Electronic Supplementary Material (ESM)
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2
Material and Methods
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a. Insects
4
b. Preparation of vegetative bacterial cells
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c. Preparation of B. thuringiensis HD73 spore solution and application of MVP
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d. Larval survival and bacterial recovery analysis upon injection experiments
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e. Preparation of primary hemocyte culture
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f. Phagocytosis assay
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g. Migration assay
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h. Apoptosis assay
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i. Lytic zone assay
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j. Phenoloxidase (PO) assay
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k. Data analysis
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Tables
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Suppl. Table S1. Cox Regression Model for larval survival of H. virescens and H. subflexa
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upon injection with different concentrations of vegetative cells of
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S. entomophila and B. thuringiensis.
18
Figures
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Suppl. Figure S1. Larval survival of H. virescens and H. subflexa on different concentrations
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of Bt spores.
Suppl. Figure S2. Larval growth rate and survival of H. virescens and H. subflexa on different
concentrations of Cry1Ac toxin (MVP).
Suppl. Figure S3. The percentage of H. virescens and H. subflexa hemocytes migrating to
vegetative cells of S. entomophila, B. thuringiensis and B. subtilis.
Suppl. Figure S4. Enzyme activity in the hemolymph of H. virescens and H. subflexa larvae
Barthel et al. Electronic supplementary material
S1
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injected with vegetative cells S. entomophila, B. thuringiensis and B. subtilis.
Material and Methods
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a. Insects
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In Jena, both strains were maintained by single-pair matings of non-siblings to avoid
31
inbreeding, and fertility and genetic variation (AFLPs) remained high and constant throughout
32
the experiment.
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whereas larvae of Hv were reared on artificial diet described in Burton et al. (1970) [1].
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Larvae of both species were held in a climate chamber (Snijders, Tilburg, The Netherlands) at
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26°C, 60 +/-10% relative humidity and 14:10 light cycle. Third to fourth instar Hv and Hs
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larvae were used in all experiments.
Larvae of Hs were reared on a corn-soy blend diet (provided by NCSU),
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b. Preparation of vegetative bacterial cells
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Bacillus subtilis and Serratia entomophila were obtained from the Department of Bioorganic
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Chemistry (MPICE, Jena, Germany), except B. thuringiensis, which was provided by Dr.
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Yannick Pauchet (Dept. Entomology, MPICE, Jena, Germany). Bacterial strains were
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cultured at 30°C and 250 rpm in lysogeny broth (LB) broth or on LB agar [2], except for
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S. entomophila which was grown in CASO medium [3]. Vegetative bacterial cells were
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obtained from overnight cultures and cell counts were estimated by optical density at 600 nm
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(BioPhotometer, Eppendorf, Hamburg, Germany). Beforehand, known numbers of colony
46
forming units (CFU) were plotted against their corresponding optical density at 600nm to
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obtain a standard curve for bacterial cell concentration of each strain.
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c. Preparation of B. thuringiensis HD73 spore solution and application of MVP
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During sporulation, the strain B. thuringiensis HD73 produces the Cry1Ac protein that is toxic
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to certain lepidopteran insects [4]. The spore solution was newly prepared beforehand using
Barthel et al. Electronic supplementary material
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the following protocol. Bacterial cells from a glycerol stock were plated on LB agar and kept
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at 30°C overnight. Subsequently, one bacterial colony was harvested and resuspended in 5 ml
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LB medium and allowed to grow at 30°C overnight on a bacterial shaker at 200 rpm. The
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following day, 100 µl of this bacterial culture was added to 50 ml HCO medium (containing
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per liter: 7 g casein hydrolysate; 6.8 g KH2PO4; 0.12 g MgSO4 7H2O; 0.0022g MnSO4 4H2O;
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0.014g ZnSO4 7H2O; 0.02 g Fe2(SO4)3; 0.018 g CaCl2 4H2O; 3 g glucose; the pH was
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adjusted to 7.2) [5]. After seven days at 30°C and 250 rpm, spores were harvested. To
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estimate their concentration, serial dilutions of this suspension were plated onto LB agar. The
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agar plates were incubated at 30°C for 48 hours and germinated bacterial colonies were
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counted (CFU/ml; colony forming units). The spore suspension was stored at 4°C and before
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each application the spore concentration was newly determined. Spore solutions of
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B. thuringiensis HD73 were only used for feeding experiments with Hs and Hv to investigate
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differences in their larval growth and survival (Fig. 1 and Fig. S1).
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In addition, we tested the efficacy of Cry1Ac toxin against both species apart from the
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effect of Bt spores. The Cry1Ac toxin used in this study was the formulated product MVP®
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(Mycogen Corporation, USA). MVP contains only the Cry1Ac toxin of Bacillus thuringiensis
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Berliner var. kurstaki, encapsulated within dead cells of Pseudomonas fluorescens [6].
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Aliquots of MVP were added to artificial diet to obtain concentrations of 0.1, 1.0 and 10 µg
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Cry1Ac/ml diet, which were provided to early 3th instar larvae of both species. Larval growth
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and survival was recorded daily for 7 days. In the control treatment, larvae were reared on
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pure artificial diet. The larval growth rate is given as average larval growth rate over 7 days
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relative to the corresponding control growth rate.
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d. Larval survival and bacterial recovery analysis upon injection experiments
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To document differences in the toxicity of entomopathogenic bacteria (B. thuringiensis and
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S. entomophila) to Hs and Hv larvae, each larva was injected with 104 or 105 vegetative cells
Barthel et al. Electronic supplementary material
S3
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of the corresponding bacterial strain. Infected larvae were kept at room temperature and the
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number of survivors was counted from 10 until 24 hours after injection. The sample size for
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each treatment varied from 30 to 44 larvae.
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To investigate whether entomopathogenic bacteria (B. thuringiensis or S. entomophila)
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and non-pathogenic bacteria (B. subtilis) survive within Hs and Hv larvae, 5x104 vegetative
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bacterial cells were injected into the haemocoel. Treated larvae were kept at room temperature
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and hemolymph samples were obtained after fixed time-points over a period of 24 hours after
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injection using a hypodermic needle. Serial dilutions of the hemolymph samples were plated
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onto LB agar. The agar plates were incubated at 30°C for 48 hours and bacterial colonies
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were counted and given in CFU/ml (colony forming units). The bacterial colonies were
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calculated on average of six hemolymph samples, i.e. from six larvae, per time point.
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e. Preparation of primary hemocyte culture
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Hemolymph was collected by bleeding uninfected 4th instar larvae of Hv and Hs using a
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hypodemic needle. Freely flowing hemolymph of approximately 30 larvae was collected into
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15ml FALCON tubes (VWR International GmbH, Darmsadt, Germany) containing 10ml of
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cold and sterile anticoagulant buffer (62mM NaCl, 100mM glucose, 10mM EDTA, 30mM
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trisodium citrate, 26mM citric acid; all chemicals were obtained from Carl Roth, Karlsruhe,
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Germany) [7]. The solution was centrifuged (2500 pm for 15 sec) and the resultant hemocyte
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pellet was resuspended in anticoagulant buffer. The number of hemocytes was determined
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using a CASY cell counter (Roche Innovatis AG, Bielefeld, Germany) and cell concentrations
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were adjusted depending on the in vitro assay (see below). These hemocyte solutions were
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directly used in all in vitro experiments and referred to as primary cell cultures.
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f. Phagocytosis assay
Barthel et al. Electronic supplementary material
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To determine whether Hv and Hs differ in the rates of phagocytosis of vegetative cells of
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B. subtilis, B. thuringiensis and S. entomophila, phagocytosis by Hv and Hs hemocytes was
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determined. Hemocytes of primary cell cultures from Hv and Hs were stained with
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Vybrant7DiO cell labeling solution (green-fluorescent carbocyanines, Invitrogen, Darmstadt,
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Germany) in a 1:200 dilution in anticoagulation buffer for 20 min at room temperature. After
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staining, 106 insect cells were added to 107 bacterial cells, which had been dyed with
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Vybrant7DiD cell labeling solution (red-fluorescent carbocyanines; Invitrogen) in a 1:200
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dilution for 20 minutes at 37°C and washed afterwards two times with DPBS (Lonza)
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supplemented with 1% BSA (AppliChem).
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The rate of phagocytosis was observed in 400µl DPBS/1% BSA and by analyzing
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samples after 7.5, 15, 30, 45, 60 and 120 minutes. To remove excess bacteria after incubation,
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hemocytes were washed two times with DPBS/1% BSA and centrifuged at low speed. The
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samples were assayed with a BD LSR II flow cytometer and the DIO+/DID+ positive
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hemocytes, i.e. DIO+ positive hemocytes which incorporated DID+ positive bacterial cells,
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were analyzed with the BD FACSDiva™ software. Only hemocytes with a larger size and a
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higher granularity than bacteria were calculated. Additionally, labeled-hemocytes and labeled-
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bacteria separately served as positive controls, whereas unlabeled hemocytes and unlabeled
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bacteria are used as negative staining controls to verify a successful staining. The assay was
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performed at least five times.
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g. Migration assay
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The migration activity of hemocytes is a measure of bacteria recognition by these hemocytes.
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To compare the migration activity of Hv and Hs hemocytes to vegetative cells of B. subtilis,
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B. thuringiensis and S. entomophila, a Boyden chamber assay was performed. A Boyden
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chamber is an in vitro method to measure the chemotactic activity of solutes on free moving
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cells e.g. hemocytes [8]. For this assay, we used BD Falcon™ 24-well multiwell plates and
Barthel et al. Electronic supplementary material
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BD Falcon™ cell culture inserts with a translucent PET (polyethylene terephthalate; VWR
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International GmbH, Darmsadt, Germany) membrane containing 8.0 µm pores. The bacteria
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were washed once with DPBS (Lonza, Basel, Switzerland) supplemented with 1% BSA
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(AppliChem, Darmstadt, Germany). The bacteria were used as a source of chemoattractant
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and 5x107 cells were diluted in 500 µl DPBS/1% BSA and placed in the lower compartment
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of a Boyden chamber. Cells of primary Hv and Hs hemocyte cultures were harvested and the
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cell count was calculated with a CASY7 cell counter in a 1:100 dilution with physiological
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saline solution (CASYTON, Innovatis AG) to discriminate between dead and living cells.
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Living Hv and Hs cells have a size of at least 6.5 µm. To reseed the hemocytes, in the upper
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compartment of the Boyden chamber 5x105 hemocytes were suspended in 300 µl
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anticoagulation buffer. After incubation for 1 hour at room temperature, the cells on the upper
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surface of the filter were completely removed by a sterile applicator (Böttger, Bodenmais,
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Germany) before 500 µl of anticoagulation buffer was added at high pressure. The pushed-
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through buffer was analyzed with a CASY7 cell counter and the living cells were calculated.
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Due to the fact that hemocytes are highly motile cells, buffer without bacteria served as the
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base line, i.e. 100% migration rate. To quantify the percentage of hemocyte migration towards
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the bacterial cells, we calculated the percentage of migrated hemocytes relative to the control.
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The assay was performed at least four times.
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h. Apoptosis assay
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To examine the influence of vegetative cells of B. subtilis, B. thuringiensis and
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S. entomophila on the viability of Hv and Hs hemocytes, apoptosis assays were conducted. Hv
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and Hs cells of primary hemocyte cultures were harvested and washed once with
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anticoagulation buffer. Bacteria were washed once with DPBS (Lonza) supplemented with
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1% BSA (AppliChem). 106 hemocytes were incubated with 107 bacteria in 50 µl
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anticoagulation buffer for 30 minutes, 1 hour and 2 hours at room temperature. Hemocytes
Barthel et al. Electronic supplementary material
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without bacteria served as a control at every time point. To remove bacteria, after incubation
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the insect cells were washed three times with TC-Buffer (140 mM NaCl, 10 mM Tris, 2 mM
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CaCl2, 1 mM MgCl2, pH 7.4) and centrifuged at low rpm. In the process of apoptosis, annexin
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(a calcium-dependent phospholipid-binding protein) binds phosphatidylserine which is
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translocated to the extracellular membrane. Therefore, the apoptosis marker Rh Annexin V-
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APC (eBioscience) was added to the hemocytes in 100 µl TC-Buffer for 15 minutes at room
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temperature. Stained hemocytes were analyzed with a LSR II flow cytometer (BD Bioscience,
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Heidelberg, Germany). The percentage of apoptotic hemocytes (annexin-positive) was
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calculated as a proportion of total hemocytes. Due to the fact that apoptosis also occurs in
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untreated hemocytes, the amount of annexin-positive cells in untreated hemocytes served as
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the base line, i.e. 0% apoptosis, thus the percentage of untreated annexin-positive cells was
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subtracted from treated annexin-positive cells. Negative percentages have to be viewed as
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proliferation of hemocytes. To rule out false positive signals, only hemocytes with a higher
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size and a higher granularity than bacteria were counted (BD FACSDiva™ software), thus
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potential annexin-positive bacteria can be excluded. Necrotic hemocytes produced by heating
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living insect cells at 65°C for 30 minutes served as positive control for the annexin staining.
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The assay was performed at least four times.
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i. Lytic zone Assay
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Petri dishes were filled with 10 ml Sörensen buffer (0.066M KH2PO4, 0.066M
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Na2HPO4*2H2O, pH 6.4) containing 0.6 mg ml-1 of lyophilized Micrococcus lysodeikticus
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ATCC no. 4698 (Sigma-Aldrich), 0.06 mg ml-1 Streptomycin sulfate (Calbiochem), PTU
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(Phenylthiourea; to inhibit melanization) with a final concentration of 1.5 % agar. Holes
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within the petri dish were made by puncturing the agar with a plastic pipette (Eppendorf
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Research 5000) and removing the agar plug by suction. Hemolymph samples (2 µl) were
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placed in each well and the plates were incubated for 24 h at 37°C. Different dilutions of
Barthel et al. Electronic supplementary material
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chicken egg white lysozyme (2, 1, 0.75, 0.5, 0.25, 0.125, 0.0625, 0.03 mg ml-1) (Sigma) were
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used as a positive control and a calibration curve was created based on these standards. Lytic
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activity in the hemolymph was determined as the radius of the clear zone around a sample
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well.
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j. Phenoloxidase (PO) activity assay
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Hemolymph phenoloxidase activity was estimated using 10 µl of hemolymph sample diluted
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in 500 µl of ice-cold sodium cacodylate buffer (0.01M Na-cacodylate, 0.005M CaCl2) and
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directly frozen in liquid N2 and stored at -80°C. Samples for PO activity measurements were
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prepared by thawing frozen hemolymph samples at room temperature then centrifuged at 4°C
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and 2800g for 15 minutes. The supernatant was removed and used for measurements where
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100µl of supernatant was added to 200 µl of 3mM L-Dopa (Sigma). Absorbance was
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measured for 45 minutes at 490 nm and 30°C, taking absorbance measurements once every
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minute. (Multiskan Spectrum multiplate reader; Thermo-Electron). As the enzymatic
195
reactions is linear from 5 – 45 min after adding the substrate (personal observation), in our
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analyses the fastest change in absorbance from 15-26 minutes (vmax) of the reaction was used.
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Data was acquired with SkanIt Software for Multiskan Spectrum version 2.1 (Thermo-
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Electron).
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k. Data analysis
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To test the effect of the treatment on larval survival post exposure, larval survival experiments
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were analyzed using the Cox proportional harzard model. To illustrate the effect of treatment
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on survival of both species, we used the Kaplan-Meier survivorship function.
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Bacterial recovery data was log-transformed and normal distribution was estimated
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using Kolmogorov-Smirnov test. Log-transformed data were assessed for significant
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differences between the species within one treatment using the Student t test (P<0.05).
Barthel et al. Electronic supplementary material
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All data presented as proportions (phagocytosis and apoptosis, not migration) were
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arcsine-square root transformed and normal distributions were estimated using Kolmogorov-
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Smirnov test. After transformation, significant treatment/species effects were assessed by
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Student t test (P<0.05). Since migration data were higher than 100%, migration data was log-
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transformed and normal distribution was estimated using Kolmogorov-Smirnov test.
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Significant differences between the species within one treatment were determined using the
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Student t test (P<0.05).
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Larval growth rate on Bt spores, phenoloxidase activity and lysozyme activity were not
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normally distributed, even after transformation, and non-parametric statistics were conducted,
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using Kruskal-Wallis test followed by pairwise comparisons with Mann-Whitney U test
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(P<0.05). Larval growth rate data upon MVP feeding were normally distributed and a 1-way
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ANOVA and Tukey’s post hoc test was carried out.
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Supplementary Tables
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Suppl. Table S1. Cox Regression Model for larval survival of H. virescens and
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H. subflexa upon injection with different concentrations of vegetative cells of
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S. entomophila and B. thuringiensis.
b1
SE2
Wald3
P4
H. virescens 104 vs. H. subflexa 104
-0.061
0.422
2.2028
0.154
H. virescens 105 vs. H. subflexa 105
-0.266
0.272
0.960
0.327
H. virescens 104 vs. H. virescens 105
-1.425
0.392
13.243
0.000
H. subflexa 104 vs. H. subflexa 105
0.978
0.318
9.465
0.002
H. virescens 104 vs. H. subflexa 104
-0.558
0.366
2.322
0.128
H. virescens 105 vs. H. subflexa 105
-0.559
0.276
4.105
0.043
H. virescens 104 vs. H. virescens 105
-1.104
0.319
11.956
0.001
H. subflexa 104 vs. H. subflexa 105
0.804
0.332
6.437
0.011
Bacterial challenge
1. S. entomophila
2. B. thuringiensis
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1
= b, regression coefficient of overall survival function
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2
= SE, Standard error of b
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3
= Wald statistic
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4
= P, Significance value for Wald statistic; Significant differences in bold
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Barthel et al. Electronic supplementary material
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Supplementary Figures
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Figure S1. Larval survival of H. virescens and H. subflexa on different concentrations of
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Bt spores. Kaplan-Meier survival plot of H. virescens (black) and H. subflexa (grey) larvae
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fed on (A) 5x104, (B) 105 and (C) 5x105 Bt spores (n=24). Statistical significance was
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determined using Cox regression survival analysis.
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Figure S2. Larval growth rate and survival of H. virescens and H. subflexa on different
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concentrations of Cry1Ac toxin (MVP). (A) Average growth rates of H. virescens (black)
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and H. subflexa (grey) larvae on different MVP concentrations after 7 days. Values on the y-
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axis represent the growth rates for all treatments relative to their corresponding control, i.e.
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artificial diet. Bars indicate means and standard errors (n=48). Statistical significance between
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the species was calculated using one-way ANOVA and Tukey’s post hoc test (n.s. = not
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significant). (B-D) Kaplan-Meier survival plot of H. virescens (black) and H. subflexa (grey)
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larvae on different MVP concentrations (n=48). Statistical significance was determined using
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Cox regression survival analysis.
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Figure S3. The percentage of H. virescens and H. subflexa hemocytes migrated to
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S. entomophila, B. thuringiensis and B. subtilis.
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Hemocytes were incubated with vegetative cells of S. entomophila, B. thuringiensis and
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B. subtilis for 1 hour. Values on the y-axis represent percentages of hemocytes, as proportions
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of total hemocytes relative to the control (base line 100% migration). Each bar represents the
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mean of three or four biological replicates and their associated SEM. Significant differences
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were determined between the two species for each time point within one treatment (not
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significant) and within one species compared to their corresponding control (Asterisks above
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the bars) using Student’s t test of log-transformed data. Significant differences are indicated
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by **P<0.01; *P<0.05.
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Figure S4. Enzyme activity in the hemolymph of H. virescens and H. subflexa larvae
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injected with vegetative cells of S. entomophila, B. thuringiensis and B. subtilis.
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Lysozyme-like activity in the hemolymph of (A) H. virescens (black) and (B) H. subflexa
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(grey) after 60 and 120 minutes. Phenoloxidase activity in (C) H. virescens (black) and (D)
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H. subflexa (grey) after 60 and 120 minutes. Bars represent the mean of 20 larvae ± S.E.M.
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Influence of wounding (PBS) and different bacterial strains on the enzyme activity was
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contrasted using a non-parametric Kruskal-Wallis test (df=17, P < 0.001). Mann-Whitney U-
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test was used for the pair comparisons between the treatments within one species. Different
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letters above the bars represent significant differences.
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