swnk plays an important role in the biosynthesis of swainsonine in Metarhizium anisopliae

Swainsonine (SW) is the principal toxic ingredient of locoweeds, and is produced by multiple fungi. A key enzyme in the SW synthesis pathway is a hybrid swnk/nrps. To analyze the role of swnk in the SW biosynthesis pathway of Metarhizium anisopliae. The concentration of SW and the swnk expression in M. anisopliae fermentation from 1st to 7th day were determined using LC–MS and RT-qPCR, respectively. M. anisopliae had the highest SW content and swnk expression on the 5th day of fermentation; Mutant strain (MT) were obtained by PEG-mediated homologous recombination (HR) which knocked out swnk in the wild-type (WT) strain. Complemented-type (CT) strain were obtained by transforming a modified PUC19 complementation vector containing the geneticin (G418) resistance gene and swnK. SW was not detected in the MT strain and reverted to its original level in the CT strain; A Psilent-1 plasmid with Benomyl (ben)-resistant that was used interfered with swnk of WT strain. The level of SW was markedly diminished in the RNAi strain. RNAi of swnk affects the formation of the cell wall in M. anisopliae. These results indicate that swnk plays a crucial role in the SW biosynthesis of M. anisopliae.


Introduction
Locoweed plants are a general term for poisonous plants of the genera Oxytropis and Astragalus in the legume family, which are widely distributed in areas such as the southwestern United States and western China. In recent years, locoweed poisoning has caused significant economic losses worldwide (Lu et al. 2016). Animal locoism has been found to be associated with the consumption of certain Oxytropis and Astragalus plants. An indolizidine alkaloid, swainsonine (SW) (Fig. 1), was frist isolated from the Australian legume Swainsona canescens (Colegate et al. 1979). Subsequently, SW was isolated from the locoweed plants and it was proved to be the main toxic component of locoweeds (Hamaguchi et al. 2007). SW can cause neurological symptoms, reproductive impairment, and damage to multiple tissues/ organs in grazing livestock, and in severe cases, death due to multiple organ failure. SW promoted apoptosis in rat primary renal tubular epithelial cells by impairing lysosomal degradation and inhibiting autophagic degradation (Wang et al. 2021a), and also induced apoptosis-like death in rat primary renal tubular epithelial cells through endoplasmic reticulum stress and MAPK signaling pathway (Wang et al. 2021b). SW can be produced by fungi including Alternaria oxytropis (Oldrup et al. 2010), Metarhizium spp. and Slafractonia leguminicola, dermatophytes and others . In China, about 33.3 million hectares are affected by poisonous grasses, among which locoweed occupies about 33% of the poisonous grass area (Yan et al. 2016). It is important to explore the toxin of locoweed (SW) for prevent and control locoweed poisoning disease. However, the endophytic fungus oxytropis of locoweed grew slowly and protoplasm preparation is difficult. We begin the investigation by using M. anisopliae as an alternative model as a starting point for understanding SW synthesis pathway. The study of the SW synthesis pathway in different fungi provides useful information for the control of animal locoism from the genetic engineering level.
Many studies have been focused on the SW biosynthetic pathway in different fungi: lysine → Saccharopine → 1,6-piperinic acid → l-Pipecolic acid → SW. The SW-producing fungi have a common SW biosynthetic gene cluster " SWN", which includes swnh1, swnh2, swnk, swnn, swnr, swna and swnt. Cook et al. (2017) compared the SWN gene cluster in a variety of SW-producing fungi. Metarhizium sp. possessed all the genes in the cluster. The catalytic enzymes encoded by these genes are involved in the biosynthesis of l-pipecolic acid to SW. The role of oxido-reductases, polyketide synthase (pks), and cytochrome p450s in the swainsonine biosynthetic pathway of A. oxytropis by proteomic analysis, and the presence of saccharopine dehydrogenase, pipecolate oxidase, aminoadipate-semialdehyde dehydrogenase in S. leguminicola were identified (Li et al. 2012). The potential enzymes in the SW biosynthesis pathway of l-lysine to l-pipecolic acid were annotated by whole-genome sequencing of A. oxytropis: saccharopine dehydrogenase, saccharopine oxidase (fad2), and pyrroline-5-carboxylate reductase (p5cr) (Lu et al. 2016). Two pathways for the generation of l-pipercoic acid were demonstrated in M. robertsii: the lysine cyclodeaminase (LCD) pathway and the lysine aminotransferase (LAT) pathway (Luo et al. 2020), It is not known whether these multiple pathways are present in other fungi.
All SW-producing fungi contain swnk and swnh2, while swna and swnt were found in a limited number of fungi (Neyaz et al. 2022). SW was eliminated in a swnk mutant strain of the entomopathogen M. robertsii, while the content of SW was restored to normal after complementation with the swnk gene . These results indicate that the swnk gene is essential for the biosynthesis of SW. SW-producing A. oxytropis isolated from locoweeds in the US contained swnk-ks, which was highly conserved (Noor et al. 2020). The pks gene annotated by Lu et al. (2016) was highly similar to the swnk gene annotated by cook et al. (2017) (Fig. S1). which was found by comparing the gene sequence and amino acid sequence. Therefore, both may be the same gene. After silencing pks1 (a key gene for melanin production in S. leguminicola) using RNAi, the fungus exhibited poorer growth and reduced production of SW, in addition to causing a change in phenotypic color of the strain from pale to dark. Based on this result, Alhawatema et al. (2017) hypothesized that off-target effects would occur if the active site of swnk was silenced.
SW has toxic effects on human cells have been revealed in many studies (Morikawa et al. 2022;Tan et al. 2021). M. anisopliae is one of the entomopathogenic fungus that most widely used for biological control of pests (Donzelli and Krasnoff 2016;Abello et al. 2018). The output of SW is not associated with its toxicity to insects . And there was no significant correlation between the content of SW and the toxicity of M. robertii to larvae  (Luo et al. 2020). Therefore, it can be inferred that disruption of the swnk gene in M. anisopliae does not affect its virulence to insects. The biosynthetic pathway of SW is slightly different in different fungi Luo et al. 2020). Metarhizium spp., a new biopesticide, is closely related to human food security. Therefore, the study for the biosynthesis of SW in M. anisopliae not only provides reference value for the prevention and control of animal locoism, but also has important significance for its safe utilization as a biological control agent.
While relevant studies have been conducted for the biosynthetic pathway of SW in M. robertsii, however the regulatory gene cluster of SW in M. anisopliae has not been verified. To confirm the function of swnk in the synthesis of SW in M. anisopliae, the trend of SW production and swnk expression in M. anisopliae ferments from day 1 to day 7 were measured. Then swnk of M. anisopliae was knocked out using homologous recombination (HR) and constructed CT strain on the basis of MT strains. Finally, the effect of the swnk gene on SW biosynthesis in M. anisopliae was determined by detecting the changes of SW production in MT and CT strains. we found the effects on SW biosynthesis and growth of M. anisopliae by conducting RNAi on swnk.

Materials and methods
Strain and fermentation culture M. anisopliae (Metarhizium anisopliae (Metsch.) Sorokīn), isolated in the soil, was obtained from Xi'an Jin Berry Biological Technology Co. Ltd., Shaanxi, China and inoculated onto Sabouraud medium (SDA) (Guo et al. 2022) and cultured at 28 °C for 10 days. Conidiums (1.0 × 10 7 ) of M. anisopliae were transferred into 300 mL SDA liquid culture medias (without agar) and fermented at 28 °C, 180 rpm, for 1-7 days. The fermentation broth was filtered through three layers of Miracloth (EMD Millipore Corp, Billerica, MA, USA) to collect the cultures and the mycelium respectively. The cultures stored at 4 °C for use and the mycelium was dried at room temperature, dried mycelium and then the mycelium was extracted with methanol (Song et al. 2019). The cultures and the extract were analyzed using HRLC-MS LC-30A+ Triple TOF 5600+ (AB SCIEX) using the methods of Luo et al. (2020) SW concentration was tested three times for each strain.

T-qPCR analysis of the swnk gene of M. anisopliae
Fungal RNA was extracted using the E.Z.N.A. Fungal RNA Kit (Omega). RNA was reverse transcribed into cDNA by utilizing PrimeScript™ RT reagent Kit (Takara). The primers for amplification of the swnk and 5.8S rRNA fragments are illustrated in Table S1. The fragments were amplified using RT-qPCR with the following conditions: 1 cycle of 95 °C for 10 min; 40 cycles of denaturation at 95 °C for 10 s, annealing at 55 °C for 90 s, and an extension at 72 °C for 32 s. Cultures were tested at 1-7 days using RT-qPCR.

Sensitivity screening of M. anisopliae
Metarhizium anisopliae was inoculated into SDA medias with 0 μg/mL, 100 μg/mL, 300 μg/mL, 600 μg/mL, and 1000 μg/mL of benomyl or G418, the growth of the colony was observed after 10 days. After the swnk gene MT is obtained, sensitivity of the MT to G418 was observed after 10 days.

Identification of the swnk gene of M. anisopliae
Fungal DNA was extracted using the CTAB method (Umesha et al. 2016). Primers for amplification of swnk (Table S2) were designed from the swnk sequence from Cook et al. (2017), GeneBank KID61008 of M. anisopliae ARSEF 549. The swnk gene was amplified from M. anisopliae DNA using L1/R1 primers.

Vector construction
The upstream and downstream fragments of the swnk gene and the benomyl (fungicide) resistance gene (ben) were inserted into pUC19 (Takara) digested with EcoR I/BamH I (Takara) using the In-Fusion® HD Cloning System (Takara) to construct a knockout construct targeting the swnk gene (Figs. S2A, S3A, S4A and Table S2).
The PCR primers Ben-L and Ben-R (Table S2) were used to amplify the ben resistance gene from pBARGPE1-BenA (Wuhan Jingxiu Scientific Biotechnology Co., Ltd., China) as a template. The primers F2/R2 and F3/R3 (Table S2) were used to amplify the upstream target fragment (swnk-i) and the downstream target fragment (swnk-ii), respectively, of the swnk gene from the genomic DNA of M. anisopliae. The swnk-i, ben, swnk-ii and the double-cut pUC19 vector were ligated using In-Fusion cloning. The swnk gene fragment was amplified using primers F3/R3 (Table S2) from the genomic DNA of M. anisopliae. The G418 gene was amplified using primers G418F/G418R (Table S2) from pSilent-Dual1. To produce a complementation vector, the swnk gene and G418 gene were inserted into the MCS of pUC19 vector using In-Fusion cloning (Figs. S2b, S3b, S4b). The swnk-1 and swnk-2 fragments were amplified using primers F1/R1, F2/R2 respectively (Table S2) from the genomic DNA of M. anisopliae. To produce a RNAi vector, the swnk-1 and swnk-2 fragments were inserted into the MCS1 and MCS2 of pSilent-1 vector, respectively, using In-Fusion cloning (Figs. S2c, S3c, S4c).

Preparation of protoplasts
The conidiums (1.0 × 10 7 ) of M. anisopliae grown on SDA medias were transferred into each 300 mL flask of SDA liquid culture medias (without agar), and incubated at 28 °C, 180 rpm for 3 days. The resulting mycelia were filtered through sterile miracloth. To the collected hyphae were added different concentrations of enzymatic hydrolysate (Sigma Aldrich) prepared with 1.2 M KCl, and hydrolyzed at 30 °C, 100 rpm 3 h. The optimal combination of enzymes and conditions were determined based on protoplast yield. Yield from different enzymes, including 1% snail enzyme, 1% cellulase and 1% lysing enzymes, and combinations of the enzymes were also tested. The enzymatically digested mixtures were filtered through a layer of sterile miracloth and two layers of filter paper into a sterile 50 mL centrifuge tube, and the protoplasts were washed extensively with 1.2 M KCl and centrifuged at 4000 rpm for 6 min at room temperature. After discarding the supernatant, 10 mL of STC Buffer (0.6 M Sorbitol; 10 mM Tris-HCl; 10 mM CaCl 2 , pH 6.5) were added and the protoplasts were gently resuspended. The mixture was centrifuged at 4000 rpm for 6 min. After discarding the supernatant, 1 mL of STC Buffer was added. The protoplasts were then centrifuged at room temperature at 3500 rpm for 6 min, which was repeated. Finally, protoplasts were adjusted to 2-5 × 10 7 /mL for subsequent experiments.

PEG mediated DNA transformation
Transformation of the protoplasts was done as described by Proctor et al. (1995). Approximately 5-10 µg of the linearized swnk knockout vector and RNAi vector were added to 50 mL centrifuge tube containing 2-5 × 10 7 /mL protoplasts, and incubated at room temperature for 20 min without shaking. Then 1-1.25 mL of 40% PTC (40% PEG 8000, 20% sucrose, 50 mM CaCl 2 , 10 mM Tris-HCl) were added to the tube (mixed thoroughly by inversion) and incubated at room temperature for 20 min without shaking. Thereafter, 5 mL of TB 3 (0.3% Yeast Extract, 0.3% acid hydrolyzed casein, 20% sucrose) containing 50 g/mL ampicillin (Sigma Aldrich) were added and shaken at room temperature overnight. The overnight protoplasts were centrifuged at 4000 rpm for 6 min. The supernatant was discarded and about 1 mL of the remaining liquid was used to suspend the remainder. The regenerated protoplasts were added to 10 mL of Bottom Agar (0.3% Yeast Extract, 0.3% acid hydrolyzed casein, 20% sucrose, 1% Agar) containing 20 µg/mL benomyl. After incubation at 30 °C for 10 h, Top Agar (0.3% Yeast Extract, 0.3% acid hydrolyzed casein, 20% sucrose, 1.5% Agar) containing 40 µg/mL benomyl was added. After 3-5 days, a single colony transformant grown on the plate was transferred to SDA medium containing 40 µg/mL benomyl. The wild type M. anisopliae was used as a control. The swnk gene mutant strain of M. anisopliae was named MT. The transformation of the complement vector was the same as described above, and 2 mg/mL of G418 was used for screening of the complement (CT).

RT-qPCR identification of MT, CT and RNAi strains
Fungal RNA was extracted using the E.Z.N.A. Fungal RNA Kit (Omega). RNA was reverse transcribed into cDNA by using PrimeScript™ RT reagent Kit (Takara). Primers F4/R4, F5/R5, F6/R6, F7/R7, F8/ R8 and ITS1/ITS4 for amplification of MT, CT and RNAi strain are shown in Table S3 and schematic representation of the obtention of the MT, CT and RNAi strain is shown in Fig. S5. The genes were amplified using RT-qPCR with the following conditions: 1 cycle of 95 °C for 10 min; 40 cycles of denaturation at 95 °C for 10 s, annealing at 55 °C for 90 s, and an extension at 72 °C for 32 s. Cultures were tested using RT-qPCR at MT, CT and RNAi strain.
Phenotypic observation and growth rate determination MT, CT, WT and RNAi strain Conidiums (1.0 × 10 7 ) of MT, CT, WT and RNAi strain were inoculated into the same position on the SDA medium (with or without 100 μg/mL Congo red) and grown at 28 °C for 10 days, after which they were measured for diameter and photographed.

SW content detection of WT, MT, CT and RNAi strains in M. anisopliae
The WT, MT, CT and RNAi strain were inoculated into SDA medium cultured at 28 °C for 10 days. Conidiums (1.0 × 10 7 ) of each strain were transferred into 300 mL flasks of SDA (without agar) culture medium and grown at 28 °C, 180 rpm, for 5th day. The mycelium and the fermentation broth were filtered through three layers of Miracloth (EMD Millipore Corp, Billerica, MA, USA) to collect the mycelium. The mycelium was dried at room temperature, dried mycelium and the cultures stored at 4 °C for use, and then the mycelium is extracted with methanol (Song et al. 2019). The cultures and the extract were analyzed using HRLCMS LC-30A+ Triple TOF 5600+ (AB SCIEX) using the methods of (Noor et al. 2020). SW concentration was tested three times for each strain.

Statistical analysis
In this study, each experiment was carried out in triplicate. Statistical analysis was performed on the measured data using the SPSS 20.0 software. The results were expressed as mean ± SEM. A one-way ANOVA was performed on each sample, *P < 0.05, indicating a significant difference between the two groups, **P < 0.01, indicating that the difference between the two groups is highly significant. Results from the cultures were used for determining the optimal time periods for swainsonine production. The mass concentration peak area for SW was compared using linear regression. The colony diameters were measured by a ruler. RT-qPCR data were analyzed using the 2 −△△CT method (Schmittgen and Livak 2008).

Detection of SW and RT-qPCR analysis of the swnk gene of M. anisopliae in different periods
To quantitate and analyze the SW content at different time points, the same volume of M. anisopliae fermentation broth and the dried mycelium was concentrated, and the level of SW was detected using a LC-MS. The standard curve was drawn according to the calculated regression equation: Y = 486.14665X + 34.36851 (R = 0.99049) (Fig. 2a). From the linear regression equation of mass concentration-peak area of SW, the SW content of M. anisopliae was calculated at 1-7 days of growth. M. anisopliae produced the highest content of SW on day 5 (Fig. 2b).
The relative expression of swnk gene in 1-7 day was detected by RT-qPCR. The swnk gene expression level of each strain was similar to the content of SW, day 5 of fermentation reached the highest expression of the swnk gene (Fig. 3).

Sensitivity screening of M. anisopliae
Metarhizium anisopliae was inoculated into SDA medias with different concentrations of benomyl or G418. After 10 days, M. anisopliae is more sensitive to 40 μg/mL benomyl and 500 μg/mL G418 (Fig. 4a,  b). After the swnk gene MT is obtained, the MT was the most sensitive of to 2 mg/mL G418 (Fig. 4c).

Production of MT, CT and RNAi strains
To determine the role of the swnk in the SW biosynthesis pathway of M. anisopliae, homologous recombination was used to knock out swnk and RNAi interference vector was used to interfere with swnk. The resulting transformant grew on SDA medias containing 40 μg/mL benomyl. The primers L8/R8, L9/ R9, L10/R10, L11/R11, L12/R12 and ITS1/ITS4 (Table S3 and Fig. S5) set was then used to identify the genomic DNA of the transformant (MT, CT and RNAi strain) with electrophoresis and confirmed by sequencing (Fig. 5). To verify the status of MT, a complement was produced by transforming the wildtype swnk and G418 gene of pSilent-Dual1 plasmid in pUC19 into the MT. The complement transformant was grown on SDA medium containing 2 mg/mL G418 and was identified as above.
The phenotypic observation and growth rate determination of WT, MT, CT and RNAi strains The WT, MT, CT and RNAi strains were grown on SDA plates (with or without 100 μg/mL Congo red) for 10 days (Fig. 6) to compare growth characteristics. The phenotypes, dry weight of mycelium and growth rate did not change significantly (Fig. 7), the hyphae of the MT and the RNAi strains were turned visibly red on SDA plates (with 100 μg/mL Congo red). Congo red (CR) specifically binds β-1,3-glucan, a component of the cell wall (Ram and Klis 2006) changing its color while hindering the formation of the cell wall. As shown in Fig. 6, the WT strains had no change in colony color and the mycelium was relatively dense and in good growth condition. In contrast, MT, CT and RNAi strains were stained with red mycelium and appeared to be loose, indicating that compared to WT strains, MT, CT and RNAi strains had increased cell wall sensitivity and were therefore susceptible to Congo red staining. The sensitivity of CT strain to Congo red was not fully recovered, which might be due to the slow recovery of damage caused by swnk deletion or some irreversibility.
Comparing the cell walls of WT and MT strains under the same conditions, transmission electron microscopy showed that the cell walls of MT and RNAi strains had diffuse perimeters with irregular shapes, incomplete edges and uneven thicknesses. In contrast, the cell wall boundaries of WT and CT were obvious and neatly arranged (Fig. 7), suggesting such mutation and RNAi in swnk affect the cell wall formation of M. anisopliae.

LC-MS detection of SW and RT-qPCR analysis of the swnk gene of MT, CT WT and RNAi strains
By detecting the swainsonine content and swnk gene expression of MT, CT WT and RNAi strains, it was found that the MT did not produce SW, and the swainsonine content of the RNAi strain was significantly reduced with detected level of 0.018 ± 0.005 μg/mL, the SW content of the CT remained the same as that of the wild-type strain, which were 0.581 ± 0.084 μg/ mL and 0.625 ± 0.056 μg/mL (Fig. 8a), respectively. The standard curve was drawn according to the calculated regression equation: Y = 89.15243X + 18.12349 (R = 0.99493) (Fig. 8b). The swnk gene expression level of each strain is similar to the content of SW (Fig. 9).

Discussion
In this study the functions of the production of SW and the expression of the swnk gene in M. anisopliae from day 1 to day 7 were investigated using gene knock out and overexpression, i.e. gain of function and loss of function approach and this level of SW were determined by LC-MS. The results show that the trend of both changes was similar and both of them reached the highest on the 5th day. By analyzing the SW content and swnk expression of MT, RT-qPCR analysis of the swnk gene. RNA was extracted, converted to cDNA, and the expression was tested at 5 days in M. anisopliae. Error bars represent the standard error of the mean (n = 3), *P < 0.05; **P < 0.01 CT and RNAi strains. We found that MT strain did not produce SW, RNAi strain of SW content was significantly reduced, and the SW content of CT strain of essentially consistent with wild-type strain. The expression of the swnk gene in each strain was consistent with the change of SW yield. However, Noor et al. (2020) found that the nucleotide sequence of swnk-ks can also be detected by PCR to identify whether fungi have the ability to produce SW.
No significant changes were found in the growth rate, hyphal dry weight and colony morphology of the strains by observing the phenotypes of MT, CT and RNAi strains of the swnk gene of M. anisopliae. However, the cell wall of the RNAi strain showed slight changes. The reason why MT strain does not have the same observable cell wall phenotype as RNAi, we speculated that MT strain may have a compensatory pathway or be related to the specificity of RNAi, the specific reason needs to be further explored. In addition, the fungal cell wall biosynthesis was also found to be regulated by the swnk gene (Zhang et al. 2023). In this study, WT, MT, CT and RNAi strains were inoculated on the medium containing Congo red dye. MT, CT, and RNAi strains of M. anisopliae were stained by the Congo red dye. And we found that the deletion of swnk gene changed the permeability of the strains, which further verified that PKS gene was closely related to the cell wall of M. anisopliae. In addition, some researchers also have found that the biosynthesis of fungal cell wall is also associated with PKS (Gow et al. 2017). However, Alhawatema et al. (2017) demonstrated that silencing the melanin PKS gene leads to a decrease in SW content. This result needs to be verified by analyzing the specificity of the silencing/knockout.
We found the swnk gene is necessary for the SW biosynthesis of all SW-producing fungi Noor et al. 2020). In this study, RNAi was first used to silence the swnk gene, and the effect was similar to the effect of knocking out the gene on SW in M. anisopliae. Creamer et al. (2021) specifically characterized the swnk, nrps, and swnk/nrps in A. oxytropis. Li and Holdom found that the growth rate of strain reaches the maximum between the 1st day and the 2nd day (Li and Holdom 1995), which is consistent with the significant increase on the 2nd day in the dry weight of mycelium measured in this experiment.
Locoweed has a strong toxic effect on animals and has caused serious damage to the world's grassland animal husbandry (Tan et al. 2021). However, Metarhizium is closely related to crop production (Anastassiadou et al. 2020). At present, it has been used as an important biological control agent for pest control in many countries. The results of this study provide basis for future study of SW biosynthesis and catalytic enzyme genes in other SW-producing fungi.

Conclusions
Our study determined that the SW content and swnk expression reached the highest when M. anisopliae was fermented to day 5, and no SW could be detected in the MT strain in which the swnk gene was knocked out by homologous recombination indicating the important role of this gene in SW synthesis. In the swnk RNAi strain, SW levels were significantly reduced and the cell wall of M. anisopliae was altered, while growth rate and cellular organelles were not significantly changed. After the swnk gene was complemented in the MT strain, the SW returned to the original level in the CT strain. The results of the phenotypic assay showed altered cell wall permeability in MT, CT and RNAi strains. These results indicate that swnk plays a crucial role in the biosynthetic pathway of SW in M. anisopliae, and the swnk may play a role in the fungal cell wall.
Acknowledgements The authors are grateful to Prof. Zekun Guo, College of Life Sciences, Northwest A&F University for his valuable help and advice during the experiment. Table S1-Primers are used to measure the expression of the swnk gene. Table S2-Primers for the swnk gene of M. anisopliae to mutation vector, complementary vector and RNAi vector. Table S3-Identification primers for the swnk gene MT, CT and RNAi strain. Figure S1-Comparison of swnk and pks genes. Figure S2-Construction of knockout vector, complement vector and RNAi vector of the swnk gene in M. anisopliae. Figure S3-Fragments of the swnk gene mutation vector, complementary vector and RNAi vector of M. anisopliae. Figure S4-Validation of the swnk gene mutation vector, complementary vector and RNAi vector of M. anisopliae and its specific fragments.