Podarcis lilfordi and P. pityusensis), encompassing their allopatric range of distribution through a noninvasive sampling, as an alternative to previous studies that implied killing specimens of these IUCN endangered and near-threatened species, respectively. Both lizard species showed a faecal microbiome composition consistent with their omnivorous trophic ecology, with a high representation of cellulolytic bacteria taxa. We also identified species-specific core microbiota signatures and retrieved lizard species, islet ascription, and seasonality as the main factors in explaining bacterial community composition. The different Balearic Podarcis populations are characterised by harbouring a high proportion of unique bacterial taxa, thus reinforcing their view as unique and divergent evolutionary entities.Gut microbial communities provide essential functions to their hosts and are known to influence both their ecology and evolution. However, our knowledge of these complex associations is still very limited in reptiles. Here we report the 16S rRNA gene faecal microbiota profiles of two lizard species endemic to the Balearic archipelago (The online version contains supplementary material available at 10.1007/s00248-022-02019-3. Recent advances in sequencing technologies and analytical methodologies are improving our understanding of the microbiome in host evolution . Early ePodarcis lilfordi and P. pityusensis, represent an interesting model to study host-microbiota associations since they are sister taxa with a nonoverlapping distribution, consisting of multiple allopatric populations restricted to coastal islands and islets of Mallorca, Menorca, and Cabrera in the case of P. lilfordi, and the main islands of Ibiza and Formentera in P. pityusensis. Podarcis lilfordi became extinct on both the main Mallorca and Menorca islands due to the anthropic introduction of foreign predators and/or competitors ),hipelago , 47. Evehipelago , 49. Inderranean . Like oterranean , and theerranean . Previouerranean , 52, 53)rbivores . Examplenoccocus , represes e.g., , 40, 56), 56Podares Table , a linead plants , or the d plants . The rel species . Within io Table , a genusP. lilfordi and P. pityusensis samples regardless of the dataset  and the input matrix (weighted or unweighted UniFrac) . Taxa from these genera were also identified as core members of P. lilfordi gut microbiota by [Alistipes, Breznakia, Oscillibacter, Ruminococcaceae UBA1819, and Rikenellaceae dgA-11 gut group. Podarcis pityusensis also showed a distinctive core taxa assemblage consistent with bacteria genera commonly found in the gut microbiome of other lizard species  [P. lilfordi and P. pityusensis lineages that started ca. 5.33 Ma ago and subsequent geographical, ecological and evolutionary divergence [Our exploratory beta diversity analyses pointed to significant differences in bacterial community composition between c) Table . Such di samples –66. The 19; e.g.,–69 and abiota by  exceptinboutsia) , 67, 70.vergence .P. lilfordi populations are restricted to coastal islands and islets of Mallorca, Menorca, and the Cabrera archipelago, which acted as refugia after the extinction of mainland populations ca. 2000 years ago due to the anthropic introduction of predators and competitors [P. lilfordi populations have led to consider them as independent evolutionary significant units [Podarcis populations could also have shaped their gut microbiota. Our exploratory PERMANOVA analyses retrieved the islet adscription and main island district as the main factor in explaining bacterial community composition. Consistently, each individual population showed a high proportion of non-shared ASVs . This result is even more significant if we consider that rarefaction curves of the samples reached the plateau stage, thus ensuring that comparisons are based on a comprehensive view of their respective ASV diversity. All these results reinforce the consideration of every single Podarcis population as a unique evolutionary entity hosting a singular gut microbiota.Extant petitors , 13. Sucpetitors . Therefont units . In thisPodarcis. In general, samples from the same population (island/islet) and collecting season showed similar composition in terms of microbial communities. In this regard, the seasonality of food has been demonstrated to partially affect the diet composition of P. lilfordi lizards [Podarcis populations inhabit unique ecological scenarios.PERMANOVA analyses based on datasets confirmed by samples from populations with matching collecting seasons: (spring + summer and summer + autumn) allowed us to estimate the effect of the seasons on the gut microbiota of the Balearic  lizards . Resourc lizards , 74. Pre lizards . Our resPodarcis in the Balearic archipelago is marked by the occurrence of allopatric processes leading to the extant isolated populations that exhibit genetic, morphological, ecological, and ethological differences, in many cases unique to one population [P. lilfordi [The evolutionary history of pulation . Such ispulation , 76. In lilfordi  could falilfordi . Environlilfordi , 78, whililfordi . In turnlilfordi , 6, 11, Supplementary file1 (PDF 964 KB)Supplementary file2 (PDF 3511 KB)Supplementary file3 (PDF 5977 KB)Supplementary file4 (PDF 4187 KB)Supplementary file5 (XLSX 1328 KB)Supplementary file6 (XLSX 20 KB)Supplementary file7 (DOCX 35 KB)Supplementary file8 (XLSX 63 KB)Below is the link to the electronic supplementary material.


Azadirachta indica (AAgNPs) and Catharanthus roseus (CAgNPs) leaf extracts in BALB/c mice. We found rapid wound healing, higher collagen deposition, and increased DNA and protein content in AAgNPs- and CAgNPs (1% w/w)-treated wounds than in control and vehicle control wounds. Skin antioxidant enzyme activities  were significantly (p < 0.05) increased after 11 days CAgNPs and AAgNPs treatment. Furthermore, the topical application of CAgNPs and AAgNPs tends to suppress lipid peroxidation in wounded skin samples. Histopathological images evidenced decreased scar width, epithelium restoration, fine collagen deposition, and fewer inflammatory cells in CAgNPs and AAgNPs applied wounds. In vitro, the free radical scavenging activity of CAgNPs and AAgNPs was demonstrated by DPPH and ABTS radical scavenging assays. Our findings suggest that silver nanoparticles prepared from C. roseus and A. indica leaf extracts increased antioxidant status and improved the wound-healing process in mice. Therefore, these silver nanoparticles could be potential natural antioxidants to treat wounds.Developing an efficient and cost-effective wound-healing substance to treat wounds and regenerate skin is desperately needed in the current world. Antioxidant substances are gaining interest in wound healing, and green-synthesized silver nanoparticles have drawn considerable attention in biomedical applications due to their efficient, cost-effective, and non-toxic nature. The present study evaluated in vivo wound healing and antioxidant activities of silver nanoparticles from  Recently, nanotechnology has been widely used to treat various diseases, such as wounds, cardiovascular diseases, diabetes, and cancer ,2. The rl damage ,8. Althol damage . It has l damage ,11. The l damage . Subsequl damage . The con process . A studyy status . Howevery status ,17 that y status . Therefoy status . Taken tAzadirachta indica (A. indica) leaf extracts are rich in naturally inherent oxygen quenchers, redox agents, and hydrogen donors that inactivate free radicals and/or trigger antioxidant enzymes to disturb the oxidation chain reaction [Catharanthus roseus (C. roseus CAgNPs) and A. indica (AAgNPs) and demonstrated their antibacterial and wound-healing properties [In this context, nanoparticles conjugated with natural antioxidants could be a possible effective intervention to promote wound-healing mechanisms. We have studied titanium dioxide nanotubes (TNT), and our data suggest that these materials have immunomodulation properties . Furtherreaction . The impreaction . Fitzmaureaction . AgNPs ireaction ,31. The reaction . Green-sreaction . In our operties . HoweverPhosphate-buffered saline, hydroxyproline, Ehrlich’s reagent, and proteinase K were purchased from the HiMedia Laboratories . Silver nitrate, quercetin, and DPPH were purchased from Sigma-Aldrich .C. roseus and A. indica were prepared and used to synthesize silver nanoparticles. Young leaves of C. roseus and A. indica were obtained from the Yogi Vemana University  premises in Kadapa, Andhra Pradesh, India. The leaves were then cleaned thoroughly to remove the debris and other organic constituents, and later dried at 37 °C. Dried leaf (10 g) powder obtained from C. roseus and A. indica was suspended in water (100 mL) and boiled at 80 °C for an hour. The filtered leaf extract was mixed with silver nitrate to prepare the silver nanoparticles, as described earlier in the study of Lakkim et al. 2020 [Leaf extracts from al. 2020 . The darC. roseus and A. indica were characterized using X-ray diffraction (XRD), Fourier-transform infrared spectroscopy (FTIR), dynamic light scattering (DLS), scanning electron microscopy (SEM) with energy dispersive X-ray analysis (SEM-EDX), and transmission electron microscopy (TEM) analyses. The detailed protocols for each technique were fully explained in our previous publication [The silver nanoparticles synthesized from lication .C. roseus and A. indica was tested by DPPH  assay. Quercetin, a known antioxidant, was used as a reference and blank using only phosphate buffer solution pH-7.4. Each experiment was performed in triplicate. The inhibition percentage was measured against blank. The percentage of DPPH radical scavenging activity was calculated according to the formula [The free radical scavenging activity of AgNPs from  formula ,36.DPPHNext, the ABTS  scavenging activity of CAgNPs and AAgNPs was performed according to the previously published methods ,38. QuerC. roseus AgNPs (1% w/w) and A. indica AgNPs (1% w/w), respectively. All respective substances in Group I, Group II, Group III, Group IV, and Group V were topically applied to the open excision wound. Following recovery from anesthesia, mice were housed in sanitized cages.BALB/c mice were purchased from Mahaveera Enterprises , and the wound experiment protocol was approved by the Institutional Animal Ethics Committee (IAEC) with CPCSEA registration no: 1841/GO/Re/S/51/CPCSEA. The mice were randomly assigned into five groups, as described in Lakkim et al., 2020 , which iWe pre-formulated 1 mg of green-synthesized AgNPs (CAgNPs or AAgNPs) ground in 1 gm of vaseline (1 mg AgNPs per 1 gm vaseline) and prepared a fine paste using a motor and pestle. This paste was used for topical application to wound surfaces on alternative days for a period of 10 days. Wound constriction was observed by monitoring the wound size on days 0, 2, 4, 6, 8, and 10. The wound closure area was measured alternatively using a digital vernier caliper. Wound-healing efficacy following treatment was represented as the wound contraction rate (WCR) percentage and calculated as described below. The percentage of wound healing in CAgNPs or AAgNPs groups was compared with controls.The percentage of wound contraction rate = original wound area—actual wound area/original wound area × 100.Biochemical assays were performed to estimate the collagen, DNA, and protein levels in the skin tissues (day 11) of BALB/c mice treated with green-synthesized silver nanoparticles. Hydroxyproline assay is widely used to estimate collagen. The hydroxyproline amount is expressed as mg hydroxyproline/g of dry skin tissue weight. The diphenylamine method for DNA estimation and Lowry’s method for protein estimation was used in wound-healing tissues of all treatment groups. Total protein was estimated by the Lowry method .Masson’s trichrome and hematoxylin and eosin (H&E) staining experiments were performed to evaluate the skin morphology and collagen deposition during wound healing in BALB/c mice treated with green-synthesized silver nanoparticles (CAgNPs and AAgNPs). Slides containing tissue sections were deparaffinized in xylene for 3 min, then immersed in graded percent of ethanol 100, 95, 80, and 70% for five minutes in each, followed by a thorough rinsing with distilled water. Further, slides were kept in hematoxylin for 1 min and washed excess stain with water. Slides were immersed in eosin for 30 s and then in graded percent of ethanol . Sections were mounted with dibutylphalate polystyrene xylene (DPX) and examined under an inverted microscope .−1 molar extinction coefficient. The moles of H2O2 degraded/mg protein/min are considered one unit of enzyme activity. GPx activity was determined using the method of Rotruck et al. with minor modifications [−3 was used to calculate enzyme activity. The enzyme activity was measured in milligrams of CDNB-GSH conjugate produced per milligram of protein/min [Changes in antioxidant enzymes, including SOD, CAT, GPx, GR, glutathione-S- transferase (GST), and lipid peroxidation markers, were determined in the wound tissue of AgNPs-treated groups, and results were compared with control. The catalase activity in wound tissue was measured using the Aebi with minor modifications . Catalasications . GPx actications . The effications . Using tications ,45. GST ications . A molartein/min .Lipid peroxidation marker in wound tissue was assessed using thiobarbituric acid (TBA), and estimated the levels of malondialdehyde (MDA) in all treated groups . The ratT-tests and two-way analysis of variance (ANOVA) were used to analyze the statistical tests. Data were represented as a mean ± SD for six replicates. A p value of less than 0.05 (p < 0.05) was considered statistically significant.Statistical comparisons were made between controls and silver nanoparticle-treated groups. Unpaired C. roseus and A. indica leaf extracts were fully explained in our previous study [p < 0.05) higher than that of control and vehicle control groups  higher hydroxyproline content in the CAgNPs-treated sample (7.7 μg), which is close to betadine treated skin samples (8 μg). Hydroxyproline in the AAgNPs-treated group (7.1 μg) was significantly (p < 0.05) higher compared to the vehicle control-treated group (p < 0.05) increased in CAgNPs- and AAgNPs-treated tissue samples compared with control vehicle control groups. The total DNA content values were 36.2, 35.5, 38.7, 42.6, and 41.4 μg per 100 mg tissue in control, vehicle control, positive control, CAgNPs, and AAgNPs groups, respectively -treated dermis exhibited complete restoration of the epithelium (squamous cell epithelium) compared with control and vehicle control groups. In addition, moderate to high granulation tissue, mild inflammatory cell infiltration, and adnexa restoration of the dermis was evidenced with CAgNPs. The AAgNPs (1% w/w) treated group also showed effective restoration of the epithelium (squamous cell epithelium) compared with control and vehicle control groups. Similarly, moderate to high granulation tissue, mild inflammatory cell infiltration, and adnexa restoration of the dermis were seen in AAgNPs-treated skin samples  higher in CAgNPs and AAgNPs-treated skin compared with control and vehicle control skin. To be specific, SOD activity in CAgNPs and AAgNPs groups was 8.2 and 8.6 U/mg protein, respectively, which was significantly (p < 0.05) higher than that in control (6.2 U/mg protein) and vehicle control (5.8 U/mg protein) samples (p < 0.05) as we found higher concentrations of H2O2 metabolized/per mg protein tissue (p < 0.05) in CAgNPs- and AAgNPs-treated groups compared with control and vehicle control groups  and anti-inflammatory properties in rats [A. indica contain a rich amount of quenchers, redox agents, and hydrogen donors that could trigger the antioxidant enzyme activities or inactivate the free radicals [A. indica, and C. roseus eventually inhibit free radical production and/or quench the elongation of peroxidation chain reactions. Our findings are consistent with the previous reports that showed that SOD, catalase, GPx, and GST activities were increased, whereas lipid peroxidation levels were decreased in wound tissue on the palm with vitamin E and α-tocopherol treatment [Another important finding from our study is that the antioxidant enzyme activities, including SOD, CAT, GPx, and GST, were substantially increased in the cutaneous wound of CAgNPs- and AAgNPs-treated groups. It is documented that the normal physiology of wound healing depends on low concentrations of intracellular ROS and ROS-mediated oxidative stress . Typical in rats . The learadicals . The phyreatment .C. roseus and A. indica plant leaf extracts. As described, UV–Vis spectroscopy revealed a sharp peak at 315–360 nm for C. roseus and 410–440 nm for A. indica nanoparticles [−1, 1632 cm−1, and 1384 cm−1. These peaks illustrate the presence of the N–H group on the surface of AgNPs, and these visible signals represent the flavonoids and terpenoids that are specifically present in leaf extracts of C. roseus and A. indica. The average particle size of CAgNPs was 110 nm, with a range of 80–250 nm, and the average particle size of AAgNPs was 60 nm, with various fields of 40–80 nm estimated by DLS. The TEM images showed that the green-synthesized AgNPs were agglomerated and embedded in a dense, thick pattern, likely acting as stabilizing chemical constituents in the leaf extracts of both C. roseus and A. indica. As presented in C. roseus and A. indica with established structure and characterization may play a vital role in improving the antioxidant status (in vitro and in vivo) that endorse wound-healing properties in mice.Previous findings from our laboratory have shown the characterization of green-synthesized silver nanoparticles from articles . The XRDC. roseus and A. indica leaf extracts are potential antioxidant compounds for treating cutaneous wounds and could be used to develop suitable wound-healing medicine or ointment.Our findings demonstrated that green-synthesized AgNPs showed efficient antioxidant properties in wound healing. The in vivo wound-healing experiments in BALB/c mice showed improved wound-healing efficacy following CAgNPs, and AAgNPs treatments and were associated with increased hydroxyproline, DNA, and protein contents. The histological evidence represented a decreased scar width, fewer inflammatory cells, well-developed collagen fibers, capillaries, and fibroblasts. Furthermore, antioxidant enzyme activities were increased, and the lipid peroxidation marker decreased in CAgNPs- and AAgNPs-treated skin samples. Our results conclude that green-synthesized AgNPs from


Diabetes mellitus is a metabolic disorder characterized by hyperglycemia due to impaired insulin secretion, insulin resistance, or both. Oxidative stress and chronic low-grade inflammation play crucial roles in the pathophysiology of diabetes mellitus. There has been a growing interest in applying natural products to improve metabolic derangements without the side effects of anti-diabetic drugs. Microalgae biomass or extract and their bioactive compounds have been applied as nutraceuticals or additives in food products and health supplements. Several studies have demonstrated the therapeutic effects of microalgae and their bioactive compounds in improving insulin sensitivity attributed to their antioxidant, anti-inflammatory, and pancreatic β-cell protective properties. However, a review summarizing the progression in this topic is lacking despite the increasing number of studies reporting their anti-diabetic potential. In this review, we gathered the findings from in vitro, in vivo, and human studies to discuss the effects of microalgae and their bioactive compounds on diabetes mellitus and the mechanisms involved. Additionally, we discuss the limitations and future perspectives of developing microalgae-based compounds as a health supplement for diabetes mellitus. In conclusion, microalgae-based supplementation has the potential to improve diabetes mellitus and be applied in more clinical studies in the future. Diabetes mellitus is a metabolic disorder characterized by high blood sugar levels or hyperglycemia that is caused by problems with insulin secretion, insulin action, or both and it is related to carbohydrate, lipid, and protein metabolism derangements ,2,3. DiaA study from 2019 reported that around 463 million people within the age range of 20–79 had diabetes mellitus and the prevalence was predicted to increase to 578 million and 700 million in 2030 and 2045, respectively . AdditioChlorella vulgaris and Arthrospira plantensis biomass, as well as astaxanthin from Haematococcus pluvialis and DHA extracted from Schizochytrium sp. [Microalgae, which include eukaryotic microalgae and prokaryotic cyanobacteria, are microscopic, unicellular, photosynthetic algae that may thrive in freshwater or saltwater ,15. The rium sp. . Microalrium sp. . Importarium sp. ,19,20,21rium sp. . TherefoDespite the challenge, research on the anti-diabetic activity of microalgae and their bioactive compounds is increasing and their potential as health supplements for diabetes mellitus is gaining traction; however, a review summarizing the progression in this topic is lacking. Therefore, we summarized the information from in vitro, in vivo, and clinical studies investigating the anti-diabetic activity of microalgae and the bioactive compounds derived from them while explaining the mechanisms involved in producing this effect. This paper also includes the limitations and future perspectives of developing microalgae-based health supplements in managing diabetes mellitus. Investigating the anti-diabetic effects of microalgae or their bioactive compounds in an in vitro setting is mainly focused on determining their ability to inhibit digestive enzymes that are associated with postprandial hyperglycemia or modulate enzymes or transcription factors involved in glucose metabolism .Arthrospira platensis, Nannochloropsis oculata, Porphyridium purpureum, and Chlorella vulgaris pose potential anti-diabetic properties by inhibiting diabetic-related enzymes and alleviating oxidative stress [e stress . In thate stress . One of e stress . In addiP. purpureum contained the highest amount of phenolic content but was limited to flavan-3-ols, whereas A. platensis had the most diverse phenolic content, such as flavan-3-ols, phenolic acids, and flavonols. Furthermore, P. purpureum and N. oculata had a substantial amount of carotenoids. For instance, P. purpureum contained carotenoids, such as zeaxanthin, β-carotene, and β-cryptoxanthin. As for the total chlorophyll content, A. platensis had the highest chlorophyll content followed by N. oculata, C. vulgaris, and P. purpureum. As for the triterpenoid content, A. platensis had the highest total content which was markedly higher compared to the other species.The author suggested that the polyphenolic and carotenoid contents of the microalgae extracts contributed to these potential anti-diabetic properties mainly due to their antioxidant, anti-inflammatory properties, and anti-apoptotic properties . PhytochA. platensis decreased the MPO release from human neutrophils in a dose-dependent manner [A. platensis exerts anti-inflammatory properties in alleviating hyperglycemia from an in vitro standpoint. A study comparing glucosidase inhibition among microalgae species isolated from the Amazon revealed that Synechococcus sp. had one of the highest glucosidase inhibitions, thus emerging as a potential anti-diabetic supplement [Synechococcus sp. methanolic extract exhibited 90.4% and 96.9% inhibitory activity against α- and β-glucosidase, respectively. The author suggested that the glucosidase inhibition by the microalgae extracts could be due to the pigments in them, such as purified C-phycoerythrin (C-PE) and phycocyanin [Additionally, another in vitro study to measure the inflammation biomarker myeloperoxidase (MPO) release from human neutrophils revealed that t manner . MPO is t manner . Therefopplement . In thatcocyanin . Moreovecocyanin .A. platensis [A. platensis could be hydrolyzed into potential bioactive peptides exhibiting antioxidant, dipeptidyl peptidase-IV (DPP-IV), and α-amylase and α-glucosidase inhibitory properties. Inhibition of DPP-IV improves blood glucose fluctuations and enhances glycemic control in diabetes. Blood glucose fluctuations are linked to pancreatic β-cell impairment; therefore, DPP-IV inhibition is an effective strategy to alleviate diabetes [Villaro et al. investigated the antioxidant and anti-diabetic properties of enzymatic hydrolysates derived from the phycobiliproteins of latensis . Proteindiabetes ,141,142.diabetes . Additiodiabetes . ResultsC. karianus in hepatocytes and adipocytes [Oxo-fatty acids (oFAs), a type of long-chain fatty acid, have been shown to demonstrate peroxisome proliferator-activated receptors (PPARs) agonist activities to regulate energy metabolism . PPARs, ipocytes . These oipocytes ,148, wheipocytes ,150. OveC. karianus are referred to as semi-potent dual PPARα/γ agonists with the potential to alleviate hyperglycemia.Moreover, the adipogenic potentials of oFAs were studied via adipocyte transcriptomic analyses. Although the focus was to compare the dyslipidemic activities of oFAs, the authors reported that oFAs enhanced insulin-sensitizing adiponectin and leptin gene expressions while suppressing pro-inflammatory cytokines gene expressions due to PPARγ activation. Moreover, IRS1 and SLC2A4 genes were upregulated resulting in improved adipose insulin sensitivity ,152. TheSeveral studies have evaluated the effects of microalgae or their bioactive compounds on a well-established diabetic rodent model characterized by high blood glucose levels. A. platensis or biomass supplementation alleviates hyperglycemia in diabetic rats mainly due to its antioxidant and anti-inflammatory activities [A. platensis extract for 3 weeks [A. platensis extract. Chemical compositional analysis confirms that the extract contains zinc (Zn), copper (Cu), iron (Fe), selenium (Se), chromium (Cr), and magnesium (Mg). According to studies, TMs provide insulin-modulatory effects [A. platensis extract. Therefore, TMs improve glucose metabolism and reduce oxidative stress and inflammation.Studies have indicated that tivities ,162. Nas 3 weeks . A total effects . In addi effects . These d effects ,166. In  effects ,168,169. effects ,168. FurA. platensis has been shown to be involved in the protection of pancreatic β-cells. Hyperglycemia causes reactive oxygen species (ROS)-mediated glucose toxicity in pancreatic β-cells that impairs their insulin-producing function, leading to insulin insufficiency [A. platensis powder for 5 or 10 days [A. platensis and its role in alleviating hyperglycemia by regulating pro-inflammatory cytokine levels.The anti-inflammatory property of ficiency . Alloxan 10 days . It was A. platensis extract improves glucose tolerance in diabetic rats by positively affecting the gut microbiota composition. Moreover, different extract types seem to exert different anti-diabetic effects. In that context, male rats that were induced diabetes by a high-fat high-sucrose diet were supplemented with 150 mg/kg/day of ethanol or water extracts of A. platensis for 8 weeks [A. platensis compared to non-supplemented diabetic rats. Moreover, an oral glucose tolerance test (OGTT) revealed that both extracts improved glucose tolerance. Interestingly, gut microbiota composition, which was also improved mainly by increasing the abundance of Oscillibacter, was indicated to play a crucial role in attenuating the adverse conditions seen in diabetic rats. These results suggest that A. platensis supplementation, especially its water extract, is efficacious in alleviating hyperglycemia, the hallmark of diabetes mellitus. The author explains that this could be attributed to PUFAs in their extracts that may modulate energy metabolism and improve pancreatic β-cell functions [In addition,  8 weeks . The resunctions . In addiunctions . In thisA. platensis supplementation could modulate energy metabolism by regulating the enzymes involved in glucose metabolism. Furthermore, this supplementation exerts anti-apoptotic properties to protect pancreatic β-cells. Supplementing 500 mg/kg of A. platensis powder twice a week for 2 months to STZ-induced diabetic male Albino rats decreased serum glucose and glycated hemoglobin (HbA1c) concentrations and increased serum insulin concentration [A. platensis may be due to its ability to inhibit PC expression. Furthermore, supplementation restored the architecture of pancreatic islets and increased glutathione concentration. Moreover, the activities of glutathione-S-transferase (GST), SOD, and catalase were increased. The upregulation of SOD, catalase, and GST mRNA expressions in these rats further corroborates those findings. These enzymes counteract the effects of ROS by neutralizing them to protect pancreatic β-cells [A. platensis supplementation decreased pro-apoptotic Bax, CASP-3, and pro-inflammatory TNF-α mRNA expressions and increased anti-apoptotic Bcl-2 mRNA expression. Additionally, p-p38, p-ERK, and p-JNK MAPKs protein expressions decreased, showing that the activation of p38- and ERK-mediated MAPK signaling was inhibited. A crucial element of the pro-apoptotic signaling pathway imposed on diabetes is the MAPK signaling cascade [A. platensis.Moreover, ntration . This stntration ,174. The β-cells . Therefo β-cells . Moreove cascade . MAPK acN. oculata supplementation was reported to have corrected hyperglycemia and hyperlipidemia in diabetic rats. A total of 10 or 20 mg/kg/day of N. oculata supplementation for 3 weeks on STZ-induced male Wistar diabetic rats resulted in increased BW, serum insulin, and high-density lipoprotein cholesterol (HDL-C), and decreased serum glucose, TG, TC, and LDL-C concentrations [N. oculata could be due to its protective effects against oxidative damage on pancreatic β-cell islets or enhanced glucose uptake by peripheral tissues which is comparable to insulin therapy on STZ-induced diabetic rats [N. oculata could have exerted the previously mentioned antioxidant effects to counteract the oxidative damage caused by STZ on pancreatic islets [N. oculata was suggested to play a role in regulating the antioxidant defense system [N. oculata could have played a role in improving the lipid profile [trations . Howevertic rats ,177. Thec islets . Moreovee system ,179. The profile .N. oculata powder has proven its antihyperglycemic effect by regulating antioxidant enzymes and pro-inflammatory cytokine levels [N. oculata. Furthermore, serum concentrations of pro-inflammatory markers, such as IL-6, NF-κB, IL-1β and TNF-α, were significantly decreased. Thus, the anti-inflammatory activity of N. oculata could also be credited for the improvement in hyperglycemia in diabetic rats. The authors suggest that carotenoids present in N. oculata could have exerted these bioactivities [Another study with a similar study design and doses of e levels . First, e levels ,182,183.tivities .N. oculata, 10% of N. gadiatana powder supplementation per day for 2 months on STZ-induced diabetic male Wistar rats also improved BW and serum glucose [N. gaditana attenuated glycosuria and polyphagia which marked its potential in reversing diabetic complications [N. gaditana supplementation, which could be due to improvement in overall energy metabolism exerted by modulating insulin secretion. Furthermore, it was reported that CAT, glutathione, and SOD enzymes were increased in the liver mitochondria and pancreatic tissues. Moreover, CAT, glutathione, SOD, and GST in the liver tissues were increased. Therefore, the liver mitochondrial MDA and carbonyl proteins concentrations were also decreased. Carbonyl proteins are markers of protein oxidation similar to how MDA is for lipid peroxidation, and these reflect the oxidative status in the rats [N. gaditana scavenges free radicals and prevents lipid peroxidation [N. gaditana [Similar to ications ,185. Thethe rats . It was the rats ,188,189.xidation . Furthergaditana ,192.C. pyrenoidosa exerts its anti-diabetic effects by modulating energy metabolism and improving gut microbiota composition [C. pyrenoidosa were supplemented to the diet-induced diabetic male rats. Both extract types decreased FBG and improved glucose tolerance denoted by the decreased area under the curve (AUC) in the OGTT. In terms of its effects on the composition of gut microbiota, C. pyrenoidosa restores the microflora balance of the intestine. The supplementation decreased the Firmicutes/Bacteroidetes ratio which is said to be a sign of improvement in the microbiota of the intestine and chronic low-grade inflammation [Blautia and Turicibacter compositions. Turicibacter is said to cause diabetes and inflammation [Oscillibacter, Parasutterella, and Ruminococcus species, were maintained by the supplementation. Ruminococcus and Oscillibacter seemed to negatively correlate with AUC FBG. However, these findings are not consistent with other studies and there is a lack of extensive characterization of the gut microbiota in diabetic rat models [C. pyrenoidosa performed better than A. platensis suggesting that the former is more efficacious at exerting anti-diabetic effect. The author speculates that the improvement in the abundance of Ruminococcus could underlie the increased hypoglycemia effect of C. pyrenoidosa compared to A. platensis. These effects could be attributed to polyunsaturated fatty acids (PUFAs) found in C. pyrenoidosa [According to a study, position . In thatammation ,194. Morammation . Moreovet models ,197. At enoidosa ,199.P. cruentum powder on diabetes due to its protective and proliferative effects on pancreatic β-cell islets. STZ-induced male Sprague–Dawley rats that were supplemented with 600, 1200, and 1800 mg/kg/day for 14 days had increased β-cell granulation and the number of pancreatic β-cells. Additionally, 1200 and 1800 mg/kg doses increased pancreatic islets of Langerhans area, but these parameters were markedly reduced by diabetes. The pancreatic β-cells produce and synthesize insulin that most importantly regulates blood glucose levels, and its impairment leads to a lack of insulin synthesis and subsequent metabolic control, leading to hyperglycemia [P. cruentum supplementation did not alleviate blood glucose levels of the diabetic rats suggesting that the doses used in this study may not be optimum to impart any hypoglycemic effect.A study has demonstrated the therapeutic effect of glycemia ,202. Preglycemia ,203,204.D. salina extract on the metabolism of STZ-induced male Sprague–Dawley rats [D. salina extract at 72, 64, 48, 40, and 24 h before sacrifice. Metabolic fingerprinting, a comprehensive analysis of metabolic profiles, was performed to assess any changes caused by the extract. The results of the study revealed significant alterations in the metabolic profiles of the diabetic rats after receiving D. salina extract. This is due to the fact that the urine fingerprinting PLS-DA scores plot shows a clear separation between supplemented and non-supplemented groups. The extract demonstrated a positive effect on most of the parameters measured, indicating potential therapeutic properties for diabetes management. In that context, there was a slight increase in BW and a decrease in liver size. The author speculates that the extract could have provided better metabolic control resulting in increased liver glycogen utilization which is a sign of improvement in Mauriac syndrome in type 1 diabetes [D. salina has antioxidant properties by reducing markers of oxidative degradation and increasing antioxidant capacity exclusively in the liver. Moreover, a-tocopherol in plasma was increased suggesting that D. salina could protect against damage from free radicals [D. salina has been reported to contain β-carotene and bioactive peptides that exhibit antioxidant activities. Nevertheless, the supplementation did not improve blood glucose levels in this study, but the TG levels were improved. This suggests that the extract exerts an antihyperlipidemic function that could alleviate complications associated with diabetes mellitus. Overall, the findings suggest that D. salina extract has the potential to positively influence the metabolism of diabetic rats.Ruperez et al. investigated the effects of ley rats . The diadiabetes . In addidiabetes ,207,208.radicals . FurtherChlorophyceae and Eustigamatophyceae families enhanced their total antioxidant capacity in adipose tissue and plasma [Administration of omega-3 fatty acids has been reported to improve diabetes by regulating lipid metabolism, insulin synthesis, and attenuating oxidative stress and insulin resistance by modulating the expressions of inflammatory and energy metabolism genes ,212,213.d plasma . Howeverd plasma . Meanwhid plasma ,217.Chlorophyceae and Eustigmatophyceae families on db/db mice at similar dosages had profound anti-inflammatory properties [Another study found that supplementation with microalgae EPA and DHA extracted from operties . In thatoperties ,219. FurC. pyrenoidosa polysaccharides (CPP) for 4 weeks were observed to have improved BW and insulin levels [Microalgae-derived polysaccharides have been studied for their antihyperglycemic activity since it slows down gastric emptying and the absorption of glucose in small intestines and protects pancreatic β-cells ,220,221.n levels . Addition levels . Addition levels . Moreoven levels . UltimatP. cruentum at 150, 300, and 450 mg/kg/day for 14 days on STZ-induced male Sprague–Dawley rats decreased food intake [P. cruentum could be a viable option in managing diabetes.In another study, the administration of extracellular polysaccharides (Eps) derived from d intake . Furtherd intake ,226,227.d intake ,229. TheMicroalgae pigments, such as chlorophylls, carotenoids (β-carotene and astaxanthin), and phycobilins  are photosynthetic pigments commonly used as dyes in the food, cosmetic, and pharmaceutical industries ,231. By H. pluvialis, explains its mechanism of action [A study carried out by Penislusshiyan et al., investigating the in vitro antioxidant activity of astaxanthin derived from f action . In thatf action ,236. Morf action . The resf action .There have not been many well-designed clinical trials examining the effects of various microalgae-based supplementations on diabetic human subjects. In addition, the trials are limited to T2DM patients which could be due to the potential of microalgae or its bioactive compounds in alleviating metabolic disorders. A. platensis supplementation has been shown to improve the glycemic profile of patients with T2DM. Doses of A. platensis as low as 1 or 2 g and/or as high as 8 or 14 g per day improved FBG and glycated protein levels such as HbA1c [A. platensis supplementation improved lipid profile which is often deranged in diabetics leading to cardiovascular diseases and death [as HbA1c ,240,241.nd death ,239,242.A. platensis demonstrates a synergistic anti-diabetic effect with metformin while improving lipid profiles in T2DM patients [A. platensis administered together with their daily metformin regimen for 3 months managed to decrease the HbA1c, FBS, TC, LDL-C, and TG, and increase the HDL-C levels better than metformin alone. This suggests that A. platensis supplementation could be an additional strategy for managing diabetes without the side effects associated with anti-diabetic drugs.Furthermore, a study has reported that patients . A totalA. platensis could be attributed to the upregulation of a crucial insulin regulatory mechanism, namely, the adenylate cyclase/cAMP pathway which causes increased cAMP levels followed by upregulation of PKA activity leading to insulin secretion [The antihyperglycemic properties of ecretion . Moreoveecretion .Applying microalgae and their bioactive compounds as health supplements to alleviate diabetes mellitus poses certain limitations. First, only one research has been carried out for most microalgae species or their bioactive compounds. Additionally, the effects of other potential microalgae species or their bioactive compounds on diabetes mellitus are unexplored. Moreover, some of the studies employed only a single dosage of microalgae or their bioactive compounds to assess their anti-diabetic properties. Furthermore, some microalgae dosages were selected based on previous studies on different microalgae species which may not have similar bioactive compound composition or concentration. Therefore, the optimum dosage for most of the test compounds is largely unknown and there is no clear verdict as to how efficacious these dosages are if they were to be considered as health supplements. In addition, the method of inducing diabetes mellitus in animal models varies among studies; therefore, the therapeutic effects of the test compounds cannot be generalized for every type of diabetes. Moreover, the disease animal models may not completely recapitulate the complex pathophysiology of diabetes, especially T2DM. Furthermore, the anti-diabetic mechanisms were only elucidated in some of the studies. Additionally, information on the digestibility or bioavailability of the specific microalgae extracts is lacking, which is crucial since an intact microalgae cell wall could hamper the bioavailability and lessen the potency of the bioactive compounds. Finally, some of the clinical trial study designs face limitations in terms of small sample size, no placebo groups, and short duration of intervention.A. platensis and astaxanthin, which have been reported as safe for consumption by governmental bodies [A. platensis does not contain toxins, deeming it safe for consumption [N. oculata, N. gaditana, D. salina and C. vulgaris extracts, fucosterol, and EPA from N. oculata have been reported to be safe in preclinical studies [Moreover, comprehensive data on the safety of microalgae consumption are not available yet. Extensive toxicological studies are limited to only some species and their bioactive compounds, especially the ones that are commonly consumed, such as l bodies ,244. Gensumption ,243,245. studies ,249,250. studies . As a reMore studies are needed to elucidate the underlying anti-diabetic mechanisms of microalgae and their bioactive compounds by means of studying the genes and proteins involved. In addition, future studies shall employ omics technology to gain valuable insights into the molecular changes imparted by a particular microalga or its bioactive compounds. In that context, novel biomarkers, therapeutic targets, and upregulated or downregulated pathways responsible for the anti-diabetic effects of the supplementation can be identified. In this review, microalgae supplementations have been shown to alleviate diabetes mellitus mainly due to their antioxidant and anti-inflammatory activities that exert protective effects on pancreatic β-cells and subsequent glycemic control. Moreover, the bioactive compounds inhibit enzymes associated with hyperglycemia. A brief summary of the potential therapeutic effects and the mechanisms of microalgae and their bioactive compounds on hyperglycemia has been illustrated in


Pseudomonas aeruginosa has become a major cause of severe infections. Due to the lack of approved vaccines, this study has presented putative vaccine candidates against it.Multidrug-resistant P. aeruginosa 24Pae112 as a reference strain was retrieved from GenBank database. The surface-exposed, antigenic, non-allergenic, and non-homologous human proteins were selected. The conserved domains of selected proteins were evaluated, and the prevalence of proteins was assessed among 395 genomes. Next, linear and conformational B-cell epitopes, and human MHC II binding sites were determined. Finally, five conserved and highly antigenic B-cell epitopes from OMPs were implanted on the three platforms as multi-epitope vaccines, including FliC, the bacteriophage T7 tail, and the cell wall-associated transporter proteins. The immunoreactivity was investigated using molecular docking and immune simulation. Furthermore, molecular dynamics simulation was done to refine the chimeric cell-wall-associated transporter-TLR4 complex as the best interaction.P. aeruginosa 24Pae112, 16 proteins (seven OMPs and nine secreted) were ideal according to the defined criteria. These proteins had a molecular weight of 110 kDa and were prevalent in ≥ 75% of P. aeruginosa genomes. Among the presented multi-epitope vaccines, the chimeric cell-wall-associated transporter had the strongest interaction with TLR4. Moreover, the immune simulation response revealed that the bacteriophage T7 tail chimeric protein had the strongest ability to stimulate the immune system. In addition, molecular docking and molecular dynamic simulation indicated the proper and stable interactions between the chimeric cell-wall-associated transporter and TLR4.Among 6494 total proteins of e.g. cell-wall-associated transporter and bacteriophage T7 tail proteins) for designing of multi-epitope vaccines (MEVs), showed the better performance compared to FliC. In our future studies, these two MEVs will receive more scrutiny to evaluate their immunoreactivity.This study proposed 16 shortlisted proteins as promising immunogenic targets. Two novel platforms ( Pseudomonas aeruginosa is a ubiquitous Gram-negative bacterium that belongs to the Pseudomonadaceae family and can survive in a variety of environments, including soil and water. Indeed, it can be easily found in almost any human- or animal-influenced habitat [P. aeruginosa is responsible for more than 5% of infectious exacerbations in patients with chronic obstructive pulmonary disease (COPD) and has been associated with increased mortality in these patients [P. aeruginosa was recognized as one of the most life-threatening bacteria and listed by the World Health Organization (WHO) as a priority pathogen for the research and development of new ways to overcome it [ habitat . Studies habitat , 3. In apatients . In 2017P. aeruginosa strains, leading to the ineffectiveness of antibiotic therapy against this microorganism [P. aeruginosa infections. To date, different approaches, such as secondary metabolites, nanomaterials, and vaccine development, have been suggested to find a suitable alternative to the currently used antibiotics against this pathogen. However, because of ongoing problems such as growing antibiotic resistance and population aging, vaccination could be more desirable and cost-effective [The overuse of antibiotics during treatment accelerates the development of multidrug-resistant organism . Therefoffective , 8.P. aeruginosa. Although many of these approaches have been tested experimentally in preclinical trials, few of them have reached the clinical phase, and none of them has been approved for marketing [Currently, numerous vaccine development methods are being tested against this superbug. For example, whole-cell vaccines , killed arketing . TherefoNeisseria meningitidis serogroup B (MenB), which is currently licensed in several countries [Present immunization strategies mainly concentrate on outer membrane proteins (OMPs) as desirable subunit vaccines since there is no risk of the pathogen reverting to its virulent form and there are few adverse effects compared to live attenuated, or killed whole-cell vaccines . Moreoveountries , 20.P. aeruginosa 24Pae112 to introduce novel and putative vaccine candidates. Also the design of new multi-epitope vaccines against this bacterium can be considered as promising subunit vaccines. The obtained results from this study provide new insights into vaccine development not only against P. aeruginosa but also against other Gram-negative bacteria.Taking into account the aforementioned, the present study aimed to investigate the whole genome sequence of P. aeruginosa were retrieved from the GenBank database (https://www.ncbi.nlm.nih.gov/genbank/) and extracted to the proteome using CLC Genomics Workbench Software  [P. aeruginosa 24Pae112 (accession number: NZ_CP053028.1), which belongs to ST235, was selected as a reference strain. P. aeruginosa ST235 is spread around the world, possibly due to selective pressure from fluoroquinolones, and became easily resistant to aminoglycosides, beta-lactams, and carbapenems through mutation and acquisition of resistance elements [In the first step, 395 completed genomes of Germany)  to perfoGermany) . After ghttps://www.psort.org/psortb/) to predict their subcellular localization [https://services.healthtech.dtu.dk/service.php?TMHMM-2.0) to identify surface-exposed regions [All proteins were uploaded to the PSORTb database (lization . In this regions .http://www.ddgpharmfac.net/vaxijen/VaxiJen/VaxiJen.html) with a cut-off value of ≥ 0.4 and ANTIGENpro (https://scratch.proteomics.ics.uci.edu/) [http://crdd.osdd.net/raghava/algpred/) with a cut-off value of ≥ 0.3 and AllergenFP (https://www.ddg-pharmfac.net/AllergenFP/) [The antigenicity of putative immunogenic proteins was predicted using the VaxiJen online tool (ci.edu/) , 27. On rgenFP/) , 29.Humo sapiens, Taxid: 9606) using the PSI-BLAST tool in the BLASTp database (https://blast.ncbi.nlm.nih.gov/Blast.cgi?SIDE=protein) [All selected proteins were analyzed to determine their sequence similarity to the human proteome (protein) . Proteinhttps://webs.iiitd.edu.in/raghava/vicmpred/submission.html) [https://web.expasy.org/protparam/) [First, the functional class of the selected proteins was determined using the VICMpred database (on.html) . Then, ttparam/) .https://www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml) and EggNOG (http://eggnog5.embl.de/#/app/home) were used to find the protein domains. CDD is a part of the NCBI’s Entrez query and provides annotation of protein sequences with the position of the conserved domain [Conserved Domain Database, CDD  was used to identify linear B-cell epitopes of all previously selected proteins with a threshold of ≥ 0.6 [http://tools.iedb.org/tepitool/), an IEDB resource, was used to predict human MHC II binding sites with a cut-off of the top 5% of peptides [The BepiPred-2.0 tool  [https://prosa.services.came.sbg.ac.at/prosa.ph) [https://zlab.umassmed.edu/bu/rama/index.pl) [http://tools.iedb.org/ellipro/) was used to identify the conformational B-cell epitopes with a threshold of ≥ 0.8 [The tertiary structure (3D) of the putative immunogenic proteins was predicted using the Robetta tool (ab.org/) . The quarosa.ph) . This sendex.pl) . ElliProof ≥ 0.8 . The preof ≥ 0.8 .https://string-db.org/) to investigate the interactions of two Hypothetical proteins with other proteins of P. aeruginosa to estimate their function. In molecular biology, STRING is a biological database and web resource for known and predicted protein-protein interactions [In this part, we used the STRING database (ractions .https://consurf.tau.ac.il/consurf_index.php) [Linear B-cell epitopes, located on the extracellular loops of seven OMPs selected from previous steps, were predicted using the BepiPred database with a cut-off value of ≥ 0.6. Additionally, the conservation of linear B-cell epitopes was determined using the ConSurf web tool (dex.php) .P. aeruginosa strain 24Pae112, including FliC (WP_124119965.1), bacteriophage T7 tail protein (WP_166796845.1), and cell-wall-associated transporter (WP_024947839.1). The tertiary (3D) structures of these chimeras were modeled using the Robetta web tool. The 3D structures were validated by the ProSA web server (https://prosa.services.came.sbg.ac.at/prosa.php) and Ramachandran Plot (https://zlab.umassmed.edu/bu/rama/).Three multi-epitope-based vaccines were generated using three different platforms obtained from https://life.bsc.es/pid/pydockweb/default/index). In addition, C-ImmSim (https://kraken.iac.rm.cnr.it/C-IMMSIM/index.php) was used to predict the simulation of the immunoreactivity of multi-epitope vaccines [http://www.ebi.ac.uk/thornton-srv/databases/pdbsum/) was used to reveal the paired residues of the chimeric cell-wall-associated transporter and TLR4 [Molecular dockings and the binding affinities of three multi-epitope vaccines to human TLR1 (PDB: 2Z7X), TLR2 (PDB: 2Z7X), and TLR4 (PDB: 3FXI) were assessed with pyDockWEB  was done using the steepest descent algorithm. Equilibrating the system was performed under 100 ps NVT at a temperature of 298 K, followed by 100 ps NPT ensembles at a pressure of 1 bar. Electrostatic interactions were calculated by PME, and the LINCS procedure was applied to constrain all bonds connecting hydrogen atoms. The final MD simulation was run for 100 ns with no restraint [Molecular dynamics (MD) simulation was done to refine the chimeric cell-wall-associated transporter-TLR4 complex using the GROMACS version 2018 simulation package. The structure was centered in a dodecahedron box and filled with water using the TIP3 water model. To neutralize the system, some molecules of water were randomly replaced by Clestraint , 46.P. aeruginosa revealed that this bacterium has a wide genome. These results indicated the plasticity of the P. aeruginosa genome due to many insertion and deletion events. Therefore, using the core proteome was not proper, and we used the whole genome of P. aeruginosa 24Pae112 instead  obtained from BPGA analysis on 395 completed genomes of  instead . The KEG instead . P. aeruBack-to-back analysis of selected proteins from the previous step, revealed that out of 241 proteins, 92 were antigenic, non-allergenic, and non-homologous to human proteins. Therefore, these proteins were selected for further analysis.P. aeruginosa genomes were selected. These proteins were involved in different functional classes, including virulence (22 proteins), cellular processes (35 proteins), metabolism (13 proteins), and information and storage (two proteins). See the According to the defined criteria, 72 proteins with a molecular weight < 110 kDa and a prevalence ≥ 75% among All selected proteins were assessed using the quartile scoring method. Finally, a total of 16 proteins (seven OMPs and nine secreted) with a contractual score ≥ 7 were considered shortlisted proteins and valuable for further investigations. The accession numbers for these proteins are as follows: Hypothetical proteins (WP_132548232.1 and WP_166796845.1), Type I fimbrial protein (WP_034004502.1), Type II secretion system secretin GspD (WP_061193930.1), Hcp family type VI secretion system effector (WP_110726056.1), TonB-dependent receptors (WP_023101627.1 and WP_125941151.1), Glycosyl hydrolase family 18 protein (WP_034004678.1), Type IVa pilus secretin PilQ (WP_132905315.1), Transporter (WP_024947839.1), OprD family porin , Type III secretion system needle length determinant (WP_210733189.1), and Peptidoglycan-associated lipoprotein Pal (WP_058148283.1). See A quality assessment of the tertiary structures of proteins showed that the predicted structures are of acceptable quality. See According to the obtained results from the STRING database, the Hypothetical protein WP_132548232.1 had co-occurrence and co-expression with peptidase C39 domain-containing protein (PA1953) and co-occurrence with ABM domain-containing protein (PA0271). On the other hand, the Hypothetical protein WP_166796845.1 is neighboring the UvrD/REP helicase N-terminal domain protein (DR97_1795). The full results of protein-protein interaction networks are presented in PEEIGGKSSNN, YNVSKASSSGG, QADANGYYPAQKDYYGCEKGVDGAC (obtained from TonB-dependent receptor), GSTSQITEKSVTGD (extracted from Transporter), and YTAPG NTRGNS (obtained from OprD family porin), with rigid (EAAAK) and flexible (GPGPG) linkers, were assessed through different arrangements, and the most antigenic arrangement was selected to generate the chimeric FliC protein. To generate chimeric proteins from the bacteriophage T7 tail (WP_166796845.1) and the cell-wall-associated transporter (WP_024947839.1), the mentioned epitopes were located in the disordered regions of these platforms. The detailed data are provided in the S2 Table. The 3D structure of the multi-epitope proteins is demonstrated in Five conserved and highly antigenic B-cell epitopes, including e.g. IgM and IgG1), Th1 cell population, and cytokines (e.g. IFN-γ and IL-2) in comparison to the cell-wall-associated transporter chimeric protein. See The pyDockWEB results demonstrated that the chimeric cell-wall-associated transporter as a multi-epitope vaccine had the strongest interactions with TLR1 , TLR2  and TLR4 . The full results of this step are presented in 2. While the SASA of TLR4 was in the range of 270 to 277 nm2  plot. This plot shows how much the protein conformation has changed during MD simulation from the initial structure. The RMSD of TLR4 and cell-wall-associated transporter was in the range of 0.2 to 0.27 and 0.2 to 0.3, respectively. Both proteins reach stability at time 10 and remain stable after that . The Roo 277 nm2 .P. aeruginosa occupies a leading role among infections caused by Gram-negative rods, particularly in critically ill and immunocompromised patients. Due to antimicrobial resistance, there are severely restricted treatment options for P. aeruginosa infection, which has become a critical and fatal problem [P. aeruginosa FDA-approved vaccines for human administration, and recent promising candidates have failed in the final steps of clinical trials [Nowadays, one of the most important challenges for physicians is the effective treatment of infections caused by Gram-negative pathogens due to the increasing antibiotic resistance in healthcare. Meanwhile,  problem , 48. To l trials .P. aeruginosa challenge and induced a Th17 response in the mouse model [P. aeruginosa-based vaccine induces preventative immunity responses against pulmonary infections [In this context, early vaccines against this bacterium targeted the lipopolysaccharide (O-antigen) and could generate satisfactory immunity. However, this protection was LPS serotype-specific, and with more than 30 subtypes of O-antigens, this strategy has met significant challenges . In addise model . Howeverse model . Moreovefections . It seemfections . In addifections .in silico studies have been conducted to introduce immunogenic targets against this bacterium. For example, Beg et al. designed multi-epitope-based vaccines against functional amyloids of P. aeruginosa (Fap) using different immunoinformatic and structural bioinformatic approaches [et al. mainly focused on P. aeruginosa’s major membrane proteins, including OprF and OprI, to design peptide-based vaccine constructs [et al. was more comprehensive, as they used a comparative subtractive proteomic analysis among 1,191 P. aeruginosa proteomes and finally left a total of twenty unique and non-redundant proteomes. However, they presented a chimeric vaccine with PADRE (pan-HLA-DR epitopes) as an adjuvant [So far, different proaches . In addinstructs . In addinstructs . Althougadjuvant  which isP. aeruginosa 24Pae112 and evaluated them from all aspects of immunoinformatics to find novel and putative immunogenic targets. In this regard, different bioinformatics tools and algorithms were used to justify the obtained results. In the case of antigenicity and allergenicity, for example, we used Vaxigen/ANTIGENpro and AlgPred/AllergenFP, respectively. However, we considered only one of them in the study since VaxiJen is the first server for alignment-independent prediction of protective antigens and has been used for antigenicity prediction in many articles on reverse vaccinology [etc. can be taken into account at the same time [In this comprehensive study, it was considered the total proteins of cinology –60. In tame time .The authors of this article believe that using multiple tools for analysis will not always yield the desired results. For example, in subcellular localization, the simultaneous use of several tools to determine the location of proteins in bacteria will be confusing due to different prediction algorithms. Furthermore, we attempted to choose experimental-based tools. However, the accuracy rate of computational tools can be considered as a limitation.The results of this study are significant, as all 16 shortlisted proteins can be obtained as subunit vaccines for further studies in animal models. These proteins are surface-exposed, have a molecular weight of < 110 kDa, and are capable of inducing both humoral and cellular immunity.P. aeruginosa [The first group of shortlisted putative vaccine candidates belong to secretion systems and transports, including (I) Type II secretion system secretin GspD (WP_061193930.1), (II) Hcp family type VI secretion system effector (WP_110726056.1), (III) Type III secretion system needle length determinant (WP_210733189.1), and (IV) Transporter (WP_024947839.1). The transport of proteins from the cytoplasm to other parts of the cell or the environment, a process known as secretion, is one of the most important prokaryotic cell functions. Prokaryotes have evolved numerous ways to transport protein cargo between locations, most of which involve the assistance of specialized protein secretion systems. Proteins taking part in secretion systems are essential for bacterial growth and are involved in several processes . Proteinruginosa , 64. TheAcinetobacter baumannii isolates [P. aeruginosa. These proteins demonstrated 46 to 57% similarity and play an important role in amino acid or organic acid uptake. The OprD has been extensively studied based on its structure, function, and involvement in the carbapenem resistance of P. aeruginosa. This protein, with an 18-strand barrel structure, forms a very narrow channel with two structural features that may contribute to its channel specificity [In the second group, four proteins, including WP_003090815, WP_217385706.1, WP_243702750.1, and WP_019681707.1, belong to the OprD family of outer membrane proteins. This family was identified for the first time during outer membrane investigations of carbapenem-resistant isolates . The Oprcificity , 67.P. aeruginosa. This protein group was previously introduced as vaccine candidates against other Gram-negative bacteria such as Escherichia coli [Klebsiella pneumoniae [Salmonella enterica serovar Typhi [et al. reported the potential of integrated PilQ/PilA (QA) antigens as a promising vaccine candidate against P. aeruginosa. Those study revealed that the chimeric protein PilQ and the disulfide-turn region of PilA trigger the production of specific antibodies in the BALB/c mouse model [In the third group, we introduced fimbrial proteins including, (I) Type I fimbrial protein (WP_034004502.1) and (II) Type IVa pilus secretin PilQ (WP_132905315.1) as a putative vaccine candidate against hia coli , Klebsieeumoniae , and Salar Typhi . Moreovese model .P. aeruginosa. TonB-dependent receptors are a family of beta-barrel proteins named for their localization in the outer membrane of Gram-negative bacteria. These complexes recognize various signals from outside of the bacterial cells and transduce them into the cytoplasm, resulting in transcriptional activation of target genes [A. baumannii [N. gonorrhoeae [K. pneumoniae [In the fourth group, TonB-dependent receptors (WP_023101627.1 and WP_125941151.1) were presented as putative vaccine candidates against et genes . This weaumannii , N. gonoorrhoeae , and K. eumoniae .P. aeruginosa. In Gram-negative bacteria, Pal is related to the integrity of the cellular envelope and interacts powerfully with the peptidoglycan layer [Burkholderia mallei and could be considered an effective target for developing a vaccine against glander [In the fifth group, peptidoglycan-associated proteins, including (I) Peptidoglycan-associated lipoprotein Pal (WP_058148283.1) and (II) Glycosyl hydrolase family 18 protein (WP_034004678.1), are among the shortlisted immunogenic targets against an layer . Consist glander .Finally, the Hypothetical protein WP_132548232.1 has co-occurrence and co-expression with peptidase C39 domain-containing proteins and co-occurrence with ABM domain-containing proteins. The peptidase C39 domain-containing protein defined by this cysteine peptidase domain belongs to the peptidase family C39 (clan CA). It is found in a wide range of ABC transporters, which are maturation proteases for peptide bacteriocins, with the proteolytic domain located in the N-terminal region of the protein . On the P. aeruginosa and suitable scaffolds for multi-epitope vaccine design and development was the significant novelty of the present study. These new platforms for vaccine design can be used not only against P. aeruginosa but also against other bacteria. The disordered regions of these proteins are proper for the implantation of conserved and antigenic epitopes from different pathogens to induce a satisfactory immune response. Cell-wall-associated transporter had the strongest interactions with TLR 1, 2, and 4. TLRs are the major mediators of inflammatory pathways, playing important roles in mediating immune responses to a variety of pathogen-derived ligands and linking adaptive immunity to innate immunity [A multi-epitope vaccine composed of a series of peptides is an ideal approach for the prevention and treatment of bacterial or viral infections . Introduimmunity . On the immunity .P. aeruginosa strain 24Pae112 to find novel and putative immunogenic targets. The results of this study are significant, as all 16 proteins presented are valuable for further investigation in animal models. These proteins were classified into six different groups according to their conserved domains, including (I) Secretion system-associated proteins and transporters, (II) OprD family outer membrane proteins, (III) Fimbrial proteins, (IV) TonB dependent receptors, (V) Peptidoglycan-associated proteins, and (VI) Unknown-function proteins . On the other hand, three designed multi-epitope vaccines showed promising results. Finally, it is worth noting that two proteins  as new platforms for vaccine design and development can be used not only against P. aeruginosa but also against other superbugs. We hope these two platforms will receive more investigation in future studies.This comprehensive study evaluated the entire genome sequence of S1 Fig(PDF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


The relationship between fruit, whole grain, and total energy consumption and the gut microbiome in the Chinese population remains unclear.We investigated the relationship between intakes of fruits, whole grains, and energy, and the diversity and composition of gut microbiota.This cross-sectional study included 167 subjects aged 40-75 years who underwent colonoscopy at Nankai Hospital in Tianjin, China. Each of the participants completed a personal history questionnaire, a 7-day dietary record, and donated a fecal sample. The V3-V4 hypervariable region of the bacterial 16S rRNAgene was amplified and sequenced using Illumina Novaseq. The relationship between diet and gut microbiota was evaluated in terms of both the overall composition and the abundance of specific taxon.Parabacteroides, Streptococcus, and Bilophila in fecal samples. Higher whole grains intake was associated with higher microbial diversity, as measured by Shannon, Simpson, and Chao1 indices. Specifically, there was a significant increase inthe relative abundance of Lachnospiraceae and a decrease in Actinobacteria with increased whole grains intake. Moreover, higher intake of total energy was associated with a lower abundance of Anaerostipes and a higher abundance of Lactobacillales and Acidaminococcus.Fruits intake was positively related to the abundance of Bacilli, Porphyromonadaceae, Streptococcaceae, Whole grains intake was positively associated with gut microbial diversity. Fruits and total energy intake were related to the abundance of specifictaxon . These findings highlight the potential importance of dietary interventions for modulating gut microbiota composition and promoting overall health. Eating whole grains is linked to a more diverse and beneficial gut microbiota composition.Acidaminococcus.Fruit and overall energy intake are associated with specific microbial groups, such as Bacilli and Our data emphasize the importance of dietary changes in controlling the makeup of gut bacteria and cultivating a healthier gut environment.The human intestinal tract harbors an extremely complex and large microbiota, with microorganisms adding up to around 100 trillion in number. This microbiota interacts with the host via various physiological processes, such as shaping the intestinal epithelium, aiding food digestion and energy metabolism, regulating host immune function, and maintaining intestinal homeostasis . Gut dysBifidobacteria and Lactobacillus, while inhibiting the growth of harmful bacteria (Prevotella and Roseburia) by attenuating oxidative stress and inflammatory responses in serum  individuals who had taken prebiotics, probiotics, synbiotics, antibiotics, or undergone radiation therapy within the past three months; 2) women who were pregnant or lactating; 3) individuals with immune deficiencies; 4) individuals with chronic or acute gastrointestinal disorders requiring medical attention, such as acute gastroenteritis, peptic ulcer, or dysentery; 5) those with infections in other areas of the body; 6) those diagnosed with familial adenomatous polyposis; 7) individuals with malignant tumors or who had undergone colorectal surgery, except for non-melanoma skin cancer; and 8) individuals with behavioral constraints, cognitive impairment, or who were extremely frail and unable to participate in the study. This study was approved by the Ethics Committee at Tianjin Nankai Hospital. All 167 subjects included in the analysis provided informed consent prior to participating in the study.Participants completed a personal history questionnaire regarding their health and lifestyle and a 7-day food record. Nutrient and total energy intakes were calculated by multiplying the food intake (in grams) by the corresponding nutrient content per gram based on the 2017 China Food Composition List .Subjects were given fecal collection tools and instructed on how to collect fecal samples. Fresh fecal samples were collected from participants, immediately snap-frozen in liquid nitrogen, and transported to the lab within 48 hours. These samples were stored at -80°C for DNA extraction and gut microbiota sequencing.Total DNA of microorganisms in fecal samples was extracted using the Stool Genomic DNA Extraction Kit , and the extracted DNA products were stored at -80°C for backup. The quantity and quality of genomic DNA were measured using the NanoDrop Spectrophotometer . The V3-V4 region of the bacterial 16S rRNA gene was amplified using specific primers 341F (5’-CCTACGGGNGGCWGCAG-3’) and 805R (5’-GACTACHVGGGTATCTAATCC-3’). The PCR products were purified using AMPure XP Beads , and the quantization was performed using the Qubit dsDNA HS Assay Kit. The Illumina Novaseq 6000 SP PE250  was used for sequencing.The data of each sample were split from the original data according to barcode sequences and primer sequences, and controlled and filtered for quality. Low-quality sequences were identified based on the following criteria: sequences less than 150bp, average mass below 20, lines containing ambiguous orders and sequences greater than 8bp, and lines containing single nucleotide repeats. Chimeric sequences were further removed.In the study, the Vsearch-1.11.1 software was used to assign effective tags to operational taxonomic units (OTUs) based on a similarity threshold of >97%. The representative sequences from each OTU were annotated using default parameters and the Green Gene database to generate lists of OTUs. The community composition of each sample was calculated at each taxonomic level . The OTUs containing less than 0.001% of the total rows in all models were removed. QIIME (version 1.7.1) was used to calculate alpha diversity indices, including Shannon, Simpson, and Chao1, while beta diversity indices were calculated using weighted Unifrac ,22. PrinAlistipes finegoldii and Alistipes senegalensis, while increased yogurt intake correlated strongly with higher abundance of Leuconostoc mesenteroides and Lactococcus lactis  based on their tertile intakes of whole grains, fruits and total energy, respectively. The Shapiro-Wilk test was used to test for normality. Normally distributed continuous variables were described using mean ± standard deviation (x ± s), and one-way ANOVA was used to compare groups. Non-normally distributed continuous variables were described using median and interquartile range, and the Kruskal-Wallis test was used for comparison between groups. Categorical variables were described using composition ratios, and the chi-square test was used to compare between groups. We examined the relationship between dietary intakes and alpha diversity metrics  using multivariable linear regression analysis. Factors associated with gut microbiota in the literature were assessed as potential confounders. For instance, higher body mass index was associated with reduced levels of s lactis . Smokingla lenta ,26. ThesP > 0.05). However, current smokers were more likely to have a low fruit intake than non-smokers (P = 0.012). Individuals having higher family income tended to consume more fruits and total energy . No significant differences were found in any of the diverse indices across groups defined by fruits and total energy intakes.The Shannon, Simpson, and Chao1 indices were used to assess the species richness and diversity in different intake groups . The resP for PCoA1 = 0.036), although no apparent clustering was observed among the groups defined by whole grains and fruits intakes.The Weighted Unifrac Distances analysis detected compositional dissimilarities (β-diversity) of the gut microbiota across different groups defined by total energy intake  and the CNS-ZD Tizhi and Health Fund (grant No. CNS-ZD2020-82).Conceptualization, Y.Z., WL.L., and Y.W.; methodology, Y.Z., WL.L., and Y.W.; software, YX.W.; validation, Y.W., J.Z., L.D., Y.Z.; formal analysis, Y.W.; investigation, Y.Z., WL.L., and Y.W.; resources, WL.L., C.L. and K.Z.; data curation, YX.W., L.D., F.S., D.W., S.M., J.Z., Y.L., W.L., K.Z.; writing—original draft preparation, YX.W., L.D., F.S., J.Z., Y.L.; writing—review and editing, YX.W., L.D., F.S., D.W., S.M., J.Z., Y.L.; Y.Z., W.L., and Y.W.; supervision, Y.Z., C.L., K.Z., WL.L., and Y.W.; project administration, Y.Z., WL.L., and Y.W.; funding acquisition, Y.Z. All authors have read and agreed to the published version of the manuscript.


Riptortus pedestris causes a significant decrease in soybean production and economic losses. To understand how these bugs adapt to different parts of the soybean plant, we studied their gut transcriptomic changes when feeding on soybean leaves, beans, and pods. We discovered that when they fed on pods and beans, there was a notable increase in the expression of digestive enzymes like cathepsins, serine proteases, and lipases. On the other hand, when the bugs consumed soybean leaves, detoxification enzymes, such as ABC transporters and 4-coumarate-CoA ligase, showed higher expression levels. These findings suggest that the dynamic regulation of digestive and detoxification enzymes enables bean bugs to successfully feed on various soybean tissues.The bean bug Riptortus pedestris (Hemiptera: Heteroptera), poses a significant threat to soybean production, resulting in substantial crop losses. Throughout the soybean cultivation period, these insects probe and suck on various parts of plants, including leaves, pods, and beans. However, the specific mechanisms by which they adapt to different food resources remain unknown. In this study, we conducted gut transcriptomic analyses of R. pedestris fed with soybean leaves, pods, and beans. A total of 798, 690, and 548 differently expressed genes (DEGs) were monitored in G—pod vs. G—leaf (comparison of insect feeding on pods and leaves), G—bean vs. G—leaf (comparison of insect feeding on beans and leaves), and G—pod vs. G—bean (comparison of insect feeding on pods and beans), respectively. When fed on pods and beans, there was a significant increase in the expression of digestive enzymes, particularly cathepsins, serine proteases, and lipases. Conversely, when soybean leaves were consumed, detoxification enzymes, such as ABC transporters and 4-coumarate-CoA ligase, exhibited higher expression. Our findings indicate that R. pedestris dynamically regulates different metabolic pathways to cope with varying food resources, which may contribute to the development of effective strategies for managing this pest.The bean bug,  Under long-term coevolution with herbivores, plants have developed various defense mechanisms, such as toxic chemicals and physical barriers, to prevent damage of phytophagous insects ,2. PlantRiptortus pedestris poses a substantial threat to soybean cultivation in Asia. Its remarkable ability to consume diverse soybean plant components—ranging from leaves to pods and beans—has significant implications for both crop quantity and quality [Zhengqing, causing significant losses in soybean production, has been attributed to R. pedestris [R. pedestris exhibits an intriguing dietary behavior. Its presence in fields precedes the maturity of soybean beans, necessitating a period of sustenance from the tender leaves of nascent soybean seedlings [R. pedestris exhibited reduced longevity and higher mortality when fed with pods and beans compared to beans alone [R. pedestris adapt to different hosts remain unclear.The bean bug  quality ,13,14. Tedestris ,16. In teedlings ,18. In ceedlings ,18. Rahmns alone . DespiteR. pedestris across various plant parts holds profound significance, and it not only unravels the ecological intricacies of pest-host interactions but also offers a gateway to pragmatic agricultural solutions. For example, our recent study revealed key lipases linked to feeding and reproduction in R. pedestris [R. pedestris adaptation to varied diets is intricate, it is plausible that beyond lipases, other genes play pivotal roles in this process. Therefore, a comprehensive exploration of gene expression patterns using omics studies becomes imperative in unearthing the underlying mechanisms.Understanding the intricate mechanisms that govern the feeding preferences of edestris . These fR. pedestris when feeding on soybean leaves, pods, and beans. Differentially expressed genes (DEGs) were identified by comparing gene expression levels within each group. We found that digestive enzymes were important for bugs feeding on beans and pods, while the detoxification enzymes were responsible for bugs adapting to soybean leaves.The gut is the main organ where digestion and detoxification occur, and it exerts an important influence on insect feeding on specific plants . In thisR. pedestris was originally collected in a soybean field in Suzhou, Anhui, China in 2019 , and reared on soybean (Glycine max L. Merr.) pods-containing plants in green house of Plant Virology Institute, Ningbo University. The soybean variety used in this study was Wandou 27. Both insects and plants were maintained at 26 ± 5 °C with a 16:8 (light:dark) photoperiod and humidity of 50 ± 5%.The R. pedestris on soybeans, three branches of leaves, twenty beans, and twenty pods were put into net cages (25 cm × 25 cm × 25 cm) separately. Then, ten adults R. pedestris were released onto each net cage. The infested leaves and pods were collected five days post infestation. The healthy bean, leaf, and pod without R. pedestris infestation were used as controls. The morphology of each sample was photographed using a Canon EOS 80D camera . The infested leaves and pods were carefully collected and cut into small pieces approximately 0.5 cm in width and 1 cm in length. The collected beans, pods, and leaves were then attached to stubs and subjected to an air-drying process at 37 °C in a baking oven. Once dried, the samples underwent gold-sputtering and were observed using an SEMTM4000 II plus microscope  [To investigate the impact of , Japan) .R. pedestris were fed with pods, beans, and leaves for a duration of four days in separate net cages (25 cm × 25 cm × 25 cm). The insect guts were dissected in a phosphate-buffered saline (PBS) solution  with a pair of forceps under a stereomicroscope . The dissected guts were washed in a clean PBS solution to avoid the contamination of other tissues. As it is difficult to remove the ingested food inside the lumen, we did not clean the gut lumen. Whole gut, including foregut, midgut, and hindgut, were collected for analysis. The collected samples were transferred into RNAiso plus  and kept on ice during the dissection period. Guts dissected from ten insects were pooled and regarded as one replicate. RNA was extracted according to the manufacturer’s protocol. The chloroform , isopropanol (Sango Biotechnology), and 75% ethanol (Sango Biotechnology) were used for RNA extraction. RNA integrity and quantity was assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system . The RNA samples were sent to Novogene  for library construction and Illumina sequencing.A group of forty 5th instar nymphs of In detail, the library construction process involved several steps. Initially, poly (A) + RNA was purified from a pooled total RNA sample of 20 μg using oligo (dT) magnetic beads. Subsequently, fragmentation of the RNA was carried out in the presence of divalent cations at 94 °C for 5 min. N6 random primers were then employed for reverse transcription, resulting in the generation of double-stranded complementary DNA (cDNA). To prepare the cDNA library, end-repair and adaptor ligation were performed. The products were then subjected to 16 cycles of polymerase chain reaction (PCR) amplification with Phusion High-Fidelity DNA polymerase, Universal PCR primers, and Index (X) Primer according to the manufacturer’s protocol. Subsequently, samples were purified using a QIAquick PCR purification kit . The prepared library was sequenced on an Illumina Nova6000 platform with 150-bp paired-end reads. For each treatment, cDNA library construction and Illumina sequencing were carried out with three biological replicates. The output data were submitted to the National Genomics Data Center under accession number: PRJCA019207.R. pedestris (https://www.ncbi.nlm.nih.gov/datasets/genome/GCA_019009955.1/ (accessed on 4 September 2022)) [The internal software Fastp version 0.23.1  was usedr 2022))  using Hir 2022)) . The lowr 2022)) .http://bioconductor.org/packages/release/bioc/vignettes/Glimma/inst/doc/DESeq2.html (accessed on 4 September 2022)) was used to identify the differentially expressed genes with default parameters. Based on these statistical analyses, genes meeting the criteria of p-value < 0.05 and an absolute value of the log2 ratio > 1 were deemed as DEGs. To reveal overall differences in gene expression patterns among different transcriptomes, R function plotPCA (github.com/franco-ye/TestRepository/blob/main/PCA_by_deseq2.R (accessed on 4 September 2022) was used to perform PCA.To investigate the transcriptomic changes in the gut under different food resources, transcripts per million (TPM) expression values were calculated using cufflink . The DESp-values were calculated according to one-sided hypergeometric test: N representing the number of genes with GO annotation, n representing the number of DEGs in N, M representing the number of genes in each GO term, and m representing the number of DEGs in each GO term. The significant pathways were defined based on a corrected p-value ≤ 0.05.The GO enrichment analyses were performed using TBtools software v1.0697 . In this® 480 Real-Time PCR System using the SYBR Green Supermix Kit . The qRT-PCR reaction program consisted of an initial denaturation step at 95 °C for 5 min, followed by 40 cycles of amplification at 95 °C for 10 s and 60 °C for 30 s. A relative quantitative method (2−ΔΔCt) was used to evaluate quantitative variation. The top ten DEGs associated with insect detoxification and digestion were selected, and the gene-specific primers used for qRT-PCR were designed using Primer 5.0 software .The gut samples of ten 5th instar nymphs were collected as described above. Total RNA was isolated with RNAiso plus. Three independent biological replicates with each repeated thrice were performed. To generate the first-strand cDNA and eliminate any genomic DNA contamination, the HiScript II Q RT SuperMix  was employed. Real-time quantitative PCR (qRT-PCR) was conducted on a Roche Light Cyclersoftware . R. pedeR. pedestris were investigated. The beans and pods became crinkled, while necrosis spots appeared on the leaves . A total of 19,006 genes were determined to be expressed in guts  captures the 37% contributing factor of variation, which well distinguished the G—bean from the G—leaf plus the G—pod. The second principal component (PC2) captures the 29% contributing factor of variation, and the G—leaf can be distinguished from the G—bean plus the G—pod in this case  A,B,D shoand pods C. The exand pods E. The exr groups G–J. Overr groups C,F. It iR. pedestris to different plant tissues is crucial for gaining insights into their behavior in the field and developing strategies to mitigate crop damage caused by these pests. In this study, we analyzed the gene expression patterns in the guts of R. pedestris that feed on soybean leaves, pods, and beans. Our findings highlight the significance of genes involved in catalytic activity, hydrolase activity, basal biosynthetic processes, and detoxification in the insect adaptation to these diverse food sources.Understanding the mechanisms underlying the adaptation of R. pedestris can successfully feed and probe on different soybean tissues, with abundant salivary sheath left on the surface of the food. However, the appearance of the salivary sheath varied on pods, beans, and soybean leaves. The palatability is closely related to the chemical composition of these food resources [R. pedestris feeding [esources ,30. The  feeding . The salR. pedestris fed on beans and pods. Both tissues are rich in amino acids and soluble proteins [R. pedestris feeding on beans and pods [R. pedestris was feeding on pods and beans indicates that the insects thrive better on beans and pods compared to soybean leaves, which is consistent with previous conclusions [Herein, we found enhanced expression of genes associated with catalytic activity, hydrolase activity, and peptidase activity when proteins ,33, and proteins ,35. Addiand pods . Furtherand pods ,38, wereclusions ,33.R. pedestris fed on soybean leaves, including ABC transporters and uricase. ABC transporters play critical roles in insect development and immunity [R. pedestris to secondary metabolites in soybean leaves. Moreover, several genes related to lipid metabolism, such as 4-coumarate-CoA ligase 1-like and RNA-binding protein NOB1, exhibited increased expression when R. pedestris fed on soybean leaves. These genes are involved in the breakdown of lipids, which are defensive metabolites against herbivores [R. pedestris to successfully feed on soybean leaves by overcoming the chemical defense mechanisms.On the other hand, components related to cellular processes, localization, and transport showed higher expression when immunity , and theimmunity ,41,42. Trbivores ,44. The R. pedestris fed on beans and pods. Similar findings have been reported in other insect species, such as the sugarcane borer , Chilo suppressalis, and Trichoplusia ni, in response to different food resources [R. pedestris fed on soybean leaves is significantly shorter than that of bugs fed on pods and beans, which is positively correlated with the concentration of amino acids and proteins [R. pedestris fed on pods and beans. These results support the previous conclusion that pods and beans are more favorable food sources for R. pedestris compared to soybean leaves [Previous studies have provided valuable references for exploring the adaptation mechanisms of herbivorous insects to their host plants. For example, Yun et al.  comparedesources ,47,48. Iproteins ,33. Addiproteins ,50,51, sn leaves ,33.R. pedestris to extend their occurrence period, leading to population growth in the field. Furthermore, we identified several candidate genes that potentially enhance the feeding behaviors of R. pedestris on leaves. These digestive genes can be employed as potential targets to minimize the population of R. pedestris in the field, thereby reducing the damage caused by these pests and preventing outbreaks of infestations.By comparing the transcriptomics of the insect gut when fed on different resources, we identified several key metabolic pathways that favor bean bug feeding on soybean leaves, pods, and beans. This enables