An insight of anopheline larvicidal mechanism of Trichoderma asperellum (TaspSKGN2)

Anopheline larvicidal property of T. asperellum has been found recently in medical science. The mechanism of actions exhibited by T. asperellum to infect mosquito larvae is the pivotal context of our present study. To infect an insect, entomopathogens must undergo some events of pathogenesis. We performed some experiments to find out the mechanisms of action of T. asperellum against anopheline larvae and compared its actions with other two well recognized entomopathogens like Metarhizium anisopliae and Beauveria bassiana. The methodology adopted for this includes Compound light and SE Microscopic study of host–pathogen interaction, detection of fungal spore adhesion on larval surface (Mucilage assay), detection of cuticle degrading enzymes (Spore bound pr1, chitinase and protease) by spectro-photometric method, Quantitative estimation of chitinase and protease enzymes, and determination of nuclear degeneration of hemocyte cells of ME (methanolic extract) treated larvae by T. asperellum under fluorescence microscope. Compound light microscopic studies showed spore attachment, appressorium and germ tube formation, invasion and proliferated hyphal growth of T. asperellum on epicuticle and inside of dead larvae. SEM study also supported them. After 3 h of interaction, spores were found to be attached on larval surface exhibiting pink colored outer layer at the site of attachment indicating the presence of mucilage surrounding the attached spores. The enzymatic cleavage of the 4-nitroanilide substrate yields 4-nitroaniline which indicates the presence of spore-bound PR1 protein (Pathogenecity Related 1 Protein) and it was highest (absorbance 1.298 ± 0.002) for T. asperellum in comparison with control and other two entomopathogens. T. asperellum exhibited highest enzymatic index values for both chitinase (5.20) and protease (2.77) among three entomopathogens. Quantitative experiment showed that chitinase enzyme concentration of T. asperellum (245 µg mL−1) was better than other two M. anisopliae (134.59 µg mL−1) and B. bassiana (128.65 µg mL−1). Similarly protease enzyme concentration of this fungus was best (298.652 µg mL−1) among three entomopathogens. Here we have detected and estimated fragmentized nuclei of hemocyte cells by fluorescence microscopy in treated larvae with different ME doses of T. asperellum, and also observed that mosquito larvae exposed to 0.1 mg mL−1 dose of ME showed maximum (100%) nuclear fragmentations of hemocytes and while 20, 45, 70 and 85% of nuclear deformities were recorded at 0.02, 0.04, 0.06 and 0.08 mg mL−1 concentrations of ME. The knowledge of this work certainly will help in understanding of mechanism of action of T. asperellum for anopheline larval killing and consequently in eradication of malaria vector.

www.nature.com/scientificreports/ and M. anisopliae have been used for several years to fight insects by many workers [4][5][6][7] , as they are natural enemies of agricultural pests and have a great role in maintaining ecological balance 8,9 . But entomopathogenecity of Trichoderma longibrachiatum and Trichoderma asperellum has been established in our laboratory as first reports in previous studies 10,11 . T. asperellum is a well known fungus in agriculture. It is regularly used in agriculture for many years both as bio control agents to curb plant pathogenic microbes and plant growth promoting agents, but anopheline larvicidal efficacy of T. asperellum (T.aspSKGN2) (GenBank Accession No. MG719999.1) is novel. The mechanisms of action exhibited by T. asperellum to infect mosquito larvae is the pivotal context of our present study. To infect an insect, entomopathogens must undergo some infection processes. To begin infection processes, at first fungal spores have to attach to the host surface by secreting adhesives 12 . Detection of fungal mucilage for attachment of spore to host surface is an important criterion to experiment fungal mechanism of infection. After successful attachment of spore, fungi have to invade insect cuticle either by mechanical process or enzymatic degradation, through formation of appresorium and then infection peg 13 . The main components of insect cuticle are chitin and other proteins 14 . Virulence of fungal pathogens can be determined by assaying enzymatic activities related to infection pathways, such as spore bound protease (Pr1), chitinases 15,16 , etc. After penetrating the host body through invasion of cuticle, the entomopathogens secrete some toxic compounds inside haemocoel or other tissues of the larval body 17 . Toxic compounds may reduce the insect Phenol Oxidase (PO) content and degenerate insect hemocytes, causing loss of insect immunity 18 . Study of cytotoxicity to insect immune cells is a parameter of assaying insecticidal efficacy. Although the mechanisms of entomopathogenecity of other entomopathogenic fungi are well known to us, the mechanism of novel entomopathogen T. asperellum, as reported by us previously 11 , has hardly been explored. There is a significant research lacuna in the way entomopathogen T. asperellum functions; its process of attachment into the outer cell of the cuticle of mosquito larvae and other pathogenicity processes. This research work takes up this research gap as its primary objective and explores further to gather knowledge about methods and mechanisms of novel entomopathogen T. asperellum. The consolidated objectives of this research work are as follows: (1) observation of host-pathogen interaction by compound light and Scanning Electron Microscopy, (2) detection of mucilage on T. asperellum spore surface at attachment site on larval surface, (3) detection of spore bound pr1 (Pathogenesis related 1 protein), chitinase and protease (caseinase) enzymatic activities of T. asperellum, iv) comparison of enzymatic activity of T. asperellum with known entomopathogens i.e. Beauveria bassiana (GenBank Accession No. KM604668.1) and Metarhizium anisopliae, observation of nuclear morphology of hemocytes cells of ME (methanolic extract) treated larvae and percentages of hemocyte degradation. Our study may provide the effective insights on mode of entomopathogenecity exhibited by T. asperellum.

Results
Mycoparasitism or host-pathogen interaction study through microscopy. Compound microscopic determination of lethality: Fungal spore treated mosquito larvae (3rd instars) were taken out at different times and stained with lactophenol cotton blue to observe host-pathogen interaction. After 5 h of interaction, spores were found to be germinated on larvae. Appresorium and infection peg were observed after 8 h of interaction. Proliferated hyphal growth on epicuticle of dead larvae was observed after 15 h of infection (Fig. 1). Scanning Electron Microscopic study revealed the hyphal proliferation on host epicuticle layer (Fig. 2) after 15 h of interaction.  www.nature.com/scientificreports/ Spore adherence on host surface. Fungal spore treated mosquito larvae were taken out at different times and stained with Ruthenium red (0.1%) to detect the spore adhesion and the presence of mucilage during the attachment process. After 3 h of interaction, spores were found to be attached on larval surface exhibiting pink colored outer layer at the site of attachment (Fig. 3) indicating the presence of mucilage surrounding the attached spores.

Production of hydrolytic enzymes by fungal pathogens. Enzymatic assay for spore-bound Pr1
Freshly harvested conidia of fungal isolates inoculated with 0.1 M Tris-HCL supplemented with 1 mM succinylala-ala-pro-phe-p-nitroanilide, exhibited yellow aqueous phase after reaction. The enzymatic cleavage of the 4-nitroanilide substrate yields 4-nitroaniline (yellow color under alkaline condition) which indicates the presence of spore-bound PR1 protein (Fig. 4). Absorbance was taken at 405 nm wave length and presented in the Table 1.
Chitinase detection (1) Detection of chitinase by plate assay method: Preselected fungal isolates showed distinct enzyme hydrolytic zone in chitin amended Czapek Dox agar plates after five days of inoculation (Fig. 5). Relative enzymatic index (REI) was calculated for each fungus and presented in the Table 2. Hydrolytic zone diameter (8.4 cm) was noted best by T. asperellum among three entomopathogens. T. asperellum exhibited highest enzymatic index value (5.20) among three ( Table 2). REI of T. asperellum was statistically same(p < 0.05) to M. anisopliae but different from B. bassiana (2) Enzymatic assay for chitinase: Fungal culture filtrate of fully grown preselected fungi (T. asperellum, B. bassiana and M. anisopliae) collected from chitin amended Czapek Dox broth were subjected to enzyme substrate reaction by adding PNG as substrate (Fig. 6). Absorbance was taken for each sample and OD values were presented in the Table 1.
Protease Detection (1) Detection of protease by plate assay method: Preselected fungal isolates (T. asperellum, M. anisopliae, B. bassiana) exhibited distinct enzyme hydrolytic zone in casein amended Czapek Dox agar plates after five days of inoculation (Fig. 7). Relative enzymatic index was calculated for each fungus and presented in the Table 2. T. asperellum exhibited highest enzymatic index value which is 2.77 amongst three entomopathogens but statistically (p < 0.05) same to other two entomopathogens. (2) Enzymatic assay for Protease: Fungal culture filtrates of fully grown preselected fungi (T. asperellum, B. bassiana and M. anisopliae) collected from casein amended Czapek Dox broth were subjected to enzyme substrate reaction by adding casein powder as substrate. Absorbance was taken for each sample and OD values were presented in the Table 1.
The t-test indicated t = 3.2181, df = 8, p value = 0.01227. The t-test (p value 0.01) showed the mean ≠ 0, so we can say that there are significant mean differences among the fungal pathogens for the OD values of hydrolytic enzymes. Box plot model (Fig. 8) proved that T. asperellum is the most enzymatically (Chitinase, Protease and Spore bound PR1) active pathogen among the three.
Quantitative estimation of chitinase and protease enzymes. Chitinase estimation The estimated concentrations of chitinases from culture filtrates of different isolates were calculated using standard curve, constructed with absorbance of different known concentrations of BSA (Fig. 9). The chitinase concentrations of three isolates were presented in the Table 3. The data ( Protease estimation The estimated concentrations of proteases from culture filtrates of different isolates were calculated using standard curve, constructed with absorbances of different known concentrations of BSA (Fig. 9). The protease concentrations of three isolates were presented in the Table 3. The data (Table 3) www.nature.com/scientificreports/ showed that protease enzyme concentration of T. asperellum was 298.652 ± 54.09 µg mL −1 ) whereas M. anisopliae exhibited the value of 263.02 ± 51.56 µg mL −1 for the same and for B. bassiana, it was 230.358 ± 42.43 µg mL −1 (p < 0.05). They are statistically different as per Duncan analysis. Moreover, the t-test indicated t = 7.6075, df = 5, p value = 0.00006235. The t-test (p value 0.0006) showed the mean ≠ 0, so we can say that there are significant mean differences among the fungal pathogens in secreting different mean cumulative concentrations of hydrolytic enzymes (chitinase and protease). Box plot model ( Fig. 8) proved that T. asperellum secretes the most concentration of hydrolytic enzymes among the three.
Determination of nuclear deformities of treated larval hemocytes. DAPI (4′,6-diamidino-2-phenylindole) stained, treated (ME of different doses) mosquito larval hemocytes and control nuclei of larva were presented in Fig. 10a-f. The treated (ME of different doses) mosquito larval hemocytes exhibited increased nuclear fragmentations in dose-dependent way ( Fig. 10b-f). Degenerative nuclei were observed from different doses of ME treated larval hemocytes, compared with control which evinced normal spherical shaped nucleus (Fig. 10a). Mean percentage of number of hemocytes with nuclear deformities grew with increasing doses in comparison with control (Table 4). Mosquito larvae exposed to 0.1 mg mL −1 dose of ME showed maximum nuclear fragmentations and 100% of hemocytes exhibited nuclear deformities in this dose. 20, 45, 70 and 85% of nuclear deformities were recorded at 0.02, 0.04, 0.06 and 0.08 mg mL −1 concentration of ME. Result of Duncan analysis to measure significant mean differences was presented in Table 4. Different letters in different rows of the table indicate that they are statistically different as per Duncan analysis (p < 0.05).
Proposed model of the fungi-larvae interaction. The proposed model of mechanism of entomopathogenic actions of T. asperellum on anopheline larvae was schematically outlined in Fig. 11.

Discussion
In this present study, insect killing mechanism of a novel entomopathogen Trichoderma asperellum has been established and concentrations of secreted cuticle degrading enzymes of T. asperellum were compared with two known entomopathogens which were B. bassiana and M. anisopliae 19,20 . To establish a successful attachment to   12 . Previous study supports that spore tip mucilage is responsible for attachment of fungal spore to the host surface 21 . Mechanism of spore adhesion on larval surface is determined by detecting the presence of mucilage at spore attachment site on host surface, using mucilage specific dye, Ruthenium Red 22 . Our study also validates that this fungus adapts to secret mucilage on spore wall for host attachment. Hyphal growth of the fungus on cuticle of mosquito larvae is observed by compound microscopy and SEM. To invade the insect cuticle, which is mainly composed of chitin and several other proteins, entomopathogens must secrete chitinase and protease enzymes 23 . Mechanism of enzymatic degradation of larval cuticle for hyphal penetrance by this fungus inside larval body is confirmed by the detection of spore bound pr1, chitinase and protease enzymes which are secreted by the fungi. In enzymatic assay, T. asperellum exhibits highest absorbance in end product of enzyme-substrate reaction confirming the presence of spore bound pr1 in appropriate amount, chitinase and protease with highest quantity amongst the three entemopathogens. In agar plate assay for protease and chitinase, highest relative enzymatic index is also recorded for T. asperellum. Highest concentrations of chitinases and protease enzymes are exhibited by T. asperellum amongst the three entemopathogens in quantitative protein estimation. The larva generally respond to the fungal infection by humoral mechanisms. After hyphal penetrance through larval cuticle, fungi secrete toxins,  www.nature.com/scientificreports/ inside the larval body, which deteriorate larval immunity to complete the infection process 24 . Phenol oxidase (PO) of insect hemolymph and cuticle generally acts as a part of innate immunity of insect against infecting microbes. Larval PO is associated with melanin biosynthesis and haemocyte production for self defense; decreased PO indicates reduction of immunity which provide favorable conditions for pathogens growth inside the insect's body 25 . In our previous study, we have reported decreased larval phenol oxidase content in ME treated larval hemolymph and cuticle, in comparison with control 11 . For mycochemistry of ME , the crude ME was fractionized, and each fraction was evaluated against anopheline larvae 11 . MF8, out of 12, was most lethal to anopheline larvae. GC-MS analysis of MF8 confirmed us that 49 compounds were present. Out of these compounds, seven compounds were recorded as insecticidal or mosquitocidal in our previous work 11 . Hemocyte, circulating immune cell, which is a vital component of larval innate immunity, destroys fungal pathogens by phagocytosis 26 .
In Drosophila larvae, hemocytes serve immunological protection by melanizing and engulfing microbes and producing antimicrobial peptides. In addition, these immune cells by phagocytosis scavenge the apoptotic cells  www.nature.com/scientificreports/  www.nature.com/scientificreports/ and play an important role during metamorphosis of this fly [27][28][29] . Here we have detected fragmented nuclei of hemocyte cells by fluorescence microscopy in treated larvae with different ME doses of T. asperellum, and also observed increased percentage of hemocyte degeneration in dose-dependent manner. Our observation reveals that the mode of action of the fungus weakens larval immunity resulting in the larval death. Furthermore, statistical analysis of data from enzymatic assays of T. asperellum, B. bassiana and M. anisopliae exhibits that data were statistically significant (p < 0.05) and there are significant different among the three entomopathogens in respect to hydrolytic enzyme secretion and we found that T. asperellum secretes highest concentrations of cuticle degrading (hydrolytic) enzymes amongst these three entemopathogens. In our previous study 11 we have showed that T. asperellum has lower LD 50 (lethal dose for 50% killing of larvae) and LT 50 (lethal time for 50% killing of larvae) value amongst other known entomopathogens. Mechanistic study and comparison of enzymatic assay with other two also validate that T asperellum can be more effective than other two well known entomopathogens in controlling mosquito larvae.   www.nature.com/scientificreports/ After meticulous examination in the laboratory this research work comes to a fruitful conclusion which would contribute to future research works in this specific field. The mechanistic study of T. asperellum exhibits following mode of infections to kill anopheline larvae: (1) Secretion of mucilage from spore for its attachment on larval surface, (2) After germination of spore, penetration of insect cuticle by secretion of cuticle degrading enzymes (spore bound Pr1, chitinase and proteases) through infection peg, and (3) After penetration, secretion of toxins inside larval body which decrease larval phenol oxidase and degenerate hemocyte cells by nuclear fragmentations causing larval immunity breakdown followed by death. Application of the fungus as a new effective bio-control agent to eradicate anopheline larvae can open up new direction of Trichoderma research as entomopathogen and play a major role in mosquito vector control and disease management programme.

Materials and methods
Isolation of fungi and their identification. In this work fungal entomopathogens such as T. asperellum

Study of mycoparasitism or host parasitic interaction through microscopy. Compound micro-
scopic study of host-pathogen interaction: Mosquito larvae (3rd instar), treated with LD 50 dose (Lethal dose for 50% killing of larvae) (2.68 × 10 7 conidia / mL) of T. asperellum (LD 50 was determined by regression analysis of the bio-assay result in our previous study) 11 were taken out from the treatment set with needle and stained with  Detection of fungal spore adhesion on larval surface (Mucilage assay). Twenty anopheline larvae (3rd instar), were exposed to LD 50 dose (2.68 × 10 7 conidia / mL) of T. asperellum spore as estimated in our early paper 11 . Larvae were taken from treatment set at each 1 h of interval in grease free slides to examine the fungal spore adhesion on larval surface. 100 µL of 0.1% Ruthidium Red (Mucilage specific stain 22 was applied to each slide to detect the presence of mucilage on spore surface at spore attachment site on larval cuticle under compound microscope. Presence of mucilage can be detected by alteration of red color of the dye into pink 31 .

Detection of cuticle degrading enzymes (Spore bound pr1, Chitinase and Protease). Spore-bound
Pr1 enzyme assay. Spore bound Pr1 activities of preselected fungi (T. asperellum, B. bassiana and M. anisopliae) were assayed following the modified protocol of Shah et al. 32 . Fungal entomopathogens were grown in PDA plates and ten milligrams of conidia were harvested after definite incubation period. Conidia of each fungus were inoculated in 1 mL of 0.1 M Tris-HCL supplemented with 1 mM succinyl-ala-ala-pro-phe-p-nitroanilide (C 30 H 36 N 6 O 9 ) (Sigma-Aldrich) and incubated for 5 min at room temperature (28 ± 1 °C). After incubation, centrifugation of the sample was done at 12,000 g for 10 min at 4 °C. The yellow aqueous phase was collected after separating conidia from the sample and transferred to wells in a flat-bottom microtiter plate. Absorbance was taken at 405 nm using ELISA (Microtiter) reader (Biorad, USA). Experiments were done in triplicates for each fungus. Buffer substrate was used as control for each set.   Enzymatic assay for chitinase and protease. To assay chitinase enzyme activity standard protocol was followed 39,40 . Chitinase produces p-nitrophenol (yellow color compound) in reaction with PNG. In a 96 well microtitre plate, 10 µL of fungal culture filtrate was taken from each set. 10 µL of 10 mM PNG (p-nitrophenyl β-D-N glucosaminide) and 30 µL of 0.1 M PBS (pH: 6) were added into it. Here PNG was used as substrate. Six replicas were taken for each fungus. The micro titer plate was incubated at 37 °C for 1 h. After that, reactions were stopped by adding 50 µL of Na 2 CO 3 in each well. Absorbance was recorded at 415 nm wave length.
To determine protease activity, modified procedure of Tsuchida et al. 41 was followed. After 5 d of inoculation 200 µL of culture filtrate was taken out from each fungal culture in separate eppendroff tube (2 mL). 500 µL of 1%( W/V) casein powder suspended in 50 mM PBS were added as substrate for each set. Then each set was incubated for 15 min in water bath at 45 °C temperature for enzyme substrate reaction. Then 1 mL of 10% TCA was added in each set to terminate the reaction. After that, each reaction mixture was centrifuged at 10,000 rpm for 15 min. 500 µL of supernatant was taken from each set in separate eppendroff tube. Then the supernatant was mixed with 1 mL of 0.4 M Na 2 Co 3 and 0.5 mL of threefold diluted Folin-ciocalteu reagent and incubated at room temperature (37 °C) in the dark for 30 min. 100 µL of each resulting solution was taken in a 96 well microtitre plate, and absorbance of developing blue color was measured at 660 nm against reagent blank using distilled water in place of culture filtrate.
Quantitative estimation of chitinase and protease enzymes. Chitinase quantification. Fungi were inoculated in 50 mL of 1% chitin amended media (as described before) and incubated at shaker B.O.D at 150 rpm and 28 ± 2 °C temperature. After 5 d of inoculation, 5 mL of culture filtrates were taken from the media and the total proteins were recovered by Acetone precipitation 42 (sample: Acetone = 1:2) in 15 mL falcons. The precipitated proteins were subjected to quantitative estimation. A standard curve was prepared with different known concentrations of BSA by spectrophotometric method 43 . Concentration of chitinase enzymes were calculated using different known concentrations of BSA as standard by MS EXCEL, 2007.
Protease quantification Fungi were inoculated in 50 mL of 1% casein amended media (as described before) and incubated at shaker B.O.D at 150 rpm and 28 ± 2 °C temperature until the colour of casein disappeared. After that 5 mL of culture filtrates were taken from the media and the total proteins were precipitated by Acetone cut 42 (sample: Acetone = 1:2) in 15 mL falcons. The precipitated proteins were quantified as described above.
Staining of nucleus of larval haemocytes and fluorescence microscopy. The Cell suspension was placed in a grease free slide and 10 µL of aqueous DAPI solution (1 µg mL −1 ) were added [44][45][46] . The solutions were mixed properly by pipetting. Solution mixture were covered with a cover slip and incubated in dark for 15 min. After that, the slides were placed under inverted fluorescence microscope (Olympus-CKX53) for observation using Q imaging software. Statistical analysis. In each experiment mean and ± SD (Standard deviation) of data were calculated. www.nature.com/scientificreports/ In addition, in case of detection of chitinase and protease in broth culture experiment different OD value for each fungus was obtained during enzymatic assays. A t-test was performed using R (Ver: 4.1.0) with combining the OD values for enzymatic assays of each fungus to analyze whether the significant mean difference was present or not. A box plot model was also represented graphically using R (Ver: 4.1.0) for further confirmation. In case of quantitative estimation of chitinase and protease enzyme assay different mean concentrations of hydrolytic enzymes for each fungus (Chitinase and Protease) were obtained. A t-test was performed using R (Ver: 4.1.0) with combining the values for hydrolytic enzymes' concentrations of each fungus to analyze whether the significant mean difference was present or not and also a Duncan analysis was performed to measure specific differences among means concentration of hydrolytic enzymes and REI of three entomopathogen. A Duncan analysis was also done to measure mean percentages of deformed nuclei of treated anopheline larvae using SPSS (Ver: 13.0).

Data availability
Data are available from www. nature. com.