Benzoquinone synthesis-related genes of Tribolium castaneum confer the robust antifungal host defense to the adult beetles through the inhibition of conidial germination on the body surface

410

411

412

Akira, S., Uematsu, S., Takeuchi, O., 2006. Pathogen recognition and innate immunity.

Cell 124, 783-801.

413

414

Arakane, Y., Muthukrishnan, S., Beeman, R.W., Kanost, M.R., Kramer, K.J., 2005.

415

Laccase 2 is the phenoloxidase gene required for beetle cuticle tanning. Proc Natl Acad Sci U S A

416

102, 11337-11342.

417

418

419

Butt, T.M., Coates, C.J., Dubovskiy, I.M., Ratcliffe, N.A., 2016. Entomopathogenic

Fungi: New Insights into Host-Pathogen Interactions. Adv Genet 94, 307-364.

420

421

422

Cloudsley-Thompson, J.L., 1988. Adaptations to Extreme Environments., Evolution and

Adaptation of Terrestrial Arthropods. Springer, Berlin, Heidelberg, 80-98.

423

424

Ferrandon, D., Imler, J.-L., Hetru, C., Hoffmann, J.A., 2007. The Drosophila systemic

425

immune response: sensing and signalling during bacterial and fungal infections. Nature Reviews

426

Immunology 7, 862-874.

427

428

Hayakawa, Y., Kato, D., Kamiya, K., Minakuchi, C., Miura, K., 2017. Chitin synthase 1

429

gene is crucial to antifungal host defense of the model beetle, Tribolium castaneum. J Invertebr

430

Pathol 143, 26-34.

431

432

Hayakawa, Y., Sawada, M., Seki, M., Sirasoonthorn, P., Shiga, S., Kamiya, K.,

433

Minakuchi, C., Miura, K., 2018. Involvement of laccase2 and yellow-e genes in antifungal host

434

defense of the model beetle, Tribolium castaneum. J Invertebr Pathol 151, 41-49.

435

436

437

Hultmark, D., 2003. Drosophila immunity: paths and patterns. Current Opinion in

Immunology 15, 12-19.

438

18

439

440

Joop, G., Roth, O., Schmid-Hempel, P., Kurtz, J., 2014. Experimental evolution of

external immune defences in the red flour beetle. J Evol Biol 27, 1562-1571.

441

442

Koyama, H., Kato, D., Minakuchi, C., Tanaka, T., Yokoi, K., Miura, K., 2015.

443

Peptidoglycan recognition protein genes and their roles in the innate immune pathways of the red

444

flour beetle, Tribolium castaneum. J Invertebr Pathol 132, 86-100.

445

446

447

Lacey, L.A., Grzywacz, D., Shapiro-Ilan, D.I., Frutos, R., Brownbridge, M., Goettel, M.S.,

2015. Insect pathogens as biological control agents: Back to the future. J Invertebr Pathol 132, 1-41.

448

449

450

Lavine, M.D., Strand, M.R., 2002. Insect hemocytes and their role in immunity. Insect

Biochemistry and Molecular Biology 32, 1295-1309.

451

452

453

Lemaitre, B., Hoffmann, J., 2007. The host defense of Drosophila melanogaster, Annual

Review of Immunology, 697-743.

454

455

Li, J., Lehmann, S., Weißbecker, B., Ojeda Naharros, I., Schütz, S., Joop, G., Wimmer,

456

E.A., 2013. Odoriferous Defensive stink gland transcriptome to identify novel genes necessary for

457

quinone synthesis in the red flour beetle, Tribolium castaneum. PLoS Genet 9, e1003596.

458

459

460

Lu, H.L., St Leger, R.J., 2016. Insect Immunity to Entomopathogenic Fungi. Adv Genet

94, 251-285.

461

462

463

Markarian, H., Florentine, G.J., Prait Jr., J.J., 1978. Quinone production of some species of

Tribolium. J. Insect Physiol. 24, 785–790.

464

465

466

Moussian, B., 2010. Recent advances in understanding mechanisms of insect cuticle

differentiation. Insect Biochem Mol Biol 40, 363-375.

467

468

469

Ortiz-Urquiza, A., Keyhani, N. O., 2013. Action on the surface: Entomopathogenic fungi

versus the insect cuticle. Insects 4, 357-374.

470

471

Pedrini, N., Ortiz-Urquiza, A., Huarte-Bonnet, C., Fan, Y., Juárez, M.P., Keyhani, N.O.,

472

2015. Tenebrionid secretions and a fungal benzoquinone oxidoreductase form competing

473

components of an arms race between a host and pathogen. Proc Natl Acad Sci U S A 112, E3651-

474

3660.

19

475

476

Rafaluk-Mohr, C., Wagner, S., Joop, G., 2018. Cryptic changes in immune response and

477

fitness in Tribolium castaneum as a consequence of coevolution with Beauveria bassiana. J Invertebr

478

Pathol 152, 1-7.

479

480

Richards, S., Gibbs, R.A., Weinstock, G.M., Brown, S.J., Denell, R., Beeman, R.W.,

481

Gibbs, R., Bucher, G., Friedrich, M., Grimmelikhuijzen, C.J.P., Klingler, M., Lorenzen, M.D., Roth,

482

S., Schroeder, R., Tautz, D., Zdobnov, E.M., Muzny, D., Attaway, T., Bell, S., Buhay, C.J.,

483

Chandrabose, M.N., Chavez, D., Clerk-Blankenburg, K.P., Cree, A., Dao, M., Davis, C., Chacko, J.,

484

Dinh, H., Dugan-Rocha, S., Fowler, G., Garner, T.T., Garnes, J., Gnirke, A., Hawes, A., Hernandez,

485

J., Hines, S., Holder, M., Hume, J., Jhangiani, S.N., Joshi, V., Khan, Z.M., Jackson, L., Kovar, C.,

486

Kowis, A., Lee, S., Lewis, L.R., Margolis, J., Morgan, M., Nazareth, L.V., Nguyen, N., Okwuonu,

487

G., Parker, D., Ruiz, S.-J., Santibanez, J., Savard, J., Scherer, S.E., Schneider, B., Sodergren, E.,

488

Vattahil, S., Villasana, D., White, C.S., Wright, R., Park, Y., Lord, J., Oppert, B., Brown, S., Wang,

489

L., Liu, Y., Worley, K., Elsik, C.G., Reese, J.T., Elhaik, E., Landan, G., Graur, D., Arensburger, P.,

490

Atkinson, P., Beidler, J., Demuth, J.P., Drury, D.W., Du, Y.-Z., Fujiwara, H., Maselli, V., Osanai,

491

M., Robertson, H.M., Tu, Z., Wang, J.-J., Wang, S., Song, H., Zhang, L., Werner, D., Stanke, M.,

492

Morgenstern, B., Solovyev, V., Kosarev, P., Brown, G., Chen, H.-C., Ermolaeva, O., Hlavina, W.,

493

Kapustin, Y., Kiryutin, B., Kitts, P., Maglott, D., Pruitt, K., Sapojnikov, V., Souvorov, A., Mackey,

494

A.J., Waterhouse, R.M., Wyder, S., Kriventseva, E.V., Kadowaki, T., Bork, P., Aranda, M., Bao, R.,

495

Beermann, A., Berns, N., Bolognesi, R., Bonneton, F., Bopp, D., Butts, T., Chaumot, A., Denell,

496

R.E., Ferrier, D.E.K., Gordon, C.M., Jindra, M., Lan, Q., Lattorff, H.M.G., Laudet, V., von

497

Levetsow, C., Liu, Z., Lutz, R., Lynch, J.A., da Fonseca, R.N., Posnien, N., Reuter, R., Schinko,

498

J.B., Schmitt, C., Schoppmeier, M., Shippy, T.D., Simonnet, F., Marques-Souza, H., Tomoyasu, Y.,

499

Trauner, J., Van der Zee, M., Vervoort, M., Wittkopp, N., Wimmer, E.A., Yang, X., Jones, A.K.,

500

Sattelle, D.B., Ebert, P.R., Nelson, D., Scott, J.G., Muthukrishnan, S., Kramer, K.J., Arakane, Y.,

501

Zhu, Q., Hogenkamp, D., Dixit, R., Jiang, H., Zou, Z., Marshall, J., Elpidina, E., Vinokurov, K.,

502

Oppert, C., Evans, J., Lu, Z., Zhao, P., Sumathipala, N., Altincicek, B., Vilcinskas, A., Williams, M.,

503

Hultmark, D., Hetru, C., Hauser, F., Cazzamali, G., Williamson, M., Li, B., Tanaka, Y., Predel, R.,

504

Neupert, S., Schachtner, J., Verleyen, P., Raible, F., Walden, K.K.O., Angeli, S., Foret, S., Schuetz,

505

S., Maleszka, R., Miller, S.C., Grossmann, D., Tribolium Genome Sequencing, C., 2008. The

506

genome of the model beetle and pest Tribolium castaneum. Nature 452, 949-955.

507

Roth, L.M., 1943. Studies on the Gaseous Secretion of Tribolium Confusum Duval II. the

508

Odoriferous Glands of Tribolium Confusum. Annals of the Entomological Society of America 36,

509

397-424.

510

20

511

St Leger, R.J., Bidochka, M.J., Roberts, D.W., 1994. Isoforms of the cuticle-degrading Pr1

512

proteinase and production of a metalloproteinase by Metarhizium anisopliae. Arch Biochem Biophys

513

313, 1-7.

514

515

516

St Leger, R.J., Goettel, M., Roberts, D.W., Staples, R.C., 1991. Pre-penetration events

during infection of host cuticle by Metarhizium anisopliae. J. Invertebr. Pathol. 58, 168-179.

517

518

Strand, M.R., 2008. The insect cellular immune response. Insect Science 15, 1-14.

519

520

521

Thomas, M.B., Read, A.F., 2007. Fungal bioinsecticide with a sting. Nat Biotechnol 25,

1367-1368.

522

523

524

Tomoyasu, Y., Denell, R.E., 2004. Larval RNAi in Tribolium (Coleoptera) for analyzing

adult development. Development Genes and Evolution 214, 575-578.

525

526

Tomoyasu, Y., Miller, S.C., Tomita, S., Schoppmeier, M., Grossmann, D., Bucher, G.,

527

2008. Exploring systemic RNA interference in insects: a genome-wide survey for RNAi genes in

528

Tribolium. Genome Biology 9.

529

530

Yokoi, K., Hayakawa, Y., Kato, D., Minakuchi, C., Tanaka, T., Ochiai, M., Kamiya, K.,

531

Miura, K., 2015. Prophenoloxidase genes and antimicrobial host defense of the model beetle,

532

Tribolium castaneum. J Invertebr Pathol 132, 190-200.

533

534

Yokoi, K., Koyama, H., Ito, W., Minakuchi, C., Tanaka, T., Miura, K., 2012a.

535

Involvement of NF-κB transcription factors in antimicrobial peptide gene induction in the red flour

536

beetle, Tribolium castaneum. Dev Comp Immunol 38, 342-351.

537

538

Yokoi, K., Koyama, H., Minakuchi, C., Tanaka, T., Miura, K., 2012b. Antimicrobial

539

peptide gene induction, involvement of Toll and IMD pathways and defense against bacteria in the

540

red flour beetle, Tribolium castaneum. Results Immunol 2, 72-82.

541

542

Zou, Z., Evans, J.D., Lu, Z., Zhao, P., Williams, M., Sumathipala, N., Hetru, C., Hultmark,

543

D., Jiang, H., 2007. Comparative genomic analysis of the Tribolium immune system. Genome

544

Biology 8.

545

21

546

Legends to figures

547

Fig. 1. SEM observations of conidia on the body surface of pupae. Day 3 pupae were infected with

548

either B. bassiana (left) or M. anisopliae (right) by the immersion in conidial suspensions. The cell

549

density of the suspension was 1 x 108 cell/ml for both fungal species. The surface of test pupae was

550

thereafter observed and photographed by SEM every 12h post challenge. The magnification was x

551

3000 throughout, and the scale bars represent 10 μm.

552

553

Fig. 2. SEM observations of conidia on the body surface of adults. Day 1 adults were infected with

554

either B. bassiana (left) or M. anisopliae (right) by the immersion in conidial suspensions. The cell

555

density of the suspension was 1 x 108 cell/ml for both fungal species. The surface of test adults was

556

observed and photographed by SEM at 48h post challenge. The pictures of both dorsal and ventral

557

body surfaces are shown. The magnification was x 1000 throughout, and the scale bars represent 30

558

μm.

559

560

Fig. 3. Developmental expression profiles of GT39, GT62 and GT63 during pupal and adult stages.

561

The mRNA levels of the three genes were determined by qRT-PCR, and shown as relative

562

abundances to those of reference gene RPL32.

563

(adult) are ages in days of respective developmental stages. Each vertical bar represents mean ± SD

564

from three biological replicates.

The numerals that follow the symbol P (pupa) or A

565

566

Fig. 4. Knockdown efficiencies of GT39, GT62 and GT63. Day 1 pupae were injected with 100 ng

567

of GT39, GT62 and GT63 dsRNA, and the mRNA levels determined in resulting day 1 adults. Each

568

bar represents mean ± SD from three biological replicate.

569

negative controls, and the significantly different values from the control marked by asterisks with p-

22

MalE dsRNA-treated animals served as

570

values.

571

572

Fig. 5. Survival of knockdown adults upon fungal infection. Day 1 pupae were treated with 100 ng

573

dsRNA of GT39, GT62 or GT63, and the resulting adults of age day 6 was examined in terms of

574

survival against the infection of B. bassiana (left) or M. anisopliae (right). The survival rates were

575

recorded every 24h up to 192 h post fungal challenge, and the results shown in Kaplan-Meier plots.

576

The conidial density used for infection was 1 x 107 cell/ml for both fungal species. The malE

577

dsRNA-treated animals were used as negative controls. The survival curves that are significantly

578

different from the control are indicated by asterisks with p-values.

579

580

Fig. 6. Conidial germination and hyphal growth on adult body surface after knockdown of GT39,

581

GT62 and GT63. Day 1 pupae were injected with 100 ng dsRNA of GT39, GT62, GT63 or negative

582

control malE. The resulting adults of age day 6 were challenged by the two fungal species, and

583

observed by SEM at 48 h post infection. Other details are the same as in Fig. 2.

584

585

586

23

Table 1. Primer pairs used for qRT-PCR.

Target gene

Forward primer sequence (5’-3’)

Reverse primer sequence (5’-3’)

GT39

TTGCTGAAGTCTACGAGAACAC

GAGCTCGATGGTGTCATTGTC

149 bp

JX569829

GT62

GCGACGATATGGGACATAACGA

GTACAAGCATTCTGGACGTAGTA

121 bp

JX569830

GT63

ACGAAGCGACCGCAAATGTTGA

ACCGTCCCAGCATCCATCAC

154 bp

JX569831

RPL32

ACCGTTATGGCAAACTCAAACG

TGTGCTTCGTTTTGGCATTGGA

183 bp

Glean_06106

Table 1. Sawada et al.

Product length

Accession #

Table 2. Primer pairs used for cDNA template preparation for dsRNA synthesis.

Target gene

Forward primer sequence (5’ to 3’)

Reverse primer sequence (5’ to 3’)

GT39

TAATACGACTCACTATAGGG-

TAATACGACTCACTATAGGG-

-GGAGGTCACCCAGAACAACT

-TGACATCCCTTGGCACATATTC

TAATACGACTCACTATAGGG-

TAATACGACTCACTATAGGG-

-AAGGTGGCACATACGATGGATA

-GCGGATTGGCATTCGGATCAAT

TAATACGACTCACTATAGGG-

TAATACGACTCACTATAGGG-

-TCAGTGGAACGTGTGGTCGAATA

-TTGCGCCCAATTCGTCACCAT

TAATACGACTCACTATAGGG -

TAATACGACTCACTATAGGG -

-ATTGCTGCTGACGGGGGTTAT

-ATGTTCGGCATGATTTCACCTTT

GT62

GT63

malE

T7 RNA polymerase promoter sequences are shown in italic.

Table 2. Sawada et al.

Product length

458 bp

375 bp

462 bp

518 bp

Hours after

Challenge

B. bassiana

M. anisopliae

12h

10

10 µm

µm

10

10 µm

µm

xx 3000

3000

xx 3000

3000

10

10 µm

µm

10

10 µm

µm

xx 3000

3000

xx 3000

3000

10

10 µm

µm

10

10 µm

µm

xx 3000

3000

xx 3000

3000

10

10 µm

µm

10

10 µm

µm

xx 3000

3000

xx 3000

3000

24h

36h

48h

Fig. 1

Sawada

et al.

48h after

Challenge

B. bassiana

M. anisopliae

Dorsal

body

surface

30

30 µm

µm

30

30 µm

µm

x 1000

x 1000

30

30 µm

µm

30

30 µm

µm

x 1000

x 1000

Ventral

body

surface

Fig. 2. Sawada et al.

Relative mRNA amount

(/RPL32)

GT39

0.06

0.05

0.04

0.03

0.02

0.01

P0

P1

P2

P3

P4

P5

A0

A1

A3

A4

A5

A7 A14 A21

A0

A1

A3

A4

A5

A7 A14 A21

A0

A1

A3

A4 A5

Adult

A7 A14 A21

Relative mRNA amount

(/RPL32)

GT62

0.05

0.04

0.03

0.02

0.01

P0

P1

P2

P3

P4

P5

Relative mRNA amount

(/RPL32)

GT63

0.6

0.5

0.4

0.3

0.2

0.1

P0

P1

P2 P3

Pupa

P4

P5

Age in days

Fig. 3. Sawada et al.

GT39

0.025

0.02

0.015

0.01

0.005

* p<0.001

0.14

0.012

0.01

0.008

0.006

0.004

0.002

* p<0.001

malE

GT39

Fig. 4. Sawada et al.

dsRNA

malE

GT62

Relative mRNA amount (/RPL32)

0.014

Relative mRNA amount (/RPL32)

Relative mRNA amount (/RPL32)

0.03

dsRNA

GT63

GT62

0.12

0.1

0.08

0.06

* p<0.005

0.04

0.02

dsRNA

malE

GT63

B. bassiana

1.00

1.00

malE

gene KD

0.80

0.60

0.40

* p<0.001

Survival rate

Survival rate

GT39 KD

M. anisopliae

malE

gene KD

0.80

0.60

0.40

* p<0.001

0.20

0.20

0.00

0.00

24

48

72

96 120 144 168 192

24

0.60

0.40

* p<0.001

Survival rate

Survival rate

malE

gene KD

0.80

malE

gene KD

0.80

0.60

0.40

* p<0.001

0.20

0.00

0.00

24

48

72

96 120 144 168 192

24

48

72

96 120 144 168 192

Hours after challenge

Hours after challenge

1.00

1.00

malE

gene KD

0.80

0.60

0.40

* p<0.001

0.20

Survival rate

Survival rate

96 120 144 168 192

1.00

1.00

0.20

GT63 KD

72

Hours after challenge

Hours after challenge

GT62 KD

48

malE

gene KD

0.80

0.60

0.40

* p<0.001

0.20

0.00

0.00

24

48

72

96 120 144 168 192

Hours after challenge

Fig. 5. Sawada et al.

24

48

72

96 120 144 168 192

Hours after challenge

48h after

Challenge

B. bassiana

M. anisopliae

malE

30

30 µm

µm

30

30 µm

µm

x 1000

x 1000

30

30 µm

µm

30

30 µm

µm

x 1000

x 1000

30

30 µm

µm

30

30 µm

µm

x 1000

x 1000

30

30 µm

µm

30

30 µm

µm

x 1000

x 1000

GT39 KD

GT62 KD

GT63 KD

Fig. 6.

Sawada

et al.

...