Jefatura de Productos Institucionales y Marketing

Beauvericin

Beauvericin when fragmented produces fragment ions at (m/z) 261 and (m/z) 522 (Logrieco et al., 1998). This was observed for beauvericin and all analogues that were fragmented in this study such as; basic beauvericin (m/z) 784, beauvericin H (m/z) 802.5, beauvericin A (m/z) 798, and beauvericin B (m/z) 812. The fragmentation patterns of two other metabolites contained in the same fraction, (m/z) 782.6 and (m/z) 820, showed no fragment ions at (m/z) 261 and (m/z) 522 which confirmed they were not beauvericin, despite being found consistently in the beauvericin-containing fractions. The mass ion (m/z) region of beauvericin and varieties is observed between (m/z) 784 to (m/z) 830.

Beauvericin A and F share the same mass ion at (m/z) 798. The fragmentation pattern for beauvericin A was as described in Figure 4.15 but not all the fragment ions reported were observed for the beauvericin A in this project. Therefore it could be a beauvericin F.

Beauvericin (m/z) 784 from this study was fragmenting like the technical beauvericin from Sigma- Aldrich. The purified beauvericin of K4B4 was demonstrating mild contact insecticidal activity whereas the technical standard did not. The source of the technical beauvericin from Sigma-Aldrich was not stated by the manufacture on the label. Common sources of beauvericin would be from B. bassiana and many species in the fungal genus Fusarium (Logrieco et al., 1998). Beauvericin in general was not reported to act as a contact insecticide. The direct lethal effect of beauvericin in insects was questioned (Khachatourians and Arora, 2001) for it was not detected in the haemolymph of 90% corn earworm larvae (H. zea) infected by B. bassiana (Champlin and Grula, 1979). When 6 µg of beauvericin was injected into the haemolymph of adult blowflies (Calliphora erythrocephala) and corn earworm larvae, the insects didn’t die (Champlin and Grula, 1979). Ingestion of beauvericin by mosquito larvae (Culex

pipiens autogenicus) however damaged the epithelium of the midgut with intensive vacuolization of

cells and visible damage to mitochondria, membranes and nuclei (Zizka and Weiser, 1993). Perhaps the excreted gut by Culex pervigilans larvae in Chapter 3 was due to beauvericin present in the mycelial

extract of K4B3. Beauvericin was also toxic to brine shrimps Artemia salina with LD50 of 2.8 µg ml-1

water (Moretti et al., 2007). Its ionophore ability, forming Na+ _{and K}+ _{complexes, enables it to increase}

the permeability of artificial and biological membranes effectively and pass through the membranes (Ovchinnikov et al., 1971). It is appropriate, therefore, to conclude not all insects are affected by beauvericin (Champlin and Grula, 1979). Its toxicity was thought to vary depending upon the insect species and stage tested (Khachatourians and Arora, 2001). In this project, the green peach aphids M.

persicae were sensitive towards fractions containing beauvericin and its different analogues through

contact. Preliminary 1D NMR showed similar structure to the technical beauvericin with some additional signals. Further 2D-NMR will have to be carried out to ascertain if the beauvericin of K4B3 is structurally different from the technical beauvericin of Sigma-Aldrich.

Apart from producing different analogues of beauvericin, K4B3 produced beauvericin and its analogue beauvericn A and B in large amounts (Figure 4.25 and 4.30). B. bassiana is a non-host specific pathogen (Strasser et al., 2000a) and hence there could be a necessity to produce a diversity of metabolites including different analogues of a given toxin. Still, the production of such high quantities of beauvericins with slight changes in structures and functional group appears costly and redundant. Individually, they were moderately insecticidal as observed in K4B3 via contact and by others via injection (Gupta et al., 1995; Kanaoka et al., 1978; Suzuki et al., 1977). They were moderate antifungal (Wang and Xu, 2012) and antibiotic (Xu et al., 2008) but were strong anti-viral agents (Shin et al., 2010). The relatively low biocidal activity of beauvericins may partly be compensated through quantity produced in K4B3. They could also be produced in abundance to work in synergy among the different analogues and along with other metabolites to elicit stronger biocidal activity (Cechinel-Filho, 2012). In a High Throughput Synergy Screening assay using 20,000 microbial extracts, beauvericin

dramatically synergized the ketoconazole fungicidal activity against diverse fungal pathogens. Ketoconazole alone shows little detectable fungicidal activity. Beauvericin might synergize with other metabolites and enhance the insecticidal activity but this will have to be tested.

Bassianolide

The metabolite detected at (m/z) 909.6 closely matched the reported bassianolide except for the difference in relative intensity of all fragment ions. As little as 7.5 µg (in 1000 ppm crude extract) of bassianolide was sufficient to kill aphids effectively via contact. No published records have reported contact activity of bassianolide. Most records on the insecticidal activity of bassianolide were from bassianolide injected into insects or added into insect feed. Fifth instar larvae of the silkworm B. mori,

died at a lethal dose of more than 5 µg larva-1 _{when injected with a water-based bassianolide}

suspension (Kanaoka et al., 1978). When bassianolide was added to the artificial diet at 13 ppm and fed to the larvae, they died after four days (Kanaoka et al., 1978; Suzuki et al., 1977). Corn earworm larvae were paralyzed shortly after injections of bassianolide into their haemolymph (Champlin and

Grula, 1979).Silkworms infected by B. bassiana were also found containing bassianolide (Suzuki et al.,

1977). The contact insecticidal activity of bassianolide (in water or organic solvent) against aphids in this study, was most likely due to its relatively hydrophilic interior and hydrophobic exterior structure (Kanaoka et al., 1978; Suzuki et al., 1977) allowing it to permeate across lipophilic insect cuticle. Fractions containing bassianolide were more insecticidal compared to beauvericins at the same concentration (Table 4.3) in this study. Though the 1D NMR analysis of bassianolide was not successful, the fragmentation pattern obtained by MSMS was sufficient to draw the conclusion that bassianolide was produced by K4B3.

Beauverolides

The presence of an immonium ion at (m/z) 139 or (m/z) 167 indicates the presence of hydroxy- methyloctanoyl and hydroxy-methyldecanoyl residues corresponding to a beauverolide (Bekker et al., 2013). The presence of additional immonium ions at (m/z) 72, 86, 86, 104, 120, 136 and 159 indicates the presence of certain amino acids, valine, leucine and isoleucine, methionine, phenylalanine, tyrosine and tryptophan respectively (Falick et al., 1993; Papayannopoulos, 1995) constructing a beauverolide (Bekker et al., 2013).

The mass ion (m/z) 544 was observed with similar fragmentation patterns as the reported

beauverolide B, C31H49N3O5: 544, 526(1), 516(25), 431(73), 413(3), 284(1), 266(4), 238(1), 279(100)

(Bekker et al., 2013; Jegorov et al., 2004). Similarly for beauverolide E, C29H45N3O5: (m/z) 516.3

(Jegorov et al., 2004) and beauverolide F, C33H45N3O5: (m/z) 564.3 (Jegorov et al., 2004).

455.3, 478.3, 481.3, 486.3, 487, 498.38, 500.3, 502.3, 504.3, 520.3, 526.5, 530.3, 538.3, 540.3, 541.29, 566.3, 580.3, 586.3, 603.3, 604.4, 625.3, 631.3, 645.3, 682.3, 700.3. The majority of beauverolides and beauverolide-suspects produced by K4B3 in submerged culture were akin to beauverolides produced by a B. bassiana in the presence of live ant muscle tissues but not brain tissues (Bekker et al., 2013) (Table 4.10).

Table 4.10. Possible beauverolides (highlighted in orange) detected from K4B3, compared to the secreted beauverolides in ex vivo culturing systems using muscle and brain tissues of ants (highlighted in yellow) (Bekker et al., 2013).

[M+H]+ _{beauverolide Brain Muscle K4B3}

482.3261 (Bekker et al., 2013)

488.2894 (Bekker et al., 2013)

500.3652 (Bekker et al., 2013) suspect

510.3574 (Bekker et al., 2013)

514.3808 (Bekker et al., 2013)

516.3432 E Figure 4.36

528.3965 (Bekker et al., 2013)

530.3588 (Bekker et al., 2013) suspect

532.3426 (Bekker et al., 2013) 544.3745 B Figure 4.29 558.3902 (Bekker et al., 2013) 560.3739 (Bekker et al., 2013) 564.3872 F Figure 4.37 576.4405 (Bekker et al., 2013)

580.3866 (Bekker et al., 2013) suspect

592.4073 C

603.4239 J suspect

608.4067 (Bekker et al., 2013)

615.466 (Bekker et al., 2013)

631.444 K suspect

Beauverolides were not pursued for further investigation in this research even though they were detected as fractions containing them did not show direct contact insecticidal activities against aphids. Various biological tests have indicated that beauverolides do not exhibit bactericidal, fungicidal or direct insecticidal effects but act more on the insect immunomodulation (modifying the immune system) (Jegorov et al., 2004; Kuzma et al., 2001). This very role of beauverolides is logical for K4B3 as the isolate is capable of infecting DBM caterpillars rapidly and efficiently. The high amount of different beauverolides detected by chance in the mycelial extract aligns with B. bassiana’s need to infect without fail as a non-host specific pathogen.

Unidentified mass ions

Fraction 4 from the LH20 experiment contained no bassianolide (m/z) 909.6 but traces of beauvericin (m/z) 784. The intriguing reason for its insecticidal activity prompted it to be fractionated on HPLC. It

showed high bioactivity at 15th _{min repeatedly. The MS scan of the fraction showed no presence of}

depsipeptide but the presence of (m/z) 196, 387, 415, 437 and 453. The mass ion (m/z) 453 has an immonium ion tryptophan (m/z) 159, but lacks the immonium ions described for a beauverolide. This has not been further pursued.