Bothrops) in southeastern Brazil, which have high genetic affinity and dwell on the mainland and adjacent islands, provide an appropriate setting to study the evolution of antipredator behavior and how different predatory stimuli can influence this behavior. The mainland Bothrops jararaca has several terrestrial and aerial predators, whereas B. insularis and B. alcatraz, restricted to two small islands, Queimada Grande and Alcatrazes, respectively, have a smaller range of aerial predators. Terrestrial predators are absent on Queimada Grande, but one potential snake predator occurs on Alcatrazes. We observed that the defensive repertoire of island snakes has not been lost, but they display different frequencies of some antipredator behaviors. The type of predatory stimuli  influenced the defensive response. Bothrops insularis most often used the escape strategies, especially against terrestrial predatory stimuli. Bothrops alcatraz displayed the highest rate of strike for both terrestrial and aerial stimuli. Our results indicate that even though relaxed selection may occur in island environments as compared to mainland environments, these pitvipers still retain their antipredator behaviors but with different response degrees to the two predator types.The reduction of predation is a potentially important factor for the evolution of the traits of an island animal species. By relaxed selection, insular animals tend to lose their antipredator behaviors. A monophyletic group of pitvipers (genus  One of the most relevant interactions between animals in a community is the prey-predator relationship. Predation drives the development of several adaptations in animals. Among these adaptations, the behavioral ones deserve attention, especially the actions that avoid or lessen predation, i.e., the anti-predator behaviors . Like maAntipredator behavior is influenced by intrinsic factors such as age, sex, reproductive condition, and experience or learning as well as extrinsic factors as habitat, temperature, and social context ,7. AmongBothrops alcatraz and the Golden lancehead Bothrops insularis are pitvipers endemic to Alcatrazes Island and Queimada Grande Island, respectively located about 30 to 35 km off the coast of the state of São Paulo, southeast Brazil  arena was set up, with an aluminum plate as a base, and Styrofoam walls. All experiments were carried out between 5 pm and 8 pm at a temperature around 23°C. For each behavioral test, the snakes were left in the arena for up to 15 minutes in order to cease exploratory behaviors and habituate to the test arena.To perform the behavioral tests, we conducted confrontations with the snakes using aerial and terrestrial predator models as stimuli . The tests were conducted at night (between 6 PM and 10 PM), under artificial lighting. For the confrontations, a 2.025 mDidelphis albiventris) as this mammal preys on B. jararaca [Athene cunicularia) with spread wings. This owl species preys on pitvipers, besides occurring on the two islands [To simulate a terrestrial predator, we used a cloth and cotton model of the white-eared opossum  and predatory stimuli . The response variables were the frequency of each antipredator behavior during the 30 confrontations with each predator model. Due to the nature of these variables and based on the experimental design, we used a generalized linear mixed model (GLMM) with Poisson distribution and log link function for each behavior. Only for body flattening a GlmmTMB model with a negative binomial distribution (nbinom1) was used. The model consisted of the additive and interactive property between the variables (species and predator). As we conducted the tests with the same individual snakes, and the sex was not of interest for our investigation, we included these two variables (ID and sex) as random variables in the models, thereby eliminating pseudoreplication in the experimental design. In addition, we performed Tukey’s test for post-hoc analysis to find significant relationships between the variables, using the "emmeans" package. All models were subjected to data dispersion analysis, homoscedasticity, and delineate tests using model diagnostic values and plots, with the help of the package "DHARMa: residual diagnostics for hierarchical (multilevel/mixed) regression models" in R (version 4.04).The fixed categorical predictor variables were snake species   . However, B. insularis exposed to terrestrial predatory stimulus had a higher rate of head-hiding behavior than B. jararaca   . In addi species . The onl= 1.000)  Tables. 0.00524) .B. jararaca and B. insularis fled more frequently when confronted with a terrestrial predator stimulus (opossum model) than with an aerial predator (owl model)    (Predatory stimulus type strongly influenced the escape behaviors. Both  0.0003)  Tables.  0.0010) . In the  0.0001) .B. alcatraz did not have its blunt flight frequency influenced by the predator type . However, B. insularis, once again, displayed higher frequencies of blunt flight compared to the other species, with approximately 54% and 70% higher likelihood than B. jararaca and B. alcatraz, respectively   .B. insularis and B. alcatraz showed no difference towards the type of predatory stimulus , but different predatory stimuli influenced the frequency of cocking by B. jararaca    . In addi0.00044)  Tables.All behaviors involving a threat signal, such as body flatten, gape, strike-ready, and strike were influenced by the type of predatory stimulus among the three pitviper species.Bothrops alcatraz had a different behavioral pattern than the other species and did not show body flattening. On the other hand, both B. jararaca and B. insularis displayed more body flattening when faced with the aerial predatory stimulus than the terrestrial one . Moreover, B. insularis flattened its body the most among the three species, mainly to terrestrial predator stimulus  (Flatten body was strongly influenced by the type of predator for two of the studied pitvipers.  0.0115)  Tables.B. insularis. Bothrops jararaca and B. insularis gaped more frequently towards the terrestrial predator stimulus , although the difference was not significant for the latter pitviper . On the other hand, in B. alcatraz gape was elicited more often by the aerial predatory stimulus   .The predator stimulus type strongly influenced gape behavior in all three species, more pronounced in 0.02352) . Further 0.0019)  Tables. B. jararaca and B. insularis, with a more pronounced reaction to the terrestrial predator stimulus than the aerial one. Although only B. insularis showed a significant difference , the p-value for the difference in the reaction of B. jararaca was marginally significant . No difference in this behavior was detected in B. alcatraz . However, this pitviper displayed the least strike-ready reaction towards the terrestrial predator stimulus among the three species  (Strike-ready behavior had a similar pattern for .000915)  Tables.Bothrops jararaca and B. insularis showed the same pattern, with more strikes towards terrestrial than aerial predators stimuli  . Among the three pitviper species, B. alcatraz displayed the highest strike behavior rates . When compared to B. jararaca the difference of 27% chance is not significant .Strike was another behavior strongly influenced by the predatory stimulus type.  0.0001) . In contor rates . The oddB. jararaca and its closest island species reacted diversely to the same predatory stimulus. Our results do not support the hypothesis that the antipredator behavior repertoire of island species was lost due to isolation. However, a quantitative difference in the defensive behaviors displayed by each snake species was evident. Bothrops insularis was the species that displayed the highest frequency of escape behaviors, mainly towards terrestrial predators. Bothrops jararaca displayed more cryptic behaviors among the three pitviper species, the more so towards aerial stimuli. On the other hand, B. alcatraz was the species most prone to bite towards both stimuli types.Our study simulating aerial and terrestrial predators demonstrated that these two contrasting predatory stimuli influenced the frequency of each category of antipredator behavior displayed by the three pitviper species. Furthermore, we showed that the mainland Bothrops species found quantitative differences in behavioral categories that seem to be adaptive reactions to variable predation pressure of aerial predators, as each of the five studied pitvipers dwells in a distinct habitat [A previous study on defensive behavior of five  habitat . However habitat . Adult s habitat . All snaBothrops jararaca was the only species that displayed changes in frequency of cocking depending on the predatory stimulus. Possibly, this influence is due to the greatest diversity of predators sympatric with B. jararaca, and cocking behavior may be important to avoid predation by a predator type that the other pitvipers do not come across [Among the escape behaviors we recorded for the three pitvipers, cocking was the most variable. Escape behaviors are strategies that increase the distance between a snake and a potential predator , and gene across . For thee across ,32.B. insularis that differentiates it most from the other two pitvipers. There is an evident predator low richness on Queimada Grande Island and experiments with plasticine snake replicas showed a significantly lower number of attacks on this island than in continental sites [B. jararaca, which dwells in areas with higher species richness, would tend to be more habituated (with a higher sensitivity threshold for response) to possible stimuli, making it more adaptive to remain motionless and evaluate the predatory stimulus rather than fleeing and thus standing out from the substrate. Consequently, the frequency of other defensive behaviors in the defensive repertoire of B. insularis is reduced compared to those of mainland B. jararaca, making fleeing the main defense strategy of this pit viper on the island. Moreover, the low predator pressure could also explain the uniform yellowish coloration of this island species, apparently out of tune with the environment. The higher frequency of escape behavior displayed by B. insularis may also be explained by is its distinctive coloration. Striped or differently colored snakes have a tendency to escape more frequently than those with spotted coloration, which tend to be more cryptic and stationary as their coloration pattern favors camouflage [B. alcatraz and B. jararaca with their chevroned pattern should display cryptic behaviors more often than B. insularis, but we found that B. alcatraz did not display any cryptic behavior.The frequent flight and blunt escape are the defensive traits of al sites . Our hypmouflage . Based oB. alcatraz. Absence of movements lessens the detection of a snake by aerial predators, as birds perceive their prey visually mostly by movements of the prey [B. jararaca displayed head hiding more often towards aerial predators than terrestrial predators. Birds of prey, such as the snake-specialist Laughing falcon (Herpetotheres cachinnans), kill their snake prey pecking at its head or tearing the head off [We found that all three pitviper species tend to use cryptic behaviors more towards aerial than towards terrestrial predator stimulus, although this was less evident for the prey . Furtherhead off . Snakes head off .B. jararaca and B. insularis displayed higher strike frequency towards aerial stimuli than to terrestrial ones. This tendency of B. insularis is likely influenced by the lack of terrestrial predators on the island, whereas for B. jararaca it could be due to greater pressure from predatory birds than from predatory mammals (29). On the other hand, B. alcatraz displayed more threatening behaviors towards terrestrial predators, possibly due to this pitviper being under greater predation pressure by terrestrial predators than the aerial ones. The large tegu lizard  is abundant on the island [B. alcatraz displayed the highest strike frequency. This species is very small and shares similarities with juvenile B. jararaca, as it has pedomorphic traits [B. alcatraz. Another important factor is that we have no knowledge of the aerial predation frequency, although there are more potential avian predators on Alcatrazes island than on Queimada Grande island, even though far less than on the mainland [B. alcatraz displayed almost no strike-ready behavior towards the predator stimuli. Possibly, higher predation risks on this small pitviper would lead to few strike-ready warnings and more strikes, a hypothesis that merit verification.Threat behavior would warn the predator that its potential prey is dangerous ,8. Withie island ,37, and e island ,39, whicc traits ,40. Snakc traits . Being m mammals . On the c traits –45, whicmainland . A behavBothrops insularis used this behavior most often among the three pitvipers. Gape behavior is used by arboreal and diurnal snake species [B. insularis is more diurnal than the two other species, which are predominantly nocturnal [B. insularis and B. jararaca displayed more gape behavior towards terrestrial predator stimulus than towards the aerial one. Gape is a display that increases the size of the snake on an x-axis, and is possibly more effective for predatory confrontations that occur on the same spatial plane. Both B. jararaca and B. insularis showed very similar patterns of threat behaviors. However, B. alcatraz showed a pattern opposite to the other two species. Tests comparing B. jararaca juveniles and adults could indicate whether this difference is also due to pedomorphic traits. Field observations showed that B. jararaca juveniles and males are more prone to strike than adult female individuals (47). Furthermore, we emphasize that Bothrops jararaca and B. insularis come from the same Bothrops population lineage, whereas B. alcatraz comes from another B. jararaca population lineage [Another threatening behavior is gape.  species  and the  species ,46. In aocturnal ,48. Both lineage .B. jararaca and B. insularis flattened more when confronted with aerial predatory stimuli than with the terrestrial one. Unexpectedly, this behavior was not recorded for B. alcatraz for any predator type stimuli. Our small sample does not allow us to say that B. alcatraz has lost this behavior, but at least it seems to be less frequent in this species. Bothrops insularis hunts during the day and B. jararaca is also observed in daylight with some frequency [B. alcatraz seems to be more secretive during the day, being usually found within a decomposing trunk or under a fallen leaf [Flatten body behavior expands the snake’s body laterally , and wherequency ,18,46. OB. alcatraz (apparently lost). The persistence of antipredator behaviors in the three studied pitvipers is consistent with the multipredator hypothesis. This hypothesis attempts to explain the persistence of antipredator behavior under relaxed selection [Antipredator behavior can be lost after the loss of a key predator, i.e., loss of a trait after relaxed selection , or it celection . Accordielection , the hypCrotalus spp.) recognition by California ground squirrels Spermophilus beecheyi (Rodentia) [Bothrops insularis and B. alcatraz were isolated from the mainland 11,000 years ago [The multipredator hypothesis has been studied in some kangaroo and wallaby species (Diprotodontia), in which the loss of all predators apparently led to a rapid loss of antipredator behavior, while the loss of only one or two predators had a limited effect on the expression of antipredator behavior for the missing species ,50,51. Todentia) ,54 indicears ago –19,55. TDidelphis for 3 million years [B. jararaca and B. insularis, remain the same while preying on rats, despite the insular species being isolated for more than 11,000 years without access to this prey type [Another explanation that may clarify the permanence of antipredator behavior is the "phantom predator past hypothesis" ,56. Thison years . These mon years ,60. Seveon years ,62. Therrey type . TherefoB. alcatraz is the least studied, and this is the first study to investigate some ecological and behavioral aspects of this small species. Further comparative behavioral studies of these three species would be useful, as well as to study other island Bothrops species recently described [Our study is the first to investigate the antipredator behavior of island pitviper species in South America and is instructive for the fields of behavioral ecology and evolution of animal behavior. The two island species are endemic and critically endangered. Among the three species, escribed ,65. ExpeS1 Fig(DOCX)Click here for additional data file.S1 TableB. jararaca) and Predator . Bold p-values indicate p < 0.05.Intercept- Species ((DOCX)Click here for additional data file.S2 TableBold p-values indicate p < 0.05.(DOCX)Click here for additional data file.S3 TableB. jararaca) and Predator . Bold p-values indicate p < 0.05.Intercept- Species ((DOCX)Click here for additional data file.S4 TableBold p-values indicate p < 0.05.(DOCX)Click here for additional data file.S5 TableB. jararaca) and Predator . Bold p-values indicate p < 0.05.Intercept- Species ((DOCX)Click here for additional data file.S6 TableBold p-values indicate p < 0.05.(DOCX)Click here for additional data file.S7 TableB. jararaca) and Predator . Bold p-values indicate p < 0.05.Intercept- Species ((DOCX)Click here for additional data file.S8 TableBold p-values indicate p < 0.05.(DOCX)Click here for additional data file.S9 TableB. jararaca) and Predator . Bold p-values indicate p < 0.05.Intercept- Species ((DOCX)Click here for additional data file.S10 TableBold p-values indicate p < 0.05.(DOCX)Click here for additional data file.S11 TableB. jararaca) and Predator . Bold p-values indicate p < 0.05.Intercept- Species ((DOCX)Click here for additional data file.S12 TableBold p-values indicate p < 0.05.(DOCX)Click here for additional data file.S13 TableB. jararaca) and Predator . Bold p-values indicate p < 0.05.Intercept- Species ((DOCX)Click here for additional data file.S14 TableBold p-values indicate p < 0.05.(DOCX)Click here for additional data file.S15 TableB. jararaca) and Predator . Bold p-values indicate p < 0.05.Intercept- Species ((DOCX)Click here for additional data file.S16 TableBold p-values indicate p < 0.05.(DOCX)Click here for additional data file.S17 TableB. jararaca) and Predator . Bold p-values indicate p < 0.05.Intercept- Species ((DOCX)Click here for additional data file.S18 TableBold p-values indicate p < 0.05.(DOCX)Click here for additional data file.S19 TableFrequencies of snake behaviors for each species and predatory model.(XLSX)Click here for additional data file.


Colletotrichum camelliae, one of the most important phytopathogenic fungi infecting tea plants (Camellia sinensis), causes brown blight disease resulting in significant economic losses in yield of some sensitive cultivated tea varieties. To better understand its phytopathogenic mechanism, the genetic information is worth being resolved.C. camelliae (strain LT-3-1) was sequenced using PacBio RSII sequencing platform, one of the most advanced Three-generation sequencing platforms and assembled. The result showed that the fungal genomic sequence is 67.74 Mb in size (with the N50 contig 5.6 Mb in size) containing 14,849 putative genes, of which about 95.27% were annotated. The data revealed a large class of genomic clusters potentially related to fungal pathogenicity. Based on the Pathogen Host Interactions database, a total of 1698 genes  were annotated, containing 541 genes related to plant cell wall hydrolases which is remarkably higher than those of most species of Colletotrichum and others considered to be hemibiotrophic and necrotrophic fungi. It’s likely that the increase in cell wall-degrading enzymes reflects a crucial adaptive characteristic for infecting tea plants.Here, a high-quality genomic sequence of C. camelliae has a specific host range and unique morphological and biological traits that distinguish it from other species of the genus Colletotrichum, characterization of the fungal genome will improve our understanding of the fungus and its phytopathogenic mechanism as well.Considering that The online version contains supplementary material available at 10.1186/s12864-023-09598-6. Colletotrichum  has significant and varied groups, such as saprophytes and endophytes in addition to plant pathogens that infect a range of plant hosts  O. Kuntze), which originated in southwestern China, is considered to be one of the most economically important crops worldwide. Due to tea products, it is ranked as the second most popular beverage in the world after water and one of the three main non-alcoholic beverages, along with coffee and cocoa. There are numerous diseases that have an impact on tea production. However, C. camelliae Massea causes tea brown blight which is one of the most prevalent and significant diseases affecting tea plants  in a large acervulus (187–290 μm in diameter) with setae, whereas the optimum growth temperature is 25℃ for C. gloeosporioides which produces small conidia [(3–6) × (2-2.5) µm] in a smaller acervulus (80–150 μm in diameter) without setae. Considering that C. camelliae has a specific host range and different morphological and biological traits as compared with C. gloeosporioides, the well-characterized but closely phylogenetically related species, this study provides new insights into genomic information for a better understanding of genome structure, evolution, and biological function of Colletotrichum fungi.As compared with the sequenced and annotated genomes of other a plants –25. On tC. camelliae reveals that it is functionally separated into coding, DRs, TRs, satellite DNAs, and noncoding RNA coding regions. Among them, the putative gene number  intermediates those of Colletotrichum fungi, ranging from 12,006 (for C. graminicola) to 18,324 genes (Colletotrichum lupini). It is worth noting that the genome of C. camelliae has 615 genes fewer than those of C. gloeosporioides and C. fructicola, which have a broad host range, while it has 3470 genes fewer than those of C. lupini with host specificity. It suggests that the fungal host range is not related to their gene numbers. We also noticed that the DRs rank at 5.89% of the whole genome of C. camelliae, which is markedly higher than those of C. gloeosporioides and C. fructicola. A large amount of DRs might contribute to expanding the genomic sizes of C. camelliae. This situation is similar to that of C. orbiculare which has a large genomic size (88.3 Mb) with high DRs (8.3%) and fewer genes  [C. camelliae encodes 98 rRNAs, which is remarkably higher than those in most Colletotrichum species such as both C. gloeosporioides and C. orbiculare that encode 58 rRNAs.Genomic organization analysis of 9 genes) . AdditioC. camelliae contains a large class of genomic clusters potentially related to the fungal pathogenicity, including transporter (540 genes), CAZy (836 genes), secreted proteins (1996 genes), CYP450 (303 genes), secondary metabolic gene clusters (71 genes), and virulence factors (571 genes). A total of 1698 (11.44% of the predicted genes) genes were annotated in the PHI database, and 541 genes were related to plant cell wall hydrolase, which is remarkably higher than those of both C. gloeosporioides (327 genes) and C. orbiculare (346 genes), as well as those of other hemibiotrophic and necrotrophic fungi, including Fusarium spp., Magnaporthe oryzae, Botrytis cinerea, and Sclerotinia sclerotiorum [C. camelliae, and these gene clusters are concluded to be important pathogenetic factors of C. camelliae, which was identified to be responsible for the fungal infection and pathogenicity. Similarly, GHs 6, 7, and 76 families play an important role in the virulence of M. oryzae [C. camelliae genome most likely reflects an important pathogenic factor to infect tea plants.The genome of rotiorum , 26–28. . oryzae , 30. SinC. gloeosporioides, including cutinase, laccase, bZIP transcription factor, calcium-translocating P-type ATPase and other pathogenicity genes [C. camellia and have high identities with those of C. gloeosporioides, suggesting that they are most likely to have the same functions. Additionally, since the ChMK1 gene has been identified to be responsible for the fungal pathogenicity causing anthracnose leaf spot as well as playing an important role in growth, cell wall integrity, and colony melanization [C. camelliae is identical with the ChMK1 gene of C. higginsianum, which is most likely to be one of the key pathogenetic factors of tea brown blight causing the cloudy symptoms. In addition, a large number of genes belong to the major facilitator superfamily (MFS), another class of pathogenic factors in pathogenic fungi [C. camelliae, which might contribute to the delivery of some virulence factors or compounds, sugars, amino acids, vitamins, drugs and other small molecules to plants and environment supporting the process of the fungal infection. Moreover, C. camelliae appears to have an even greater capacity (with 71 gene clusters) for secondary metabolite production, similar to those of C. gloeosporioides (76 gene clusters), fewer than those of C. higginsianum (84 gene clusters), higher than those of both C. orbiculare (54 gene clusters) and C. graminicola (53 gene clusters), and much higher than those identified from other fungal genomes such as M. oryzae (32 gene clusters) and F. graminearum (37 gene clusters). In addition, C. camelliae encodes a large number of cytochrome P450 proteins, accounting for 2.04% of the total genes, remarkably higher than those in C. gloeosporioides (1.06%) and C. orbiculare (0.74%), respectively. These proteins are related to both primary and secondary metabolisms, and the production of mycotoxins, and detoxification [C. camelliae and C. gloeosporioides revealed that both species were placed together in the same cluster based on the phylogenetic analysis. This finding reveals that the synteny between C. camelliae and C. gloeosporioides is remarkably higher than that between C. graminicola and C. higginsianum (35%) and that between C. orbiculare and C. fructicola (40%) . These results suggest that C. camelliae most likely evolved from C. gloeosporioides through the expansion of some specific pathogenicity-related gene clusters, which might be related to its specific host range and pathogenicity differing from other Colletotrichum fungi.With knockout-backup approaches, other pathogenic factors have been identified in ty genes –34. All nization , and theic fungi  were detC. camelliae was sequenced by the Pacbio RSII platform, and its pathogenicity-related gene clusters were annotated. A large class of plant cell wall hydrolases was detected, indicating the number remarkably higher than those of C. gloeosporioides and C. orbiculare, as well other hemibiotrophic and necrotrophic fungi. The expansion of cell wall degrading enzymes most likely reflects an important adaptive trait to infect tea plants. Moreover, other pathogenic factors including cutinase, laccase, bZIP transcription factor, calcium-translocating P-type ATPase, and other pathogenicity genes are also detected in C. camelliae, suggesting that they have high identities with those of C. gloeosporioides, sharing the same functions. Both synteny relationship and phylogenetic analysis revealed that C. camelliae is closely related to C. gloeosporioides, while it contains a large class of diverse genes or other functional clusters separating C. camelliae from other Colletotrichum fungi.A high-quality genome of C. camelliae has a specific host range and unique morphological and biological traits compared to other Colletotrichum fungi. Thus, characterization of the fungal genome will provide us with substantial data and a theoretical basis for studies on the transcriptome, proteome, and metabolome as well as enrich the database of Colletotrichum.Collectively, C. camelliae inducing brown leaf blight was isolated from tea plants cultivated in Hubei Province, China [A phytopathogenic strain LT-3-1 of e, China . Fungal Genomic DNA was extracted using the SDS-based DNA extraction method as previously described . ExtractC. camelliae was predicted with AUGUSTUS v2.7 [C. gloeosporioides (accession No. QFRH00000000) to predict the coding genes, repetitive sequences, and non-coding RNAs. The coding genes were generated using PASA after the predicted data generated with both tools were integrated with EVM. The interspersed repetitive sequences and the tandem repeats were predicted using the RepeatMasker (http://www.repeatmasker.org/) [The genomic information of TUS v2.7 , 42 and er.org/)  and the er.org/) , respecter.org/) , rRNAmmeer.org/) , and BLAer.org/) , respect− 5, minimal alignment length percentage larger than 40%) with the sequences reported in seven databases, including Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG)[Gene function was annotated by BLAST search , Clusteres (KEGG), while tes (KEGG). PathogeC. gloeosporioides (accession No. WVTB00000000), Colletotrichum higginsianum (accession No. MWPZ00000000), Fusarium graminearum (accession No. CABDWO000000000), and Verticillium dahliae (accession No. ABJE00000000), were selected for comparison with the genomic data of C. camelliae. Genomic alignment was performed using the MUMmer and LASTZ tools [The genomes of TZ tools , 52, andTZ tools . The phyTZ tools .Below is the link to the electronic supplementary material.Supplementary Material 1Supplementary Material 2


Plasmodium cynomolgi is a nonhuman primate parasite that causes malaria in humans and is transmitted by the Anopheles mosquito. Macaques, the natural hosts of P. cynomolgi, are widely distributed in Asia, especially in Southeast Asia. Anthropogenic land-use changes and wildlife habitat reduction due to local environmental changes, deforestation, urban expansion, and construction increased the frequency of human-macaque-vector interactions and facilitated the emergence of zoonotic malaria, causing an exponential increase in the infection rates in this area. Although microscopic tools are the gold standard for malaria diagnosis, they have very low sensitivity. Therefore, disease control and prevention require rapid, sensitive and accurate diagnostic tests.P. cynomolgi. Laboratory validation determined the method’s sensitivity and specificity compared to the nested PCR method. The lower limit of detection was 22.14 copies/μl of recombinant plasmid per reaction. The combination method represented 81.82% sensitivity and 94.74% specificity compared to the nested PCR.This study aims to develop a diagnostic method using a recombinase polymerase amplification (RPA) combined with a lateral flow (LF) strip method to specifically diagnose P. cynomolgi.The diagnostic testing developed in this study combines a recombinase polymerase amplification (RPA) and a lateral flow (LF) strip, offering rapid high sensitivity and specificity. Further development of this technique could make it a promising method for detecting  P. cynolmogi is zoonotic protozoa that cause malaria in both macaques and humans. In the past few years, the human infection rate from this parasite has risen throughout Southeast Asia. We have developed molecular testing for diagnosing P. cynolmogi based on a recombinase polymerase amplification (RPA) combined with a lateral flow (LF) strip. The rapid, sensitive, and portable of our testing represents a promising assay for the P. cynomolgi diagnosis method. Anopheles mosquito. It is caused by over 200 species of protozoa from the genus Plasmodium. Four Plasmodium species, Plasmodium ovale , P. vivax, P. malariae, and P. falciparum, are the leading cause of malaria in humans. However, numerous simian Plasmodium species have been reported to infect humans naturally. Among these, P. cynomolgi, a predominant malaria parasite found in Southeast Asia Old-World monkeys, naturally causes zoonotic infections in humans [P. cynomolgi are long-tailed and pig-tailed macaques , commonly found in many Southeast Asian countries [P. cynomolgi human infection in 2011 [Malaria is a hemoparasitic disease in humans and nonhumans transmitted via the infected female n humans ,2. The dountries . After t in 2011 . Land-us in 2011 .P. cynomolgi are asymptomatic, highly sensitive diagnostics are required for disease control and prevention in malaria-endemic areas [Anopheles mosquito and a significant source of gametocytes for local mosquito vectors, increasing the rate of malaria transmission. As the morphological and biological characteristics of P. cynomolgi are similar to those of P. vivax, misdiagnosis is highly likely with microscopic examination, the gold standard for malaria diagnosis [As nearly all patients infected with ic areas ,7. Parasiagnosis –11. Moreiagnosis ,13. Receiagnosis ,15.RPA is an isothermal nucleic acid amplification method ideal for limited resource areas due to its low-resource requirements. This system requires a combination of three enzymes: polymerases, single-stranded DNA-binding proteins, and recombinases to amplify the target DNA sequences at a constant temperature . The DNAP. cynomolgi. Specific primers were designed using the sexual 18sRNA gene as a template or amplification and the LF strip for the readout. We evaluated the specificity and sensitivity of this assay by testing it on macaque blood samples.In this study, we developed a method combining RPA and LFD to detect This research was approved by the Department of National Parks, Wildlife and Plant Conservation, Thailand, for study in a protected area, collection of the blood samples and released of wild macaques (Permit Number: 0909.204/14187). All procedures were carried out in accordance with the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, U.S.A., and were approved by the Animal use and care committee of Kasetsart University Research and Development Institute, Kasetsart University, Thailand (ID: ACKU59-SCI-011).P. cynomolgi (GenBank Acc. No. AB287288). The database was set as RefSeq representative genomes. The product and primer sizes were set as 120–300 bp and 30–35 nt, respectively. Other parameters were set as default. The primers with target-specific labels were tagged with Biotin and Fluorescein-5-isothiocyanate (FITC)   and syntP. falciparum, P. vivax, P. malariae, P. ovale, and P. knowlesi, used as specificity controls, were provided by Dr. Porntip Chavalitshewinkoon-Petmitr of the Department of Protozoology, Faculty of Tropical Medicine, Mahidol University.The genomic DNA (gDNA) from human malarial parasites, M. arctoides at the Pa La U waterfall, Huahin district, Prachuap Kiri Khan Province, wild M. leonina at the Khao Yai National Park, Nakornratchasima Province, and wild M. fascicularis at Chang Island, Mu Ko Ranong National Park, Ranong Province. The gDNA was extracted directly from samples using GeneJET Whole Blood Genomic DNA Purification Mini Kit  and stored at −20°C until use. In our previous study, all gDNA samples showed negative results for dengue virus (DENV), Zika virus (ZIKV), Chikungunya virus (CHIKV), Leptospira spp., and Burkholderia pseudomallei using molecular detection [Plasmodium species, a nested PCR approach was employed, following the previously described method [Plasmodium-positive samples underwent further examination using species-specific primers to identify P. knowlesi, P. coatneyi, P. cynomolgi, P. inui, and P. fieldi, as outlined in a previous study [P. cynomolgi RPA-LFD assay.Ninety-three blood samples were collected from wild macaques by Dr. Wirasak Fungfuang in three areas of Thailand from July 2017–July 2019, including samples from wild etection . To analP. cynomolgi samples and P. inui macaque blood samples. The amplification reaction mixture contained 1× Phusion HF Buffer, 0.2 mM deoxynucleoside triphosphates (dNTPs), and 0.5 μM of each Plasmodium spp.  primer [DNA was amplified using the gDNA of ) primer . ReactioRPA was performed in a 50 μL reaction mixture using the TwistAmp basic kit  according to the manufacturer’s instructions. All the reaction components , except DNA templates and 14 mM magnesium acetate, were prepared as a master mix distributed into a 0.2 mL Eppendorf tube strip containing a dried enzyme pellet. Then, after adding 5 μL of each DNA template to the mixture aliquots, 2.5 μL of magnesium acetate was pipetted into the tube lids and centrifuged into the reaction mix using a benchtop centrifuge. The reaction tubes were immediately incubated at 39°C for 20 min on a heating block.The RPA-amplified products were visualized based on the calorimetric signals on the nucleic acid LFD strips, which were fabricated by Kestrel BioSciences Thailand Co. Ltd. Briefly, 10 μL of RPA-amplified products were mixed with 90 μL of running buffer  in a fresh Eppendorf tube. Subsequently, the LFD strip was immersed into the mixture and incubated for 2 min at room temperature. The solution migrated along the LFD strip through the conjugate pad coated with gold nanoparticles labeled with anti-FITC. The complexes were captured by the antibiotin embedded on the test line and goat antimouse IgG on the control line. After 2 min, the strip with one visible line in the control area was considered negative, while the strip with two visible lines in both control and test areas was considered positive.Plasmodium species  and the P. cynomolgi and P. inui plasmid DNA were used as templates for detection. A DNA-free template was included as a negative control. All amplicons were evaluated using AGE and LFD, and the results were compared.To evaluate the specificity of the assay, gDNA from patients infected with five human 6–22.14 × 10−1 copies/μL) of P. cynomolgi plasmid DNA as templates for RPA under the same conditions specified before. All RPA-amplified products were screened by AGE and LFD, and the results were compared. Meanwhile, quantitative PCR (qPCR) with the same primers and templates were performed as the control for RPA.To set the sensitivity and the lower detection limit of the RPA-LFD assay, we used ten-fold serial dilutions , 0.5 μl of each primer (10 μM), 1 μl of plasmid DNA and 8 μl of nuclease-free water. The reactions were conducted in triplicate on CFX96 Touch Real-Time PCR Detection System . The thermal cycling program included an initial denaturation step at 95°C for 60 seconds, followed by 45 cycles of denaturation at 95°C for 15 seconds and annealing/extension at 60°C for 30 seconds. To serve as a negative control, DNase-free water was used as a non-template control in all reactions. A sample was considered positive if the qPCR threshold cycle (CT) value of the plasmid DNA crossed the zero baseline before the 40th cycle. After the 45Plasmodium-positive blood samples from wild macaques determined by nested PCR were used to assess the performance of the RPA-LFD assay. Two hundred μL of the blood samples, kept in sterile containers, was immediately used to extract gDNA using the GeneJET Whole Blood Genomic DNA Purification Mini Kit . RPA was performed, as previously described, using 5 μL of DNA for each reaction. The RPA-amplified products were screened by LFD, and the results were compared with the nested PCR using species-specific primers to detect P. cynomolgi as described previously [P. cynomolgi) and a negative control (DNA-free template) were included to rule out false positives or false negatives .P. cynomolgi and other Plasmodium species. The assay showed specificity to P. cynomolgi DNA without any cross-reactivity with other Plasmodium species   , which wilution)  and qPCRilution) . The ideilution) .P. cynomolgi using the nested PCR method, while the RPA-LFD assay detected nine positive samples (30%) with two false negatives, making its sensitivity at 81.82%. Of the 19 samples indicated as negative by the nested PCR method, one tested as false positive by RPA-LFD assay, yielding a specificity of 94.74%. The Cohen’s kappa index of the RPA-LFD assay was 0.78, significantly consistent with the reference method  and pig-tailed macaques (M. nemestrina), the natural definitive hosts, are found [P. cynomolgi-infected patients and animals is critical to prevent and manage this disease in the endemic regions [Close human–animal-vector encounters because of land-use changes increase the epidemiological risk of developing zoonotic infectious diseases, including malaria –26. The re found . The accP. vivax and P. cynomolgi [Light microscopy-based techniques are the gold standard for malaria diagnosis. However, their accuracy and sensitivity depend on the examiner’s skill and the sample’s parasitemia levels. . Moreoveynomolgi –11. Althynomolgi , 15. RPAynomolgi . This teP. cynomolgi RPA-LFD assay. We established the RPA-LFD system and evaluated its diagnostic validity using P. cynomolgi noninfectious and infectious blood samples. The sexual 18sRNA gene RPA primers were designed following the RPA guidelines: 30–35 nucleotides in length, GC content between 40–51.4%, low potential for homo/heterodimers specific, and yield a 144 bp DNA product. The assay demonstrated high sensitivity (81.82%) and specificity (94.74%) compared to the nested PCR, which was used as the reference test. This assay could detect 22.14 copies/μl of recombinant plasmid per reaction. Our P. cynomolgi RPA-LFD could detect 3–6 parasites/μl, relying on the copy number of 18sRNA gene per parasite varying from four to eight in Plasmodium species. [18sRNA gene primers showed a lack of cross-reactivity with other Plasmodium species. These results reveal that the sensitivity and specificity of this system are ideal for P. cynomolgi diagnosis testing.To our knowledge, this is the first report showing the successful development of a species.  The testspecies. . EvaluatThe complex and time-consuming sample preparation step for reducing DNA polymerase inhibitors is the limitation of this technique. The result should be interpreted immediately within 2 minutes to avoid false positive results due to contamination. Future work could design a suitable sample extraction and minimize the chance of contamination.P. cynomolgi in simian blood samples. This method is a rapid, portable, and accurate tool for detecting P. cynomolgi infections. The result of this assay can be interpreted by the naked eye. Comparison of the results of this assay with that of nested PCR showed its high sensitivity and specificity. Based on these findings, this RPA-LFD assay is a potential diagnostic test for detecting P. cynomolgi.In conclusion, we developed the RPA-LFD system for the specific diagnosis of


Agastache genus is part of the Lamiaceae family and is native to North America, while one species, Agastache rugosa (A. rugosa), is native to East Asia. A review on the phytochemistry and bioactivity of Agastache genus was last performed in 2014. Since then, a lot of progress has been made on the characterization of the phytochemical and pharmacological profiles of Agastache species. Thus, the purpose of this paper is to present a summary of the findings on the phytochemistry and biological effects of several Agastache species, including both extracts and essential oil characterization. We performed a comprehensive search using PubMed and Scopus databases, following PRISMA criteria regarding the study selection process. The available data is focused mainly on the description of the chemical composition and bioactivity of A. rugosa, with fewer reports referring to Agastache mexicana (A. mexicana) and Agastache foeniculum (A. foeniculum). Agastache species are characterized by the dominance of flavonoids and phenolic acids, as well as volatile compounds, particularly phenylpropanoids and monoterpenes. Moreover, a series of pharmacological effects, including antioxidant, cytotoxic, antimicrobial, anti-atherosclerotic, and cardioprotective properties, have been reported for species from the Agastache genus.The  Agastache Clayton ex Gronov is a small genus of the Lamiaceae family that consists of 22 species known under the common name ‘giant hyssop’. Agastache species (sp.) are native to North America, with one species originating from East Asia. Agastache sp. is used as a medicinal and ornamental plant and is known in traditional medicine as a natural remedy against pain, hypertension, and gastrointestinal diseases . On’ leaves .A. ‘Arcado Pink’ leaves (92.2%) -β-D-glucopyranoside, salicylic acid-2-O-[6′-O-(E)-feruloyl]-β-D-glucopyranoside, chavicol-1-O--β-D-glucopyranoside, and other derivatives [In fact, several active compounds isolated from ivatives .A. rugosa is a component of GHJGS (gwakhyangjeonggi-san), an herbal plant mixture used in traditional Korean medicine. It has anti-inflammatory and antioxidant effects in vitro in RAW 264.7 macrophages. In lipopolysaccharide (LPS)-stimulated macrophages, GHJGS significantly reduced the production of proinflammatory cytokines such as TNF-α (tumor necrosis factor-α), IL-6 (interleukin-6), and prostaglandin E2 [andin E2 .A. rugosa essential oil exhibited in vivo anti-inflammatory activity in an adjuvant-induced arthritis rat model, reducing the level of pro-inflammatory markers such as TNF-α, IL-1, and IL-6 [A. rugosa contains a variety of bioactive chemicals with anti-inflammatory potential and could be beneficial for the treatment of pathologies associated with an inflammatory process.Furthermore, and IL-6 . These fA. rugosa ethanolic extract and tilianin exhibited anti-lipogenic and anti-adipogenic effects on 3T3-L1 adipocytes. Tilianin caused a dosage-dependent decrease in lipid accumulation, whereas A. rugosa extract consistently decreased lipid accumulation regardless of dose, demonstrating that both tilianin and A. rugosa extract have anti-obesity effects in vitro. The researchers also found significant decreases in the levels of factors and proteins responsible for activating the process of adipogenesis, such as PPAR, C/EBP, SREBP1, and others [A. rugosa ethanolic extract decreased lipid accumulation by 20.4% in 3T3-L1 adipocytes [d others . Anotheripocytes .A. rugosa aqueous extract exerted an in vitro anti-atherosclerotic effect by inhibiting the proliferation of aortic vascular smooth muscle cells (VSMC). Also, by increasing the levels of the cyclin-dependent kinase inhibitors p21WAF1/CIP1 and p27KIP1, A. rugosa extract produced cell cycle arrest at the G0/G1 transition phase. Although both rosmarinic acid and caffeic acid were found in high concentrations in A. rugosa extract, only rosmarinic acid showed specific action on the inhibition of DNA synthesis and cell cycle progression during VSMC proliferation [Lee et al. showed that feration .A. rugosa extract was also confirmed by in vivo studies. Thus, the administration of A. rugosa methanolic extract lowered plasma cholesterol levels in mice. The study found that A. rugosa extract therapy reduced the accumulation of macrophages in atherosclerotic lesions. To better understand the mechanism of action, the study also investigated the effects of tilianin on cultured human umbilical vein endothelial cells (HUVECs). The results showed that tilianin reduces TNF-K-induced VCAM1 expression by 74% in HUVECs, an early phase in atherosclerosis pathogenesis. Also, tilianin administration significantly reduced the activity of NF-kB, another important factor in the atherosclerosis process [The in vitro anti-atherogenic effect of  process .A. rugosa volatile oil administration had a hypocholesterolemic effect in vivo, significantly decreasing LDL and triglyceride levels in mice. The essential oil was also effective in reducing the oxidation of LDL and up-regulating the expression of LDL receptors. Also, the administration of A. rugosa volatile oil down-regulated the expression of two transcription factors in the intrahepatic metabolism of cholesterol, SREBF-1 and SREBF-2, named sterol regulatory element binding factor [The study performed by Jun et al. concluded that g factor .A. mexicana is traditionally used in herbal Mexican medicine for the treatment of hypertension [A. mexicana extracts and their main components using in vitro and in vivo models.rtension ,83. GiveA. mexicana exhibited vasorelaxant and antihypertensive effects when administered to male Wistar rats. Tilianin produced relaxation in concentration- and endothelium-dependent and independent manners in rat thoracic aortic rings. The mechanisms of action involved were NO/cGMP pathway modulation and the opening of K channels [A. mexicana callus extract has vasorelaxant activity on the aortic ring. Interestingly, the callus extract with the lowest tilianin level had the best vasorelaxant effect when compared with the wild plant. This could be due to the presence of various secondary metabolites that are most likely only formed in in vitro cultures [50 (53.51 mg/kg) for antihypertensive effect and LD50 (6624 mg/kg) were determined [Tilianin isolated from channels . Furtherchannels . Anothercultures . Regarditermined .A. mexicana. The dichloromethane extract produced a considerable vasorelaxant effect ex vivo in a rat aorta ring model. Moreover, the isolated ursolic acid showed antihypertensive action in hypertensive rats. Thus, the available studies support the empirical use of A. mexicana as an antihypertensive drug in Mexican traditional medicine [Some authors suggested that ursolic acid could also be responsible for the antihypertensive activity of medicine .A. rugosa extract also displayed procoagulant activity, while the isolated main components of A. rugosa extract, acacetin and tilianin, had significant anticoagulant activity in vitro [Agastache sp. extracts in the management of bleeding disorders.Noteworthy, Cao et al. (2017) showed that in vitro . FurtherA. rugosa extract, showed antidiabetic potential in vitro. Acacetin increased glucose absorption in cultured muscle cells via an insulin-independent pathway, GLUT4 translocation. Moreover, acacetin-induced GLUT4 translocation was mediated via intracellular reactive oxygen species (ROS) generation [Acacetin , one of the main constituents of neration .A. mexicana. Tilianin administration (60 mg/kg body weight) for 10 days induced a significant antihyperglycemic effect at day 6. The administration of tilianin decreased IL-1b and IL-18 expression in adipose tissue, two pro-inflammatory mediators that can affect insulin sensitivity [Another study aimed to determine the antidiabetic effect of tilianin (Acacetin 7-glucoside) in diabetic rats. Tilianin was isolated from the methanolic extract of sitivity .Agastache sp. extracts could be investigated in terms of antidiabetic potential, corroborating the presence of tilianin and acacetin, two active compounds with antidiabetic potential.Thus, A. rugosa, has a hepatoprotective effect in vivo against fulminant hepatic failure caused by D-galactosamine and lipopolysaccharide in mice. Acacetin pretreatment significantly increased the survival rate, reducing the level of seric aminotransferase, a biomarker for hepatocellular injury. Acacetin pretreatment also reduced the level of the pro-inflammatory tumor necrosis factor-α (TNF-α) and increased the level of IL-6, a pleiotropic cytokine involved in hepatic homeostasis. The study concluded that acacetin inhibits D-galactosamine/lipopolysaccharide-induced hepatocellular damage, which could be relevant in future therapeutic applications for treating liver illnesses [A. rugosa extract in rats, acacetin was found in high concentrations in urine and feces, thus representing the main active metabolite [A. rugosa, tilianin, and rosmarinic acid were found in very low concentrations in urine and feces, indicating that these compounds are extensively converted to acacetin in rats. The hydrolysis of acacetin-O-glucoside (tilianin) to acacetin appears to play a significant role in the metabolism of acacetin-derivative secondary metabolites in A. rugosa [Agastache sp. extracts warrant further investigation of the hepatoprotective potential in vivo.Cho et al. found that acacetin, isolated from aerial parts of llnesses . It is itabolite . Apart f. rugosa . Corrobo. rugosa , AgastacA. rugosa essential oil, has hepatoprotective potential in acute hepatic failure. The intraperitoneal administration of β-caryophyllene prior to the induction of hepatic failure led to a reduction in the level of serum aminotransferase, tumor necrosis factor-α, and interleukin 6 [Using the same animal model, Cho et al. demonstrated that β-caryophyllene, a sesquiterpene compound from leukin 6 .Agastache sp. One study found that thymoquinone has a hepatoprotective effect on acetaminophen-induced acute liver injury. Administration of thymoquinone in rats reduced the levels of ALT and AST and also restored the levels of GSH, which has a significant role in the detoxification of acetaminophen. The authors also concluded that the hepatoprotective effect of thymoquinone is mediated through suppression of the MAPK signaling pathway [Thymoquinone is an active aromatic compound found in  pathway .Agastache sp. extracts and volatile oils against acute liver injury.Taken together, these studies promote the future investigation of the hepatoprotective potential of A. rugosa extract showed anti-osteoporotic potential both in vitro and in vivo. A. rugosa ethanolic extract stimulated the differentiation of MC3T3-E1 pre-osteoblasts [A. rugosa extract reduced the expression of NO-synthetase and inhibited NO production in ROS 17/2.8 osteoblasts. Excess NO production has been associated with osteoblast apoptosis and bone loss. Thus, A. rugosa leaf extract prevents NO-mediated apoptosis in vitro and may be beneficial in the prevention of osteoporosis and other inflammatory bone conditions [eoblasts . Also, Anditions .A. rugosa ethanolic extract reversed postmenopausal osteoporosis induced in an animal model by ovariectomy [Deferribacteres, Proteobacteria, and Bacteroidetes and decreasing the number of Firmicutes bacteria [A. rugosa aqueous extract reduced ovariectomy-induced bone loss in mice by suppressing RANK ligand—a marker of the osteoclast differentiation. The extract inhibited the expression of cellular oncogene fos (c-Fos) and nuclear factor NFATc1, key factors in osteoclastogenic transcription. The results support the possibility of using the aqueous extract of A. rugosa in the prevention and treatment of post-menopausal osteoporosis by suppressing osteoclastogenesis [In vivo, riectomy . It has bacteria . Similarogenesis .A. mexicana has been traditionally used in Mexican folk medicine for the treatment of pain. Decoction of A. mexicana aerial parts is empirically indicated for various pain disorders such as stomachache, colic, and intestinal pain, as well as to enhance digestion [A. mexicana extracts and volatile oils in the treatment of nociception.igestion . HoweverA. mexicana ssp. mexicana and ssp. xolocotziana volatile oils showed anti-nociceptive activity in vivo, reducing abdominal pain in an animal model. The effect of the volatile oils from both subspecies could be explained by the presence of limonene and pulegone. Limonene demonstrated the strongest anti-nociceptive action in the study. It also significantly reduced intestinal inflammatory cytokines in a drug-induced ulcerative colitis model, providing gastroprotection against gastric injury. The results of the study support the traditional use of A. mexicana in the treatment of gastrointestinal conditions, showing the therapeutic potential of monoterpenes in the treatment of ulcers, colitis, and abdominal pain [Agastache sp. essential oils [nal pain . Anotherial oils .A. mexicana extracts in hexane, ethyl acetate, and methanol inhibited the abdominal constriction generated by 1% acetic acid injection. Hexane and ethyl acetate extracts showed the highest antinociceptive efficacy. Although methanol extract was obtained in major yield, its antinociceptive effect was inferior to that of hexane and ethyl acetate extracts, suggesting less participation of the polar compounds in this type of nociception [iception .A. mexicana extracts could be partially explained by the presence of ursolic acid, a triterpene found in concentrations of 130.7 mg/g (ethyl acetate extract) and 20.3 mg/g (methanolic extract). Ursolic acid exhibited significant antinociceptive potential in different animal models, including the formalin test and the writhing test. Its efficacy was demonstrated in both neurogenic and inflammatory nociception, while its mechanism of action could involve the presence of cGMP and TRPV1 receptors [A. mexicana extracts , along with acacetin and ursolic acid, showed anti-nociceptive activity using the writhing test in mice [One study indicated that the antinociceptive effect of  in mice .A. mexicana as a pain reliever is sustained by in vivo studies that confirmed its antinociceptive effect in different types of nociception models.Thus, the traditional use of A. mexicana ssp. xolocotziana extract displayed spasmolytic properties on guinea pig ileum pre-contracted with KCl, while A. mexicana ssp. mexicana had a spasmogenic effect. Both subspecies are used in folk medicine for abdominal pain and other gastrointestinal diseases, but when administered in vivo, only ssp. xolocotziana has been shown to exhibit a spasmolytic effect. The mechanism of action could involve nicotinic receptor agonism and blockade of calcium channels. Qualitative and quantitative variances in the composition of each subspecies could be accountable for their distinct pharmacological effects [ effects .A. mexicana extract, may be responsible for the spasmolytic effect of A. mexicana [Using the same animal model and inducing ileum contractions with KCl, another study found that acacetin and ursolic acid, two active components from mexicana .A. rugosa 70% ethanolic extract (100 mg/kg/day) showed gastroprotective effects in an acute gastritis mouse model induced by administration of HCl/EtOH (clorhidric acid/ethanol). The gastroprotective effect was explained by the increased secretion of gastric mucus and the inhibition of the infiltration of inflammatory cells. At the molecular level, A. rugosa affected some signaling pathways involved in the cellular inflammatory response (PI3K/AKT and NF-kB). These results confirm the use of Korean mint as a medicinal plant in the treatment of digestive disorders such as dyspepsia, nausea, and vomiting [vomiting .A. mexicana is traditionally used as a natural remedy for the treatment of anxiety disorders [A. mexicana extract and tilianin, one of the major bioactive compounds of the extract. The results showed that methanolic crude extract  and tilianin  had anxiolytic effects when administered to mice. The mechanism of action could involve GABAA receptors, considering that the anxiolytic effect did not occur in the presence of flumazenil. These results demonstrated that tilianin is one of the main components of the methanolic extract responsible for the anxiolytic activity of A. mexicana and requires further research involving clinical studies [isorders . Thus, r studies .A. mexicana ssp. mexicana and xolocotziana exhibited anxiolytic-like activities at low doses (0.1 to 10.0 mg/kg), but at larger doses (above 100 mg/kg), they showed sedative activity that impacted general activity and motor coordination. The aqueous extract of ssp. mexicana appears to be more potent than that of ssp. xolocotziana, as it causes anxiolytic-like effects and sedative effects at lower doses [A. mexicana water extract are more consistent with an anxiogenic-like property than an anxiolytic-like property [A. mexicana ssp. mexicana and ssp. xolocotziana in traditional medicine as anxiolytic, sedative, and tranquilizing agents.Other researchers found that aqueous extracts of er doses . Interesproperty , but theA. rugosa essential oil produced significant changes in EEG activity, stimulating brain wave activity and increasing spectral edge frequency by 50% of alpha spectra values. Also, exposure to volatile oil reduced the absolute and relative theta power spectra. These EEG changes suggest that A. rugosa essential oil can be used in aromatherapy for positive psychophysiological conditions [Furthermore, a study showed that exposure to nditions .Agastache sp. extracts and volatile oils, and they demonstrated good antioxidant activity against different free radicals.Several studies evaluated the in vitro antioxidant effects of A. rugosa extract had DPPH  and ABTS ) radical scavenging activity [A. rugosa also displayed radical scavenging potential against DPPH, with an antioxidant activity of 8.79 µg/mL [A. rugosa leaf extract manifested its antioxidant activity by increasing the expression of the HO-1 (heme oxygenase 1) protein, an enzyme that protects against oxidative stress. The study showed that A. rugosa extract protects RAW264.7 macrophages against H2O2-induced oxidative stress and could be useful for the treatment of pathologies associated with cellular oxidative stress [A. rugosa, increased HO-1 expression and prevented the formation of reactive oxygen species in RAW 264.7 cells [Studies showed that activity ,81. The 79 µg/mL . Regardie stress . Further.7 cells .A. rugosa honey presents ferric-reducing antioxidant power of 3.61 ± 0.02 µmol TE/g  [50 = 0.77 ± 0.02 mg/mL), with a significant positive correlation between the total antioxidant capacity and the total phenol content of the tested bee product [valents)  and DPPH product .A. foeniculum essential oil and extracts exhibited good antioxidant activity. The essential oil was effective against DPPH radicals, inhibiting 77.88% of DPPH radicals at a concentration of 10 μL/mL [A. foeniculum extract showed DPPH radical scavenging activity, cupric-reducing antioxidant capacity, and ABTS radical scavenging activity [10 μL/mL . A. foenactivity ,27,37. TA. aurantica flowers extract is comparable to previously published values from different Agastache flowers [The DPPH scavenging activity of  flowers .p ≤ 0.05) as well as the total concentration of phenolic compounds . There was also a strong association between antioxidant activity (DPPH assay) and flavonoid concentration . A stronger link was also shown between antioxidant activity, as determined by the ABTS assay, and the total amounts of hydroxycinnamic acids and phenolic compounds [Plant phytochemical composition influences antioxidant activity. Typically, phenolic chemicals contribute significantly to antioxidant activity. The antioxidant activity, as evaluated by the DPPH assay, was shown to be highly correlated with the level of hydroxycinnamic acids . Acacetin, one of the major compounds isolated from the extract, also exhibited xanthine oxidase inhibitory action (IC50 = 0.58 M), which is lower compared with allopurinol, routinely used as a xanthine oxidase inhibitor. Thus, A. rugosa extract could act as a functional component and natural medicine for the treatment of hyperuricemia and gout [and gout .O, two active compounds isolated from the methanolic extract of A. rugosa leaves, had inhibitory activity on MAO-A and MAO-B, two enzymes implicated in the degradation of amines, including dopamine. Thus, acacetin and its derivative could be further tested for their potential as MAO inhibitors, with application in the treatment of some neurological disorders, including Parkinson’s disease [Lee et al. showed that acacetin and its derivative, acacetin 7- disease .A. foeniculum extract inhibited α-amylase and α-glucosidase, enzymes related to carbohydrate digestion. Amylase inhibitory activity was highly correlated with hydroxycinnamic acid concentration and moderately correlated with flavonoid concentration [A. foeniculum extract is needed.ntration . The inhA. rugosa flowers and leaves was found to have moderate to high antimicrobial activity. A strong in vitro inhibitory effect against Staphylococcus aureus (S. aureus—MIC 21 µg/mL) and Escherichia coli (E. coli—MIC 21 µg/mL) was noticed in the case of the essential oil from flowers, while the essential oil from leaves strongly inhibited E. coli (MIC 9.4 µg/mL). Both products were able to inhibit biofilm formation [S. aureus and E. coli was also confirmed by another study, which in addition reported antimicrobial activity against two other Gram-negative bacteria . The antibacterial capacity could be attributed to estragole, the major compound of essential oil, but also to other trace volatile compounds such as terpenoids, including dl-limonene, linalool, β-caryophyllene, and germacrene-D [E. coli was also susceptible to the action of A. rugosa methanolic extract, together with Aeromonas hydrophila, Aeromonas salmonicida, Staphylococcus haemolyticus, and Cronobacter sakazakii [The essential oil obtained from ormation . The antacrene-D . E. coliakazakii .A. foeniculum essential oil had a moderate effect on some Gram-positive  and Gram-negative bacteria , with MICs ranging from 0.157 to 2.50 µL/mL. [A. foeniculum aqueous extract showed in vitro properties against several bacterial strains  [Antimicrobial activity evaluation showed that 0 µL/mL. . Regardiumoniae) .A. rugosa essential oil fraction and estragole exhibited antifungal activity against Aspergillus and Candida species (MICs ranging from 2.5 and 10 mg/mL). Also, essential oil and estragole displayed synergism with ketoconazole against Blastoschizomyces capitatus, a rare fungus that causes serious infections in immunocompromised patients [A. rugosa and ketoconazole against Candida species . However, this synergism was not confirmed for the combination of estragole and amphotericin B [C. albicans was also susceptible in vitro to the action of A. foeniculum essential oil [Shin et al. found that patients . These rericin B . C. albitial oil .A. rugosa against Trichophyton species. Synergistic effects were noticed between A. rugosa essential oil and estragole with ketoconazole against the studied Trichophyton species, with fractional inhibitory concentration indices that ranged from 0.05 to 0.27 [Another study evaluated the antifungal capacity of essential oil from  to 0.27 .A. mexicana ssp. xolocotziana volatile oil had antifungal activity against a panel of fungal strains, including Aspergillus sp., Penicillium sp., and Eupenicillum sp., with MICs ranging from 0.3 to 30 g/mL [Juárez et al. showed that  30 g/mL .Agastache honey in comparison to other commercial honeys. Agastache honey showed superior antifungal activity against dermatophytes (T. mentagrophytes and T. rubrum) and C. albicans. Agastache honey contained several volatile compounds with antifungal activity, whereas other honeys contained few antifungal compounds. Phenol-2,4-bis, estragole, nonanoic acid, and ethyl ester were found as chemical markers in Agastache honey. Agastache honey products could be developed for topical use against fungal skin infections [Other researchers investigated the antifungal properties of fections .A. foeniculum volatile oil displayed strong insecticidal activity against Tribolium castaneum and Rhyzopertha dominica [A. foeniculum was found to be effective as a natural pesticide against Oryzaephilus surinamensis and Lasioderma serricorne [A. foeniculum volatile oil can be used to manage and reduce the negative impacts of modern insecticides.dominica . Moreoverricorne . These fA. rugosa extract against photoaging induced by UV-B radiation in HaCaT keratinocytes. The results of the studies showed that A. rugosa extract inhibited the activity of elastase and hyaluronidase, enzymes associated with the skin aging process [+ [A. rugosa extract reduced the level of NO, an important intracellular pro-inflammatory mediator [Some in vitro studies investigated the protective potential of  process . Also, t process , and ABTocess [+  free radmediator ,108, andmediator ,108,109,A. rugosa extract’s anti-UV-B protective properties are augmented when using probiotic fermentation [entation ,109. Botentation ,109.A. rugosa extract in an animal model. The extract was given orally to hairless mice at doses of 100 or 250 mg/kg/day for 12 weeks, along with UVB exposure. UVB-induced wrinkle formation, epidermal thickening, erythema, and hyperpigmentation have been histologically improved. The extract also restored skin moisture by improving skin hydration and transepidermal water loss, increasing hyaluronic acid levels, and significantly increasing collagen density [A. rugosa extract could be used as a preventive and therapeutic agent against photoaging and support the extract’s cosmetic value.Later, other researchers investigated the anti-photoaging effect of  density . Thus, tGlycyrrhiza glabra, Agastache rugosa, and tilianin significantly reduced neutrophil levels in the lungs and bronchoalveolar lavage fluid in an animal model of chronic obstructive pulmonary disease, thus showing potential in the management of this severe pathology [A herbal mixture containing athology .A. mexicana ssp. mexicana volatile oil produced relaxation of the trachea smooth muscle, attenuating carbachol- and histamine-induced contractions. The study also suggested that the mechanism of action involved calcium channel blockade and that estragole and D-limonene could be responsible for the relaxant effect of the essential oil [A. mexicana extract had a relaxant effect on rat-isolated trachea contracted with carbachol [A. mexicana extract and volatile oil could be further investigated in terms of their anti-asthmatic potential.tial oil . Also, Aarbachol . Thus, AAgastache genus, a small genus of aromatic plants from the Lamiaceae family. Studies on Agastache sp. have seen significant progress in the last decade. Studies evaluating the chemical composition and therapeutic potential pointed out that Agastache sp. represent valuable natural sources of bioactive compounds. Agastache sp. extracts contain mainly flavonoids  and phenolic acids . Tilianin (acacetin-7-O-glucoside) and its aglycone, acacetin, were identified as major compounds in the hydroalcoholic extracts of Agastache sp. Many of the therapeutic effects of the extracts have been attributed to the presence of these secondary metabolites. Some of the most important pharmacological effects of the extracts include anti-adipogenic, anti-atherosclerotic, cardioprotective, anti-diabetic, anti-osteoporotic, and hepatoprotective properties. The anti-atherosclerotic and cardioprotective effects of the extracts have been explained by the presence of tilianin, a glycosidic flavonoid with therapeutic potential in the cardiovascular field. Tilianin exhibited anti-lipogenic, anti-atherosclerotic, antihypertensive, and anticoagulant activity. Although these are promising results, most of the studies used in vitro models. To better understand the therapeutic potential of Agastache sp. extracts and their main components, in vivo studies are needed. Subsequently, standardization of the extracts of tilianin and providing a specific concentration of the active compound could enhance therapeutic efficacy and guarantee the quality of the extracts. Another option would be the structural modeling of tilianin with the development of structural analogues and the investigation of structure-activity relationships.In this paper, we have performed a comprehensive review of the literature on the phytochemical profile and pharmacological properties of several species from the Agastache sp. contain mainly phenylpropanoids (estragole and methyl eugenol) and terpenoids . Estragole is the major volatile compound reported in most of the studies; however, there are many chemotypes within the same species. Some chemotypes are characterized by the dominance of phenylpropanoids, while others are characterized by the dominance of terpenes. Thus, depending on the chemical composition, Agastache sp. volatile oils have demonstrated different pharmacological effects: cytotoxic, antimicrobial, anti-nociceptive, anti-inflammatory, and anti-atherosclerotic. Estragole also exhibited cytotoxic, antibacterial, and antifungal effects. Future research directions include testing the cytotoxic effect of volatile oils and estragole on other cancerous cell lines  and investigating the cytotoxic potential of other Agastache species. The cytotoxic effect has been determined so far only for A. rugosa and A. foeniculum.The volatile oils obtained from Agastache sp. volatile oils. Also, the dosage and mode of administration of the volatile oils are not always clearly mentioned. The extraction of the active volatile compounds from the essential oils and the investigation of their pharmacological effects may considerably enhance the therapeutic potential of the volatile oils.In the few studies conducted on animals, the authors do not report the adverse effects or toxicity of Agastache species has seen a lot of progress in recent years, there are still many scientific gaps that require investigation. Of the 22 species of the genus, only a few of them have been characterized phytochemically and pharmacologically. Despite the promising therapeutic potential of some Agastache sp. extracts, volatile oils, or their active components, clinical studies are lacking. Moreover, some pharmacological properties have been tested only in vitro, and most of them were attributed to crude extracts and not to active compounds. Thus, the investigation of the pharmacological effects using animal models, the testing of the active compounds, and subsequently the standardization of extracts from these compounds represent important research directions.Although research on Agastache species are aromatic plants considered natural sources of important bioactive compounds, representing potentially effective clinical drug candidates. Compared with other genera, Agastache genus is less investigated; thus, our present work gives a scientific foundation for future research and medicinal applications on the plants of this genus.In conclusion,


Oryza granulata, possess unique characteristics that distinguish them from other Oryza species. For instance, O. granulata characteristically has a GG genome and is accordingly classified as a basal lineage of the genus Oryza. Here, we deployed a versatile hybrid approach by integrating Illumina and PacBio sequencing data to generate a high-quality mitochondrial genome (mitogenome) assembly for O. granulata. The mitogenome of O. granulata was 509,311 base pairs (bp) with sixty-seven genes comprising two circular chromosomes, five ribosomal RNA (rRNA) coding genes, twenty-five transfer RNA (tRNA) coding genes, and thirty-seven genes coding for proteins. We identified a total of 378 simple sequence repeats (SSRs). The genome also contained 643 pairs of dispersed repeats comprising 340 palindromic and 303 forward. In the O. granulata mitogenome, the length of 57 homologous fragments in the chloroplast genome occupied 5.96% of the mitogenome length. Collinearity analysis of three Oryza mitogenomes revealed high structural variability and frequent rearrangements. Phylogenetic analysis showed that, compared to other related genera, O. granulata had the closest genetic relationship with mitogenomes reported for all members of Oryza, and occupies a position at the base of the Oryza phylogeny. Comparative analysis of complete mitochondrial genome assemblies for Oryza species revealed high levels of mitogenomic diversity, providing a foundation for future conservation and utilization of wild rice biodiversity.Wild upland rice species, including  Oryza, 25/27 are wild species, while 2/27 represent the cultivated species [Oryza are sub-classified into 11 genome types. These genotypes include six diploids , and five tetraploids  [Oryza, O. granulata belongs to the O. meyeriana complex that characteristically possesses the GG genome and falls within the lower cladogram in the Oryza phylogeny [Oryza to facilitate the breeding of highland rice represents an enormous challenge because the optimum growth and output of most species within the genus Oryza inherently ocurrs under damp and even aquatic conditions. O. granulata is a resilient shade- and drought-tolerant upland wild rice species and poses durable resistance to economically essential pests and pathogens compared to other cultivated and non-cultivated rice species [Oryza species [Oryza characteristics and assuring global rice safety. However, human disturbance and habitat fragmentation made many natural populations of O. granulata endangered and some even went extinct [Of the 27 species recognized in the genus  species ,2,3. Depnd KKLL) ,5. Withihylogeny ,6. The w species . Genomic species ,13,14, s extinct . Althoug extinct ,17, effiOryza mitogenomes are common in the mitogenomes of land plants [O. granulata mitogenomes in public repositories, including the NCBI GenBank database. Efforts are needed to generate the mitogenomic data of O. granulata in order to comprehend how it evolved and how to conserve and utilize its precious genetic diversity.The nuclear, mitochondrial (mitogenome), and plastid genomes are the three relatively independent genetic components typically found in a plant cell. Mitochondria are double-membraneous and ubiquitous cellular organelles that are the primary sites for oxidative metabolism and energy transformation in eukaryotic cells, and are often referred to as the “powerhouse” or “energy factory” of cells . As semid plants ,24,25,26d plants ,28. Thesd plants ,30. Compd plants ,32 and cd plants  genomes,O. granulata. We assembled and annotated the full O. granulata mitogenome. Additionally, we examined the gene content, genome structure, and evolutionary phylogeny of O. granulata with other species in the genus Oryza. We performed a comparative mitogenome analysis of Oryza species to obtain vital genome information, including variable genomic regions, conserved regions, and incidence of mitogenome reshuffling or rearrangements. Additionally, we looked into the transfer of genes between O. granulata’s chloroplasts and mitogenomes. Such a mitogenome contains information that can support the development of efficient molecular markers, carry out genetic engineering, and explain the phylogenetic and evolutionary relationships among the Oryza species.In the present study, we integrated data from third-generation PacBio sequencing and second-generation Illumina sequencing of O. granulata plants sampled from Menghai County, Yunnan Province, China. Briefly, seeds harvested from O. granulata plant growing in Menghai County, Yunnan, were planted in the greenhouse at Kunming Institute of Botany, Chinese Academy of Sciences. Fresh, healthy, and intact leaves harvested from the individual seedlings were immediately frozen in liquid nitrogen and later transferred into a −80 °C laboratory freezer before DNA extraction. Total DNA was extracted using a modified CTAB method [Genome sequence data were generated using DNA extracted from dozens of B method . NanoDroOryza sativa L. spp. japonica cv. Nipponbare (Nip) mitogenome (Accession Number: NC011033) [https://github.com/broadinstitute/pilon, (accessed on 8 June 2023)) using Illumina data with the default parameters. Candidate mitochondrial genome contigs were identified by a BLASTn search [Raw reads generated with PacBio SMRT technology were mapped to the complete C011033)  using BWC011033)  and SAMtC011033) . The mitC011033)  using thC011033)  ). The tRNA and rRNA were annotated using tRNAscan-SE v1.21 [The mitochondrial genome was manually annotated with Mitofy, an online homology-based prediction tool  (http://SE v1.21  and RNAmSE v1.21 . Genome SE v1.21  and editThe simple sequence repeats (SSRs) in the assembled mitogenome were identified using a Perl script MISA . The minThe prediction of C to U RNA editing sites in the mitogenome PCGs was performed using the Deepred-mt . The DeeO. granulata chloroplast genome sequence was retrieved from the NCBI GenBank (Accession Number: KF359920) [F359920) , and BLAF359920)  was usedF359920)  by calliF359920) . O. rufipogon (RUF) and O. sativa L. spp. japonica cv. Nipponbare (Nip) ).We selected two additional species, re (Nip)  to conduv2.11.0+  program,O. rufipogon, O. sativa L. spp. japonica Nipponbare, O. sativa L. spp. indica 9311, O. coarctata, O. minuta, Zea luxurians, Zea mays, Sorghum bicolor, Eleusine indica, Triticum aestivum, Phoenix dactylifera, Glycine max, Vitis vinifera, and Arabidopsis thaliana) were downloaded from the NCBI database and used for phylogenetic analysis with Ginkgo biloba selected as the outgroup  [O. granulata mitogenome’s features are similar to features previously reported for Oryza mitogenome, especially for Nip [O. minuta [To obtain a complete mitogenome for e of Nip  and cove~550 kb)  over 860 for Nip , RUF [57. minuta . The twoO. granulata mitogenome encoded thirty-seven protein genes, five rRNA genes, and twenty-five tRNA genes  of the O. granulata mitogenome, and protein-coding sequences comprised only 6.42%  of the total length. The non-coding sequences of the O. granulata mitogenome were almost 93.58%, which is higher than the average of (89.46%) the coding sequences previously reported for angiosperm [The NA genes , totalingiosperm .O. granulata mitogenome, with dimeric repeats being the most abundant (Simple sequence repeats (SSRs) are units of sequence repetition that can be one to six base pairs long . Due to abundant , while mabundant . O. granulata mitogenome. These dispersed repeats comprised 303 pairs of forward repeats, 340 pairs of palindromic repeats, and 340 palindromic repeats; the longest palindromic repeat of 3100 bp was recorded as the length of the longest forward repeat  [O. granulata mitogenome was 509,311 bp, and its genome size is somewhat close to other Oryza species, such as Nip  [O. coarctata  [O. minuta  [Camellia sinensis (800~1200 kb) [Arabidopsis thaliana  [Ginkgo biloba  [O. granulata mitogenome was 43.52% and fell in line with the GC content reported for the above species, indicating tight conservation in GC content during evolutionary course of angiosperms.Plants receive life-sustaining energy from mitochondria and possess larger and more sophisticated mitogenomes than animals due to the abundance of repetitive sequences . NumeroungHui63) . The len,520 bp) , Shuhui4,520 bp) , O. coar,065 bp) ,70, and ,022 bp) , but dif1200 kb) ,72, Arab,808 bp) ,74, and ,544 bp) . The GC O. granulata. Besides these protein-coding genes, tRNA and rRNA genes identified in the O. granulata mitogenome were also in multiple copies. In contrast, the O. granulata mitogenome has lost rpl10, rpl16, rps3, rps10, rps11, rps12, sdh3, and sdh4 genes. Before being lost from the mitogenome, we speculated that a significant proportion of these genes, if not all, may have functionally moved into the nucleus [sdh genes are in tandem with the gene loss pattern observed in other species within the genus Oryza [Generally, there is no positive correlation between gene numbers and the proportion of protein-coding genes identified in plant mitogenome due to the high incidence of gene loss and accumulation of multiple gene copies resulting from the rearrangements of repeat sequences ,77,78. W nucleus . These tus Oryza .O. granulata mitogenome consisted of A or T nucleotides, making mitogenomes richer in A/T content than G/C content. Multiple research findings suggested that plant mitogenomes are rich in A/T content [O. granulata mitogenome in AT repeats likely accounted for the high A/T content. The current investigations showed that the predominant proportion of 515 pairs (80.09%) of the dispersed repeat sequences identified in the mitogenome of O. granulata ranged between 30–100 bp, which is comparatively higher than the proportion of 30–100 bp types of dispersed repeat sequences reported in many plants. Plant mitogenomes are not a single circle but a complex and dynamic variety of shapes [O. granulata mitogenome.The plant mitogenome contains many repeat sequences which the SSRs have extensively used as bio-markers to identify and assess genetic variation in species . Charact content ,81. The f shapes . In planf shapes ,84. AddiO. granulata mitogenome, which is remarkably similar to those in other terrestrial plants [rpl5 and rps1 genes only have one RNA editing site, indicating that the rpl5 and rps1 genes are conserved.The mitogenomes of higher plants contain large amounts of RNA-editable genes and have been shown to play core roles in the initiation of essential steps in the expression of genes . It belol plants ,26,91. MM. polymorpha) to 10.85 percent (P. dactylifera) of sequences originating from plastids [O. granulata organellar genomes and found that the O. granulata mitogenome integrated 57  MTPTs. In angiosperm, tRNA-coding genes are often translocated from the chloroplast genome to the mitochondrial genome [Dynamic intercellular phenomenons, including horizontal gene transfer (HGT), and intracellular gene transfer (IGT), are vital fundamental parameters that are known to drive the exchange and the integration of organellar and nuclear genomes and DNA fragments into nuclear genomes to create NUMTs and NUPTs in plants ,93. Studplastids . Here, wl genome .Oryza species are non-conservative, indicating the likely occurrence of rapid genome recombination throughout the lengthy evolutionary process. Contrary to the outstanding level of conservation seen in animal mitogenomes [Oryza.Mitogenomic collinearity results showed that sequences of the three ogenomes ,96, signogenomes . TherefoOryza species and ten other plant species’ mitochondrial genomes was consistent with the taxonomic data associated with those species. These results demonstrate the potential for using data from organelle genomes in plant phylogeny research [O. granulata mitogenome and comparative genomic analysis provides a solid foundation for future Oryza plant-relatedness studies. Furthermore, the current study will serve as a valuable alternative methodology for investigating plant diversity and evolution.The phylogenetic tree inferred based on six research . The ava