworm (FAW), is an important agricultural pest native to the United States that has spread and become a serious threat to a variety of other countries [S. frugiperda was first reported in the Yangtze River Valley, bordering Burma, in 2019 [S. frugiperda becomes imperative to preventing dramatic loss of crops, and conventional management of S. frugiperda relies on the use of chemical agents [Bacillus thuringiensis toxins [ountries ,2. The sountries ,4. In Ch in 2019 . This in in 2019 ,7. Contrl agents ,9,10,11 s toxins ,13. Conss toxins ,15, alteDoublesex (dsx) acts as a regulatory element that controls sexual dimorphism in insects [Dsx was first found to produce male- and female-specific proteins via alternative splicing that regulate sex differentiation in Drosophila melanogaster [dsx homologues were reported to contribute to sexual dimorphism and polymorphism in a number of different insect species, including honey bees, wasps [dsx is initiated in the early embryonic stage and persists until maturity to regulate the development of sexual dimorphic traits [Fdsx and Mdsx dictate sex determination in multiple insects, i.e., somatic sexual differentiation of dsx is fundamentally conserved among insects, the sex determination cascade in S. frugiperda is still not fully understood. insects . Dsx wasnogaster . Later, s, wasps ,18, flies, wasps ,21,22,23s, wasps ,25,26, as, wasps ,28. Sexus, wasps . Expressc traits ,31. Althdsx through genetic approaches have recently been reviewed [dsx by RNA interference led to female-like genitalia development in Nasonia vitripennis males [Aedes aegypti [dsx in Lepidoptera. Knocking out Plutella xylostella dsx resulted in malformation of external genitalia and decreased hatchability of eggs [Spodoptera litura, adult male dsx mutants showed smaller testes and an inability to mate with wildtype females [Ostrinia furnacalis also verified the vital role of dsx in sexual dimorphism [dsx reduced the size of reproductive organs and fertility in Apis melifera [dsx knockout combined with a gene drive system has rapidly spread the disrupted dsx allele to elimination of Anopheles gambiae population after 7–11 generations in laboratory settings [Functional studies of reviewed . Silenciis males  and disr aegypti . Most re of eggs . In Spod females . A recenmorphism . CRISPR/melifera . In addisettings .Dsx is a highly conserved downstream gene dictating sex differentiation at the bottom of the sex determination cascade [Bombyx mori comes from the female-specific factor, PIWI-interacting RNA (piRNA), located on the W chromosome [Fem to target and cleave the downstream gene Masculinizer (Masc), which encodes the zinc-finger protein masculinizer (MASC) [Masc is transcribed from the Z chromosome and plays an important role in masculinization and dosage compensation and regulates the formation of Mdsx in the male sex determination cascade [Olfactory Receptor (OR) and Pheromone Binding Protein (PBP), are associated with sexual dimorphism in O. furnacalis [OR and PBP also confirmed that knockout of dsx interferes with expression of sexually dimorphic genes [Factors involved in the sex determination cascade include primary signals, executors, and transducer master regulators . Dsx is  cascade . The priromosome . In the r (MASC) . Masc is cascade ,42. The rnacalis . Researcic genes ,36,44.dsx determines sex differentiation at the bottom of the pathway [B. mori, the functional importance of dsx was verified, and its potential application in SIT was proposed [A. gambiae, such as a sex distorter targeting Fle and a gene drive targeting dsx, which caused progressive decreased ratio of female and eventually collapsed population [dsx would help to test the feasibility of using dsx as a target gene for SIT.SIT is a classical genetic pest control strategy used to suppress pest populations in the field by releasing insects carrying sex-specific lethal genes or sex-specific sterilized genes. In insects,  pathway . In B. mproposed . Novel ppulation ,46,47. ESfdsx regulates sex determination and is involved in fecundity and fertility of S. frugiperda. The objective of this study was to investigate the functional role of dsx in S. frugiperda. Through the CRISPR/Cas9-mediated gene-editing system, exons encoding common female- and male-specific transcript of dsx were designed for specific knockouts. Impacts on both morphology and physiology were observed.In this study, we hypothesized that alternative splicing of S. frugiperda strain used in this study was collected from Dongyang, Zhejiang Province, China. Larvae were kept in the lab within acrylic boxes and fed with artificial diet. The main components of artificial diet were yeast extract, wheat bran, vitamin C, sucrose, and agar as described previously [The eviously . The strS. frugiperda was extracted with Trizol reagent , and cDNA was synthesized using a GoScriptTM reverse transcription kit  according to the manufacturers’ instructions. The dsx of S. frugiperda was identified through blast against the amino acid sequences of D. melanogaster (GenBank accession number NP_001287220.1) and B. mori (GenBank accession number NP_001036871.1) against S. frugiperda in NCBI. Primers were designed using the Primer 3 website  to amplify exons 2 to 5 flanking the coding regions of female- and male-specific regions of Sfdsx  [CSfdsx, FSfdsx, and MSfdsx sites following the rule of 5′-GG-(N)18-NGG-3′ on dsx. The CSfdsx, FSfdsx, and MSfdsx gRNA sequences were designed with a length of 20 bp and aligned with S. frugiperda genome sequence (ASM1297921v2) to determine their specificity. The sgRNAs were sub-cloned and ligated to the pJET1.2 vector . sgRNAs were synthesized using MEGAScript T7  in vitro following the manufacturer’s instructions. The TrueCutTM Cas9 Protein  was purchased commercially and stored at −80 °C for experimental use.Specific target sites on exons harboring alternative splicing of er 2014) . We desiS. frugiperda adults were sexed and paired in a transparent plastic bag. Collected eggs were injected with 300 ng/μL Cas9 protein mixed with 300 ng/μL sgRNA within 1 h of oviposition under microscope . After injection, the eggs were incubated at 25 °C for 4 days until hatching and transferred to containers with artificial diet.To collect eggs for microinjection, five pairs of Genomic DNA of the pupal shell was extracted using phenolic chloroform and precipitated with isopropanol sodium acetate by incubation with protease K . Primers were designed to amplify the spanning region of the three knockout sites using Hieff Canace Gold High Fidelity DNA Polymerase , following these reaction conditions: 98 °C 3 min pre-denaturation; 98 °C 10 s, 55 °C 20 s, 72 °C 30 s, altogether 35 cycles. After 72 °C 30 s of final extension, the product was connected to the pJET1.2 vector  and sent to Sangon Biological Company for sequencing. The phenotype of the mutant was photographed using a micro-imaging system .The phenotypic impact on morphology of pupae and adults, including external genitalia, testes, and ovaries, was imaged under a stereo microscope . The number of injected eggs used for gene knockout, hatching rate, pupation rate, and sex ratio of molted adults were recorded and summarized in dsx mutagenesis on adult fecundity and sterility, two-day-old dsx mutants were paired with opposite-sex adults. CSfdsx, FSfdsx, and MSfdsx mutants were paired with opposite-sex WT virgin females or males, while WT pairings were used as a control. Each pair was maintained in a single plastic bag supplemented with 10% honey water. Eggs were collected, and their number was recorded each day for ten days post-pairing. Afterward, egg hatching rate was recorded for each pair. Experiments were performed three times with three to five pairs for each treatment.To investigate the impact of CSfdsx males and females, FSfdsx females, and MSfdsx males. cDNAs was synthesized using 1 μg total RNA as template using a GoScriptTM reverse transcription kit  according to the manufacturers’ instructions. To investigate the effects of Sfdsx mutagenesis on the OR and PBP genes, RT-qPCR was performed to examine the relative expression of OR1, PBP1, and PBP2 after Sfdsx was mutated. ß-actin was the internal reference [After sex-specific mutagenesis, total RNA was extracted from the whole body of adults using Trizol reagent , including wildtype females and males, eference . The reaSfdsx among different developmental stages, total RNA from S. frugiperda eggs, day one first to sixth instar larvae, wandering stage larvae, pupae, female adults, and male adults was extracted using Trizol reagent . A total of 5 μg RNA was used as a template, and cDNA was synthesized with a GoScriptTM reverse transcription kit . Quantitative real time PCR was performed to examine the temporal expression of Sfdsx, and ß-actin was the internal reference. Relative gene expression was calculated following the 2−ΔΔCT method [t-test. Column and error bars stand for mean ± SEM in all cases. p < 0.05 was considered a significant difference.To investigate the temporal expression of T method . StatistSfdsx was 196,795 bp, consisting of six exons with two sex-specific splicing patterns. The length of Sfdsx female-specific ORFs was 780 bp, encoding 259 amino acids. The length of male-specific ORFs was 801 bp, encoding 266 amino acids. The predicted alternative splicing patterns are shown in Sfdsx against homologous amino acid sequences in D. melanogaster (GenBank accession number NP_001287220.1) and B. mori (GenBank Accession number NP_001036871.1).The genomic sequence length of CSfdsx, FSfdsx, and MSfdsx sgRNAs, respectively. The hatching and mutant ratios for each treatment are recorded in CSfdsx, FSfdsx, and MSfdsx treatments, respectively  and B. mori (GenBank Accession number NP_001036871.1) were used as query to identify the putative coding sequence of Sfdsx. We cloned female- and male-specific regions of Sfdsx and identified one male-specific and three female-specific dsx transcripts in S. frugiperda  to combat this global invasive pest in a sustainable and environmentally friendly way.In this study, our combined results support our hypothesis that alternative splicing of


Staphylococcus aureus is an important pathogen for keratitis, a vision-threatening disease. We aimed to investigate the genotyping, antibiotic susceptibility, and clinical features of S. aureus keratitis, and to explore the possible role of Panton–Valentine leucocidin (PVL), a major virulence factor of S. aureus. We recruited 49 patients with culture-proven S. aureus keratitis between 2013 and 2017 at Chang Gung Memorial Hospital, Taiwan. PVL gene, multilocus sequence type (MLST), staphylococcal cassette chromosome mec (SCCmec), and pulsed-field gel electrophoresis (PFGE) were performed. Antibiotic susceptibility was verified using disk diffusion/E test. There were 49 patients with S. aureus keratitis; 17 (34.7%) were caused by methicillin-resistant S. aureus (MRSA) and 9 (18.4%) isolates had PVL genes. The predominant genotyping of MRSA isolates was CC59/PFGE type D/SCCmec VT/PVL (+). All methicillin-sensitive S. aureus (MSSA) and approximately 60% MRSA were susceptible to fluoroquinolones. No significant differences in clinical features, treatments, and visual outcomes were observed between MRSA/MSSA or PVL(+)/PVL(−) groups. In Taiwan, approximately one third of S. aureus keratitis was caused by MRSA, mainly community-associated MRSA. Although MRSA isolates were more resistant than MSSA, clinical characteristics were similar between two groups. Fluoroquinolones could be good empiric antibiotics for S. aureus keratitis. Staphylococcus aureus is a critical and commonly isolated human bacterial pathogen. Methicillin-resistant Staphylococcus aureus (MRSA), which is described as strains resistant to all available penicillin and other β-lactam antibiotics, warrants particular attention because of its limited treatment options. MRSA has been considered a hospital-acquired pathogen, but its prevalence in otherwise healthy patients without identified risk factors has reportedly increased. MRSA infections are classified as healthcare-associated MRSA (HA-MRSA) and community-associated MRSA (CA-MRSA); both strains are clinically, microbiologically, and genetically different , CC45/PFGE type AK/SCCmec IV (n = 1), ST573 (CC1)/PFGE type U/SCCmec IV (n = 1), and ST239/PFGE B1/SCCmec III (n = 1). Of the 17 (88.2%) MRSA isolates, 15 that carried type IV or V SCCmec belonged to CA-MRSA based on molecular definition. We did not report the genotyping of other MSSA isolates here because they had quite diverse molecular characteristics [We identified 49 cases of eristics .S. aureus isolates were susceptible to vancomycin, teicoplanin, and tigecycline. A higher ratio of MSSA isolates was significantly more susceptible than MRSA to several antibiotics, including clindamycin (p = 0.009), erythromycin (p = 0.072), and all four fluoroquinolones (p = 0.001). When we further stratified S. aureus isolates by the presence of PVL gene, PVL (+) MRSA isolates seemed to be more susceptible to four fluroquinolones, but less susceptible to erythromycin and clindamycin than PVL (−) MRSA isolates, but no significant differences were noted.As illustrated in S. aureus keratitis. No differences were observed in the distributions of age (p = 0.875), sex (p = 1.0), or source defined by epidemiology (p = 0.074) between MRSA and MSSA groups. Notably, 9 of 17 (52.9 %) patients with MRSA keratitis had no HA factors.S. aureus keratitis. No differences in other local and systemic risk factors were discovered between MRSA and MSSA groups as well as PVL (+) and PVL (−) groups.The most common predisposing factor for both MRSA and MSSA keratitis was ocular surface diseases and previous ocular surgery, which accounted for more than half of p = 0.178), infiltration size (p = 0.284), or presence of hypopyon (p = 0.286) were identified between the MRSA and MSSA groups.As shown in S. aureus keratitis were initially treated with topical levofloxacin (0.5%) or a combination of fortified antibiotics (vancomycin at 25 mg/mL and amikacin at 25 mg/mL). In 15 patients, the medication regimen was changed to 25 mg/mL vancomycin after obtaining the culture results. The rate of treatment modification did not differ significantly between the MRSA and MSSA groups (p = 1.0). Seven patients, three with MRSA and four with MSSA keratitis, required surgical interventions. Three patients, one with PVL (+) MRSA, the other with PVL (−) MRSA, and another with MSSA keratitis, received a patch graft with glycerol-preserved cornea for perforated ulceration. The other four patients (one PVL (+) MRSA and three MSSA keratitis) received amniotic membrane transplantation or tarsorrhaphy to promote re-epithelialization. No significant differences were identified in the rate of surgical intervention (p = 0.681), admission (p = 0.347), healing time (p = 0.648), or final visual outcome (p = 0.612) between the MRSA and MSSA groups , centrally located corneal ulcer (p = 0.038), elder age (p < 0.001), and higher LogMAR VA at presentation (p < 0.001) were associated with higher final LogMAR VA . All patients with A groups . There wS. aureus keratitis and replenished the lack of genotyping and antibiotic susceptibility of fluoroquinolones in our previous study [S. aureus keratitis was caused by MRSA, around 90% of which belonged to molecular-defined CA-MRSA. The prevalence of the PVL gene was approximately one-fifth of S. aureus isolated from keratitis. The susceptibility of fluroquinolones was 100% and about 60% in MSSA and MRSA isolates, respectively. No significant differences in clinical features and outcomes were identified between MSSA and MRSA or PVL (+) and PVL (−) S. aureus.This study updated the information regarding us study . Our finS. aureus keratitis, 26 infections were caused by MRSA, 12 (46.2%) of which had no healthcare factors, i.e., CA-MRSA by epidemiological definition [In our previous 5-year 2006–2010) study of 59 patients with –2010 stumec such as type IV or V are the most common elements in all major clones: ST8/SCCmec IV (USA300) in the United States [mec V in Asia [mec IV in Australia [mec IV in Europe [mec IV/PVL (−) (designated as Asian-Pacific clone) and ST59/PFGE type D/SCCmec VT/PVL (+) (designated as Taiwan clone) [The prevalence of CA-MRSA clone lineage differs by geographic area, but PVL and small SCCd States , ST59/SC in Asia , ST93/SCustralia , and ST8n Europe . In Taiwn clone) ,27. Recen clone) . which wmec VT, which belonged to the Taiwan clone of CA-MRSA. The genotype of the other PVL (+) MRSA was ST8/PFGE type A/SCCmec IV . USA 300 is rare in Taiwan, accounting for 0.51% (27/5308 between 1995 and 2005) of MRSA [In our study, nine PVL (+) isolates were identified: seven MRSA and two MSSA. The genotypes of six PVL (+) MRSA were CC59/PFGE type D/SCC of MRSA ; the clo of MRSA .S. aureus keratitis in Taiwan. If the infection does not respond well to fluroquinolones, fortified vancomycin, the last resort for MRSA, could be considered. In the present study, MSSA isolates were susceptible to all tested antibiotics; it is no wonder that MRSA isolates were more resistant to certain antibiotics than MSSA. Notably, around 60% MRSA (even higher for PVL (+) MRSA) were susceptible to fluoroquinolones, which are the empiric antibiotics most frequently prescribed by ophthalmologists. Several studies have reported that the rate of in vitro resistance to fluoroquinolones has been increasing in MRSA keratitis ,31,32. TS. aureus keratitis between the PVL (+) and PVL (−) groups, which contradicts the finding of a study by Sueke et al. [S. aureus keratitis, nine (9.5%) of which were PVL (+) (eight MSSA and one MRSA); they reported nonsignificant trends for healing and treatment time, ulcers, and scar size, but they indicated that the overall clinical score and rate of surgical intervention were greater in the PVL (+) group than PVL (−) group with the effect being unrelated to MICs [S. aureus corneal infection because of a strain-dependent effect [S. aureus keratitis in the current study. This discrepancy may have been caused by our comparing PVL (+) and PVL (−) isolates of all S. aureus keratitis, not CA-MRSA keratitis alone; however, further analysis of two ST59 CA-MRSA clones could not be performed because of the small sample size. In addition, the presence of PVL gene does not imply that the toxin is produced in vivo [In this study, we did not observe differences in clinical features and outcomes of e et al. . They in to MICs . The dist effect . It has t effect . Therefot effect ,39, altht effect ,40. In at effect . However in vivo . MoreoveS. aureus keratitis in Taiwan. In addition, it opens perspectives for further experimental studies such as using enzymatic inhibitors assays, chromatographic characterization, comparative proteomic and gene expression mechanisms to compare PVL from different S. aureus strains.The present study had some limitations. First, although we prospectively collected the isolates, we retrospectively reviewed the clinical data. Second, the relatively small sample might have affected the analysis of statistical significance. Third, in vitro susceptibility based on the serum systemic standards does not always correlate with clinical response, because no susceptibility standards for topical therapies have been established. Fourth, considering the different microbiological characteristics in differing geographic areas, the findings of the present study should not be generalized to other regions or populations. However, this study compensated for the lack of genotyping in our previous study ,7 and prS. aureus keratitis were caused by MRSA, and the prevalence of PVL gene in all S. aureus isolates from keratitis was 18.4% (9/49); CC59/PFGE type D/SCCmec VT/PVL (+) was the predominant clone. All MSSA isolates and more than 60% MRSA isolates were susceptible to fluoroquinolones. Ulcer location and size, advanced age, and poor VA at presentation were significantly associated with poor visual outcomes, whereas neither the presence of PVL nor methicillin resistance was a significant prognostic factor. This study provides the updated information including genotyping, antibiotic susceptibility, and clinical features of S. aureus keratitis, that may help clinicians to handle such patients.In conclusion, our 5-year study determined that 34.7% (17/49) of S. aureus isolates were prospectively collected from patients with ocular infections in the microbiology laboratory of the Chang Gung Memorial Hospital, a 3700-bed medical center in Northern Taiwan, from January 1, 2013, to December 31, 2017. In total, 49 patients with S. aureus keratitis were identified. Medical records of these cases were retrospectively reviewed. Data collected included demographic information, medical and ocular history, systemic and local predisposing factors, clinical features, treatment, presenting and final visual acuity (VA), and length of follow-up. Healthcare-associated criteria were defined on the basis of the definition proposed by the Centers for Disease Control and Prevention Active Bacterial Core Surveillance sites  and wereS. aureus keratitis. The antibiotic treatment regimens were subsequently modified on the basis of culture results, susceptibility testing, and clinical response. Surgical interventions were indicated in some cases given the clinical conditions. The healing time was recorded once the infiltration had subsided and the epithelial defect healed. If data were available, VA was recorded at least 2 months after the keratitis had subsided and stabilized. Snellen VA values were converted into logMAR units for statistical analysis. VA—assessed through counting fingers, hand movements, light perception, and no light perception—was recorded as logMAR following the schedule suggested by Scott et al. [Empirical or fortified antibiotics were administered hourly to treat t et al. .S. aureus isolates to antibiotics including cefoxitin, clindamycin, erythromycin, trimethoprim/sulfamethoxazole, teicoplanin, and vancomycin was routinely assessed using the disk diffusion method in our microbiology laboratory, following the guidelines of the Clinical and Laboratory Standards Institute antimicrobial susceptibility testing standards. Cefoxitin was used instead of oxacillin/methicillin to test for β-lactam antibiotic resistance. Moreover, an E-test  was used to determine the susceptibility of isolates to fluoroquinolones—including ciprofloxacin, levofloxacin, gatifloxacin, and moxifloxacin—that were not included in the antibiotic susceptibility profiles in our microbiology laboratory.The antimicrobial susceptibility of all S. aureus isolates were tested by the presence of PVL genes. The PCR amplification of the lukS-PV and lukF-PV genes encoding PVL components was described previously [SmaI digestion, multilocus sequence typing (MLST), and SCCmec typing. PFGE was used to fingerprint the MRSA clinical isolates and PVL (+) MSSA according to the procedures described in a previous study [S. aureus MLST database (https://pubmlst.org/saureus/ (accessed on 30 August 2022)) and were defined as sequence types (STs) accordingly. The SCCmec typing was determined by a multiplex PCR strategy described previously [mec type VT was determined by a particular primer described elsewhere [mec and type IV or V SCCmec were classified as HA-MRSA and CA-MRSA, respectively [All eviously . ENREF 6us study . The crius study  were useus study ; the alleviously . SCCmec lsewhere . Based oectively .p < 0.05.The continuous variables are presented as mean ± standard deviation, and the categorical variables are presented as the number of cases with a percentage. The intergroup differences in the variables were determined using Fisher’s exact test for analyzing nominal variables and the Mann–Whitney U test for continuous variables. Pearson correlation analysis was used to determine factors associated with visual outcome. Statistical significance was defined as


Saussurea costus is from medicinal plants and have therapeutic properties that were recorded in a variety of medical functions. The usage of biomaterials in the synthesis of nanoparticles is an essential strategy in green nanotechnology. Iron oxide nanoparticles (IONPs) were composed in the stage of  solution by using the aqueous extract of Saussurea costus peel in an eco-friendly method to evaluate their antimicrobial property. The properties of the obtained IONPs were evaluated using a scanning (SEM) and transmission (TEM) electron microscope. The mean size of IONPs discovered by Zetasizer varies between 100 and 300 nm, with a mean particle size of 295 nm. The morphology of IONPs (γ-Fe2O3) was determined to be nearly spherical and prismatic-curved. Moreover, the antimicrobial property of IONPs was assessed with nine pathogenic microbes, revealing that the nanoparticles have antimicrobial activities with Pseudomonas aeruginosa, Escherichia coli, Shigella sp., Staphylococcus sp. and Aspergillus niger, with possible applications in the therapeutic and biomedical fields. Plant-derived medicines continue as a valuable resource, particularly in developing countries, for treating severe ailments. Around 60–80 percent of people worldwide mostly use folk medicine in treating diseases . A wide Saussurea costus,  also identified as "Al-Kost Al-Hindi" in Arab countries, belongs to the Asteraceae family, which has many species in India, Pakistan, and parts of the Himalayas . Th. ThSaussand apps .Staphylococcus sp., Bacillus subtilis gram-positive, Klebsiella pneumonia, Shigella sp., Pseudomonas aeruginosa, Escherichia coli gram-negative, and Aspergillus niger, Alternaria sp., and Rhizopus sp. Fungi, obtained from the biology department faculty of sciences, university of princess Nourha bint Abdulrahman  UV-spectrophotometer . The size and morphology of biogenic IONPs were determined using a transmission electron microscope (TEM), a JEOL JEM-1011 , and field emission scanning electron microscopy  at 200 kV and 30 kV voltage, respectively. A zeta size tool was used to assess the average size distribution and the zeta potential .Aspergillus niger, Alternaria sp., Rhizopus sp., and bacteria forms, the gram-positive Staphylococcus sp., Bacillus subtilis, and gram-negative community Klebsiella pneumonia, Shigella sp., Pseudomonas aeruginosa, and Escherichia coli, have been examined (5 mL). On every agar plate, sterile swabs are placed at irregular intervals. Three of them were properly separated. The sterile cultivation agar surface was used to create wells (holes) with a diameter of 4 mm per plate. Use a metal cork to bore the hole. The aseptic use of 0.2 mL of extract at every hole was performed in the room. The excerpts can be dispersed and cultivated in the agar medium after 1 hour at room temperature. The negative control used in comparison was aseptic pure water. The plates were kept for 24–48 hours at 37°C and 25°C. Inhibition zones were identified as distinct regions surrounding the pools [The anti-microbial efficient acts of iron nanoparticles were calculated using a well-gar dispersion strategy. They were cultured on Mueller-Hinton Agar at a concentration of 108/ml for bacterial growth and potato agar for fungal growth. Three fungi forms, examined . To meashe pools .S. costus were added to the bacterial growth at MIC levels, and the cultures were kept at 37°C for 1 hour. Treated cells were centrifuged at 6000 rpm for 10 minutes at 4°C. PBS was used to wash the pellets at pH 7, and they were then fixed for 1 h in glutaraldehyde 8% and Sorensen’s phosphate buffer (SPB). To fix the samples, 4% osmium tetroxide (OsO4) and H2O (1:1) volume were mixed. The specimen was examined using FE-SEM  after it had been washed and dehydrated for 24 hours.Treated bacteria were examined using FE-SEM to observe any modifications in the bacterial shape and to identify the exact mechanisms of action of IONPs with each examined microbe. IONPs from Saussurea costus peel extract were used a biosynthetic approach to generate IONPs from mineral solutions with a concentration of 1 mM. The iron oxide nanoparticles (Fe3O4) produced by using a mixture of  and S. costus root as a reducing agent resulted in a dark brown color of the solution, which had previously been yellow. The color of the combination changed in a time-dependent manner for 24 hours at dark room temp. As an indicator of iron oxide formation, the dark brown color was steady and evident.S. costus roots by using Zetasizer , one gram-positive (Staphylococcus sp.) and three gram-negative , with inhibition zones ranging from 5 mm to 11 mm  Tables . IONPs hto 11 mm . SEM was. IONPs (Fe3O4) created by combining  and S. costus root as a reducing agent caused a dark brown color of the mixture, which had previously been yellow. The dark brown colour indicates iron oxide formation, as reported in several studies, including IONPs generation via controlled bio-precipitation from Mansoa alliacea leaf extract [Aloe vera [An environmentally friendly procedure was employed in this research to produce iron oxide nanoparticles , indicating that large biomolecules, such as enzymes or proteins, were related to the synthesized IONPs. Our findings agree that the existence of protein or enzyme molecules, which was also affirmed using UV-visible spectra with the biosynthesized Ag-NPs –42. Thesductases . Our finPseudomonas aeruginosa (11 mm), followed by gram-positive; Staphylococcus sp. (9 mm), gram-negative; Escherichia coli (9 mm), and Shigella sp. (6 mm), as well as fungi; Aspergillus niger (5 mm). The antimicrobial property of IONPs could be linked to the oxidative stress system caused by reactive oxygen species (ROS), which could destroy biomolecules like DNA and proteins [P. guajava leaf extract, hematite nanoparticles were discovered to be powerful antibacterial components against Gram-positive and Gram-negative bacteria [L. siceraria was found to be effective against A. hydrophila (gram-positive) and E. coli (gram-negative) [S. typhi, S. aureus, S. enterica, and K. pneumonia) out of nine [P. chrysogenum (28.67 mm), accompanied by A. niger (26.33 mm), T. roseum (22.67 mm), and A. alternata (21.33 mm) [L. monocytogenes (12 mm) and two fungi, A. flavus (13 mm) and P. spinulosum (14 mm) was moderate [Nanoparticles had greater antimicrobial properties than plant extract and Fe ions against gram-positive, gram-negative bacteria and fungi. The zone of inhibition was greatest against gram-negative proteins , 45. Folproteins . A numbeproteins . After bbacteria . When chegative) . The mos of nine . Hematit1.33 mm) . The antmoderate .Couroupita guianensis fruit extract had effective bactericidal against tested human pathogens [The IONPs have antimicrobial properties . Both grathogens . IONPs dathogens .E. coli, as well as invade and concentrate in the cytoplasm, causing vacuole creation and cell wall alterations [Bacterial cells were unharmed in the absence of IONPs, but after NP treatment, the vast majority of cells had reduced diameters and various stages of membrane injury, with some lysed. Electron microscopy revealed that IONPs can attach directly to the cell wall of erations , 53. IONerations . Antibioerations . Bacterierations .2O3) from an aqueous extract of S. costus root and a combination of . The synthetic IONPs had a positive potential and were spherical and prismatic curve-shaped, with a mean size of 295 nm. S. costus contains the natural substances that are in charge of the bio-reduction mechanism. Antimicrobial activities of IONPs were discovered against gram-negative P. aeruginosa, E. coli, and Shigella sp., as well as gram-positive Staphylococcus sp. and fungi such as A. niger. The creation of iron oxide nanoparticles from S. costus root is an eco-friendly, simple, effective, and low-cost biological method that may be recognized as a strong antibacterial substance. More focus and investigation are currently needed to control the size and shape of nanoparticles and determine the exact mechanism of IONP formation by green plants.We described the production of iron oxide nanoparticles (γ-Fe