Entomopathogenic nematode Steinernema carpocapsae surpasses the cellular immune responses of the hispid beetle, Octodonta nipae (Coleoptera: Chrysomelidae)

Accepted Manuscript Entomopathogenic nematode Steinernema carpocapsae surpasses the cellular immune responses of the hispid beetle, Octodonta nipae (Coleoptera: Chrysomelidae) Sanda Nafiu Bala, Abrar Muhammad, Habib Ali, Youming Hou PII:

S0882-4010(18)31243-9

DOI:

10.1016/j.micpath.2018.08.063

Reference:

YMPAT 3148

To appear in:

Microbial Pathogenesis

Received Date: 9 July 2018 Revised Date:

29 August 2018

Accepted Date: 29 August 2018

Please cite this article as: Bala SN, Muhammad A, Ali H, Hou Y, Entomopathogenic nematode Steinernema carpocapsae surpasses the cellular immune responses of the hispid beetle, Octodonta nipae (Coleoptera: Chrysomelidae), Microbial Pathogenesis (2018), doi: 10.1016/j.micpath.2018.08.063. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Entomopathogenic nematode Steinernema carpocapsae surpasses the cellular immune

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responses of the hispid beetle, Octodonta nipae (Coleoptera: Chrysomelidae) Sanda Nafiu Bala 1, 2, Abrar Muhammad 1, 2, Habib Ali 1, 2, Youming Hou 1, 2* 1

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State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops,

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Fujian Agriculture and Forestry University, Fuzhou, Fujian China 2

Fujian Province Key Laboratory of Insect Ecology, Department of Plant Protection, Fujian Agriculture and Forestry University, Fuzhou, Fujian, China

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*Corresponding author and present address: You-ming Hou, Department of Plant

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Protection, Fujian Agriculture and Forestry University, 15, Shangxiadian Road, Cangshan

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District, Fuzhou, Fujian, 350002, China. Phone: +86 591 8376 8654.

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E-mail: [email protected].

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Abstract: The Nipa palm hispid, Octodonta nipae (Maulik) is an important invasive pest of

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palm trees particularly in Southern China. How this beetle interacts with invading pathogens

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via its immune system remains to be dissected. Steinernema carpocapsae is a pathogenic

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nematode that attacks a number of insects of economic importance. The present study

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systematically investigates the cellular immune responses of O. nipae against S. carpocapsae

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infection using combined immunological, biochemical and transcriptomics approaches. Our

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data reveal that S. carpocapsae efficiently resists being encapsulated and melanized within

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the host’s hemolymph and most of the nematodes were observed moving freely in the

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hemolymph even at 24 hours post incubation. Consistently, isolated cuticles from the parasite

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also withstand encapsulation by the O. nipae hemocytes at all-time points. However,

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significant encapsulation and melanization of the isolated cuticles were recorded following

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heat treatment of the cuticles. The host’s phenoloxidase activity was found to be slightly

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suppressed due to S. carpocapsae infection. Furthermore, the expression levels of some

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antimicrobial peptide (AMP) genes were significantly up-regulated in the S. carpocapsae-

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challenged O. nipae. Taken together, our data suggest that S. carpocapsae modulates and

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surpasses the O. nipae immune responses and hence can serve as an excellent biological

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control agent of the pest.

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Keywords:

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phenoloxidase; Octodonta nipae.

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1. Introduction

response;

Steinernema

carpocapsae;

Antimicrobial

peptides;

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Nipa palm hispid, Octodonta nipae (Maulik) (Coleoptera: Chrysomelidae), is an

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invasive species of palm trees of the Palmae family in Southeast Asian countries [1,2]. It is

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native to Malaysia and was first identified as a forest invasive pest in Hainan province of

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China [3]. It was reported to have spread to other provinces including the Fujian province in

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2007 [4]. The palm trees that were reported to be damaged by the attack of this pest include

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the queen palm, Syagrus romanzoffiana (Chamisso) Glassman) [5], California fan palm,

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Washingtonia filifera (Linden ex. Andre), Canary island date palm, Phoenix canariensis

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(Chabaud) [6,4], Chinese windmill palm, Trachycarpus fortune (Hooker) H. Wendland) and

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rattan, Calamus manan (Miquel) [7]. However, adults and larvae of this pest cause the most

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remarkable damages by feeding on unopened central epidermal parenchyma and leaf fronds,

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which leads to scorching and stunted growth and, in some situation, subsequent death of the

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whole tree [8,9,10,11].

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The most common control method is the use of insecticides. However, other control

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agents have reportedly been used [5]. These include Tetrastichus brontispae (an

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endoparasitoid of O. nipae) and the entomopathogenic fungus, Metarhizium anisopliae

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[12,13,14,15,16]. Entomopathogenic nematodes (EPNs) are biological control agents for

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different economically important agricultural pests from laboratory to field levels[16]. The

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most widely used EPNs are from the families Steinernematidae and Heterorhabditidae. EPNs

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on many crops[17] such as adult western corn rootworm Diabrotica virgifera [18], flea

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beetles Phyllotreta spp. [19][20]and Colorado potato beetle Leptinotarsa decemlineata found

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in leaf surfaces [21].It is generally known that insects defend themselves against bacterial,

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fungal and nematode infections through their innate immune system which is categorized into

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cellular and humoral systems [22][23]. The cellular immune reaction includes phagocytosis,

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lysis, nodulation and encapsulation, whereas the humoral immune reaction includes

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hemolymph-localized melanization, inducible antimicrobial peptides synthesis, production of

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reactive intermediates of oxygen and nitrogen [24]. Thus, the defence reactions against

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entomopathogenic nematode infections occur via encapsulation and melanization (proPO

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activation system) [25,26]. These processes are carried out by immune cells called

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hemocytes. They include prohaemocytes, granulocytes, plasmatocytes, lamellocytes, crystal

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cells, oenocytoids and spherulocytes. However, these cells vary in insects, but can be

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identified by their morphology, function, and molecular markers. For instance, granulocytes

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and plasmatocytes are involved in cellular defense response in Lepidoptera, whereas

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plasmatocytes and lamellocytes are found in Drosophila [27]. In O. nipae, the phagocytosis

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of Escherichia coli was detected by both granulocytes and plasmatocytes at 12 h post-

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injection [28]. Similarly, changes in the number of granulocytes and plasmatocytes were

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observed in cellular encapsulation of Heterorhabditis bacteriophora and Steinernema glaseri

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in Polyphylla adspersa third instar larvae [29].

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Previous experiments demonstrated how the entomopathogenic nematode S. feltiae

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escapes the immunological detections of G. mellonella, avoiding the host’s hemocyte

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encapsulation and at the same time inhibiting its proPO system activity[30][31][32][33].

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These immune-evasion mechanisms are caused by mimetic properties of the body surface of

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Steinernematids, due to the presence of some lipid compounds on its body cuticle. Similarly,

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Mastore et al. [32] have reported the ability of S. carpocapsae to modulate the proPO system

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activity of red palm weevil, by interfering with its melanization process, thereby overcoming

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the immune responses of the pest.Recently, researches have been focused on insect immune

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genes responses to entomopathogenic nematodes and their symbiotic bacteria interactions due

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to

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[25][34][35][36][37][38][39][40]. Expression of immune genes encoding antimicrobial

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peptides (AMPs) were mainly reported in the symbiotic nematode H. bacteriophora [34][41].

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In addition, bacteria isolated from nematodes were also shown to regulate AMPs in

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Drosophila [39]. Similarly, it was reported that symbiotic form of S. carpocapsae caused high

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up-regulation of drosomycin, attacin-A, attacin-B and attacin-C at both 6 and 24 h after

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infections [37].

generated

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transcriptome

analyses

especially

in

Drosophila

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In O. nipae, defence mechanisms against the parasitoid T. brontispae were exploited

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at molecular and physiological levels by Tang et al. [40], Meng et al. [28], and Zhang et al.

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[42]. Recently, the effects of parasitism on mRNA levels of attacin and defensin gene

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families were determined in O. nipae [24]. This resulted in the up-regulation of all AMPs at

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all-time points except for defensin 2B, which was down regulated at 12 hours post parasitism.

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Currently, the effects of entomopathogenic nematode infections, particularly S. carpocapsae,

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on the cellular and immune gene responses of O. nipae largely remain speculative. The

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present study therefore evaluated the phenoloxidase activity and encapsulation level of O.

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nipae larvae infected with S. carpocapsae and H. bacteriophora. We further investigated the

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inhibition properties of S. carpocapsae body cuticle on the phenoloxidase activity,

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encapsulation and expression of APM genes in O. nipae larvae.

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2. Materials and Methods

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2.1. Insect and nematode culture

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ACCEPTED MANUSCRIPT Octodonta nipae adults were collected from Hainan island, China (coordinate:

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20°2.7498′ N, 110°20.5002′ E and Elevation: 14 m = 45 ft.) and reared with small piece of

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fortunes windmill palm, Trachycarpus fortunei (Hook) in the laboratory according to

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methods previously described by Hou et al. [8]. The insects were maintained at 25 ± 1°C, 80

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± 5% RH, and photoperiod of 12: 12 (Light: Dark). The entomopathogenic nematodes

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Steinernema carpocapsae and H. bacteriophora were obtained from Guangdong Institute of

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Applied Biological Resources, China [43]. Nematodes were cultured using the last instar

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larvae of greater wax moth, Galleria mellonella [44]. Infective juveniles (Ijs), which involve

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the nematodes plus their mutualistic bacteria, were stored in distilled water at 13°C and were

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used in all experiments within 15 days of emergence from the host. Prior to the experiments,

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nematodes were kept at 25 °C for 30-60 min [45]. All experiments were carried out in

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triplicate.

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2.2. Octodonta nipae larvae infection assay

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Bioassay was conducted to determine the survival of the third instar larvae of O.

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nipae by applying 10 µl of S. carpocapsae suspension containing 0, 10, 25, 50 and 100 Ijs, to

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a larva, respectively, in 96-well plates (Costar®, Corning Incorporated Corning, New York

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14831, USA) according to Dobes et al. [46] with minor modifications. Thirty larvae were

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placed individually in each well plate containing 1×2 cm tissue paper with small piece of

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fortunes windmill palm, T. fortunei. The survival rates were checked from 8 to 48 hours after

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treatment at 4 hours intervals. For the control treatment, 10 µl distilled water containing no

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Ijs was used.

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2.3. Haemolymph collection

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Haemolymph was collected by cutting a proleg of last instar larva of O. nipae with a

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pair of scissors, and was further cold-treated and surface sterilized with 70% ethanol as

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described by Balasubramanian [47] with little modifications. Haemolymph collections were

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processed by low-speed centrifugation (1476 g for 3 min at 4°C) to eliminate haemocytes and

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tissue debris. The supernatant (haemolymph) was used immediately or stored at −20°C for

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future use.

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2.4. Nematodes encapsulation assay

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In-vitro host immune responses were studied according to previous reports [48][49]

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with little modifications. About 10 Ijs in 10 µL of sterile distilled water was mixed with 20

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µL of haemolymph from O. nipae larvae in 120 µL of Grace’s insect medium and incubated

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at 25 °C. Cellular responses were observed using stereo-microscope (Nikon SMZ745T

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Stereo, Camera; Nikon DS-fi2). Observations (free movement, encapsulation and

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melanization) were made at 1, 8 and 24 h post-bleeding. H. bacteriophora and S.

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carpocapsae were used against O. nipae haemocytes.

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2.5. Prophenoloxidase activity assay

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To determine the effects of the presence of S. carpocapsae and H. bacteriophora Ijs

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on the activity of O. nipae prophenoloxidase system, haemolymph was obtained from O.

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nipae larvae as described above. The haemolymph was diluted to 5:50 µl (v: v) with 50 mM

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phosphate buffer (pH 8.6), and centrifuged at 1700 g at 4 °C, for 1 min to obtain the

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supernatant. A 0.004 g of L-Dopa (8 mmol/L L-Dopa in 10 mmol/L 50 mM phosphate buffer

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pH 8.6) was dissolved in 10 ml of PBS (150 mM NaCl, 2.7 mM KCl, 1.8 mM

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KH2PO4 and 10.1 mM Na2HPO4; pH 8.6). Finally, 20 µl nematode suspension +

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30 µl supernatant + 100 µl L-Dopa was used for the assay. For control treatment, only 20 µl

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of PBS was added in place of the nematode suspension. The relative activity of

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phenoloxidase was measured with a spectrophotometer (SpectraMax, Molecular Devices

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Corporation, CA) at A 490 nm 5 min−1, at 20 °C [24, 27,29]. The experiment was performed

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in three replicates.

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2.6. Isolation of S. carpocapsae body cuticle The Ijs was suspended in 20 volumes of extraction buffer (10 mM Tris–HCl, 10 mM

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EDTA, 1 mM PMSF, pH 7.2) and then crushed for 60 seconds at 65 Hz using a tissue lyser.

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It was then homogenized in a glass homogenizer, washed several times in PBS and the level

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of purification was checked by light microscopy.

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2.7. Invitro encapsulation assay of S. carpocapsae isolated cuticles

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This bioassay was conducted as described above, with minor modifications. The

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ability of host hemocytes to encapsulate the parasites’ normal isolated cuticles (NCT), heat

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killed cuticle (HKCT) (heating suspension in a microwave at 1000 W for 2 min) and

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synthetic microbeads was carried out in vitro according to a previous protocol [50]. All

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samples were washed in PBS and resuspended in Grace’s insect medium before the

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experiment. After 30 min from the hemocytes adhesion to the substrate, targets were added to

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cell cultures at a concentration of about 10–15 units/well. The encapsulation process was

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examined at 2 and 8 h post incubation and observations were made under microscope.

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2.8. Phenoloxidase activity of S. carpocapsae isolated cuticles

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To determine the effects of the presence of NCT and HKCT of S. carpocapsae on the

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activity of the host proPO system in vitro, the kinetics and hemolymph dilutions were the

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same as described above with some modification. Ten to fifteen cuticle fragments + 30 µl

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supernatant + 100 µl L-Dopa was used. For the control treatment, 30 µl of PBS was added

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instead of the supernatant. The phenoloxidase activity was measured using a

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spectrophotometer (SpectraMax, Molecular Devices Corporation, CA) at A 490 nm 5 min−1,

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at 20 °C. The experiment was performed in three replicates.

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2.9. Quantitative real time PCR (qRT-PCR) of selected AMPs

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To determine the effects of S. carpocapsae infections on the mRNA expression level

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of AMPs in O. nipae larvae, two types of AMPs, attacins and defensins were selected. Third

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above. Total RNA was extracted from five larvae each at 8, 16 and 24 hours post treatment

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using TRIzol reagent (Invitrogen, Carls-bad, CA) according to the manufacturer's

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instructions. The concentration and integrity of the RNA were determined using a NanoDrop

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2000 (Thermo Fisher Scientific Inc., Waltham, MA). cDNA was synthesized using

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TransScript® II All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (One-Step

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gDNA Removal) (TransGen-TransScript, Beijing, China). qRT-PCR was performed in

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triplicate for each biological replicate with 20 µl reaction volume containing 1 µl of 500 nM

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primers (Table S1), 1 µl of 10-fold diluted cDNA, 8 µl of sterilized water and 10 µl of

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FastStart universal SYBR Green Master Mix (Roche) (Roche, Basel, Switzerland) in an ABI

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7500 System and procedure was performed as previously reported [24]. All calculations were

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performed using the accompanying ABI 7500 system software with ribosomal protein S3

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(rpS3) as a reference gene [40]. The primers sequences are provided in Table S1.

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2.10. Statistical analysis

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All analyses were performed using IBM SPSS Statistics version 22 (IBM

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Corporation, New York, 10504-1722, United States) (SPSS, RRID: SCR_002865). Analyses

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for survival experiments were carried out using Kaplan-Meier tests. Percentages of free

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moving, encapsulated, melanized nematodes were calculated based on the number of

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recovered nematodes versus the number of inoculated nematodes.. The data were arch sin and

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square-root transformed for percentage of melanized nematode before analysis. One-way

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ANOVA was used to analyze the percentage data at different time points among the

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nematode species. When ANOVA showed a significance effect (P ˂ 0.05), means were

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compared using least significance differences (LSD). The level of mRNA expression of

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AMPs was transformed by Logarithmic function. For the level of mRNA expression of

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AMPs and Phenoloxidase activities, data analysis was performed using the Student’s t-test;

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differences between mean values were analyzed and considered significant when P < 0.05 or

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considered extremely significant when P < 0.0001 with respect to the control values.

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3. Results

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3.1. Survival of O. nipae larvae infected with S. carpocapsae

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We treated O. nipae larvae with different concentrations of 0, 10, 25, 50 and 100 S.

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carpocapsae Ijs per larva to make comparisons to determine the minimum concentration of

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the Ijs required to infect the larvae as well as the best time points for RNA isolation. Figure 1

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reveals significant differences in the survival rates of O. nipae larvae at the various

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concentrations of S. carpocapsae (χ2 = 31.88, df = 4, P = 0.001, Log-Rank Test). Based on

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the result, the concentration of 100 Ijs per larva was selected to be used for subsequent

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infection of the larvae for RNA isolation at 8, 16 and 24 hours post treatment.

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3.2. S. carpocapsae survives the O. nipae innate immune responses To investigate the effectiveness of the O. nipae humoral immune responses against S.

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carpocapsae, the nematodes (Ijs) were incubated in the hemolymph obtained from O. nipae

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larvae and observations were made for free movement, encapsulation and melanisation at 1 h,

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8 h and 24 h post incubation using H. bacteriophora Ijs susceptibility as a control.

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Remarkably, most of the S. carpocapsae Ijs were free moving; very few were encapsulated

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while none were found melanized at all the incubation time points (Table 1). To demonstrate

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the active nature of the hemolymph, most of the H. bacteriophora Ijs incubated under the

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same condition as the S. carpocapsae Ijs were almost completely encapsulated and very few

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were free moving, with melanisation being recorded at 24 h post incubation (Fig. 2 A-F;

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Table 1). Taken together, our data demonstrate that S. carpocapsae survives the active innate

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defence system of the O. nipae larvae.

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3.3. S. carpocapsae infection does not affect the prophenoloxidase activity of O. nipae To further dissect the cellular mechanism behind the insignificant effect of the O.

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nipae immune responses to the S. carpocapsae Ijs, we assayed for prophenoloxidase activity

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of the insect in the presence and absence of the nematode. The results reveal a slight decrease

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in the relative phenoloxidase activity of the host in the presence of S. carpocapsae IJs as

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compared to the control, and this was statistically insignificant (figure 3). However, in the

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presence of H. bacteriophora IJs as a positive control, a significant increase in the activity of

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the enzyme was observed which consistently agrees with its obvious susceptibility to the

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immune responses of O. nipae. This suggests that S. carpocapsae does not interfare with the

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O. nipae phenoloxidase activity to resist the host’s immune responses.

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3.4. Steinernema carpocapsae heat killed isolated cuticles were encapsulated and

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melanized by O. nipae hemocytes

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The S. carpocapsae cuticles were isolated for in vitro analysis of their recognition by

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the O. nipae immune system (Fig. 4). When NCT were co-incubated with microbeads, there

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were no hemocyte attachments both at 2 h and 8 h after treatments as shown in Figures 5A

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and 5C. All microbeads were subsequently recognized by hemolymph of O. nipae larvae and

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were encapsulated and melanized. The NCT of S. carpocapsae were shown to possess the

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mimetic properties which prevented them from being recognized by the hemocytes of the O.

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nipae larvae. However, when these cuticles were subjected to heating, the HKCT were

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recognized, encapsulated (Fig. 5B) and subsequently melanized (Figure 5D) by the

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hemocytes of O. nipae larvae. This reveals that the factor that prevents effective immune

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responses against S. carpocapsae is on the cuticle which either disguises the pathogen as self

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or inhibits the host immune responses.

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3.5 Steinernema carpocapsae normal isolated cuticles strongly inhibited phenoloxidase

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activity of O. nipae The effects of S. carpocapsae NCT and HKCT on O. nipae larvae immune system

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were determined by analyzing their inhibition capacities to the relative phenoloxidase activity

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(F2,6 = 27.37, P = 0.002). Both NCT and HKCT inhibited phenoloxidase activity of O. nipae

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compared to the control treatment (Fig. 6). However, relative increase in phenoloxidase

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activity were recorded in HKCT compared to control (t16 = 5.57, P = 0.001). The stronger

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inhibition effect was noted with normal isolated cuticle compared to control treatment (t16 =

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8.58, P = 0.001). Collectively, these results further support the involvement of the S.

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carpocapsae in modulation of the O. nipae cellular and humoral immune responses.

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3.6. Steinernema carpocapsae induces up-regulations of AMPs in O. nipae

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Host species evolve different defence strategies against their invading pathogens.

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Most insect species use antimicrobial peptides (AMPs) for such counter attack. For this

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reason, we investigated the expression levels of five selected AMPs genes (including attacin

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2509, attacin 25810, attacin 5152, defensin 3241 and defensin 4664) in O. nipae that were

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challenged with S. carpocapsae by qRT-PCR. The results indicate that all the AMP genes

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were remarkably upregulated at all-time points, especially at 24 h after infections, except for

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Defensin 2A (Fig. 7A-7E). Thus, infection by symbiotic S. carpocapsae elicits dynamic

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responses in O. nipae larvae immune system. When compared with control treatment, we

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found strong expressions of Attacin C1 (t4 =2.07, P = 0.001), Attacin C2 (t4 =1.72, P = 0.010)

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and Attacin C3 (t4 =2.93, P = 0.030) at 24 h post S. carpocapsae infections. However, these

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AMPs were slightly upregulated at both 8 h and 16 h after S. carpocapsae infections. For

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Defensin 2A, we found higher transcript abundance upon nematodes infection early at 8 h (t4

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=1.817, P = 0.001) and lower but significant transcriptions at 16 h (t4 =3.34, P = 0.034) and

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24 h (t4 =1.59, P = 0.045) post infections. Contrarily, Defensin 2A was found significantly

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down regulated at 16 h (t4 =1.346, P = 0.024) and 24 h (t4 =1.58, P = 0.040) after nematodes

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infections and fairly upregulated at 8 h (t4 =1.28, P = 0.272) post infections.

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4. Discussions In this study, we investigated the immunological resistance capacity of O. nipae

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larvae challenged by S. carpocapsae. It was reported from a previous study that the immune

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responses of an insect due to nematode infection depend on the insect species and the kind of

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nematode involved [23]. Here, S. carpocapsae escaped recognition by O. nipae hemocytes,

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especially at early hours of infection as evident by high percentage of free moving Ijs of the

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nematode (Table 1). Significant encapsulation of H. bacteriophora Ijs was observed and was

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seen to be higher at all-time points compared to S. carpocapsae. Additionally, the latter

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parasite also escaped being melanized by the host’s hemocytes throughout the observation

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periods which further explains why its lowest concentration of 10 Ijs per insect was able to

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destroy all the treated hosts within 48 hours (Fig. 1).

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Because steinernematids have been reported previously to possess immune-evasion

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factors on their body surfaces which give them the mimetic property of being recognized as

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self by the immune system of many insects [33][34][35] and that a previous study showed

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that the success in parasitization of S. carpocapsae is aided by its body surface cuticle [38],

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we isolated S. carpocapsae cuticles and performed in vitro encapsulation and melanization

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assays using the hosts’ hemolymph. Consistently, the normal cuticles were neither

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encapsulated nor melanized at all observation time points. Complete melanization was

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however observed at 8 h after incubation when the isolated cuticles were heat-treated prior to

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treatment with the host’s hemolymph (Fig. 5). Manachini et al. [51] also reported that

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infection of the red palm weevil Rhynchophorus ferrugineus with S. carpocapsae suppressed

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the parasite encapsulation capacity of the host. However, further studies are required to

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unveil the active component(s) on the body surface cuticle that is responsible for the

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resistance to the host’s counter attack. In addition to encapsulation and subsequent melanization reactions, the proPO system

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is one of the most important immune response mechanisms that fight against pathogens like

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nematodes [52]. The phenoloxidase enzyme (zymogen) was believed to be activated by

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serine protease that resulted in encapsulation and melanization of invaders by hemocytes. In

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this study, the relative PO activity of O. nipae challenged with S. carpocapsae was

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significantly lower than that of H. bacteriophora-challenged O. nipae and the normal control

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treatment, suggesting that S. carpocapsae overcomes O. nipae immune responses by

306

modulating the activity of its PO enzyme system. This is in line with some previous reports

307

that S. feltiae escape their hosts’ immune responses through inhibition of the hosts’ PO

308

activities in R. ferrugineus [23,24,43]. Furthermore, the suppression of PO activity in both

309

living and dead Steinernematids and their symbiotic bacteria by A. segentum and Pieris

310

brassicae has also been reported [39,40]. On a contrary view, the serine protease secreted by

311

S. carpocapsae and not the body cuticle was shown to be responsible for the inhibition of

312

host proPO system [55].

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Expression of immune related genes in insect hosts in response to some pathogen

314

infections results in the activation of different immune pathways [35]. In Drosophila,

315

response to entomopathogenic nematodes results in the activation of immune deficiency

316

pathway, which in turn induces the transcription of certain AMP genes; infection with

317

Heterorhabditis-Photorhabdus complex induced the expression of metchnikowin, diptericin,

318

drosomycin and attacin genes mainly at 24 h after infections [33]. In the same vein, symbiotic

319

H. bacteriophora was found to induce transcription of AMP genes, while injection of

320

Photorhabdus alone fails to induce the expression of these genes in Drosophila [35]. In this

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study, AMPs were shown to be upregulated after infection with symbiotic S. carpocapsae

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ACCEPTED MANUSCRIPT except for Defensin 2A. Infection with similar symbiotic S. carpocapsae nematodes were

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reported to caused expression of drosomycin, attacin-A, attacin-B and attacin-C at both 6 and

324

24 h post infections in Drosophila [37]. Similarly, when O. nipae pupae were injected with

325

EGFP-expressing Escherichia coli, the AMPs were up-regulate at early and late parasitism

326

hours except for Defensin 2B, which was down-regulated after parasitism [24]. Generally, S.

327

carpocapsae body surface is involved in the suppression of encapsulation reactions and

328

inhibition of PO activities in O. nipae larval immune system. The body cuticle likely

329

manipulates hemolymph proteins which in turn protect them from opsonization and

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encapsulation by hemocytes of the insect host [56]

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5. Conclusion

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In conclusion, this study provides the first data on the immune interaction of O. nipae

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to entomopathogenic nematodes. The study presents the involvement of S. carpocapsae body

334

cuticle in the successful invasion of O. nipae larvae immune system. This is because of the

335

suppression of encapsulation reactions and inhibition PO activities by both whole nematodes

336

and isolated cuticles. Therefore, this body cuticle was speculated to have down regulated the

337

expression of one of the APM genes examined in this study. Future research will focus on

338

applying symbiotic bacteria alone to further confirm the inhibition of O. nipae larvae immune

339

response is due to symbiotic nematode or its isolated bacteria and also on dissecting the

340

molecular mechanism behind the S. carpocapsae resistance to the host’s immune responses.

341

Acknowledgements

342

We thank Prof. Richou Han and his associate Dr. Xun Yun for providing us with

343

Entomopathogenic nematodes for the experiment. This work was supported by grants from

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the National Key R & D Program of China (2017YFC1200605) and Fujian Science and

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Technology Special Project (2017NZ0003-1-6).

346

Conflict of interest: The authors have no conflict of interest to declare

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ACCEPTED MANUSCRIPT Authors Contributions:

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SNB: Experimental Design and data collection, analysis and interpretation.

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HA: Data collection, analysis and interpretation; manuscript writing

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AM: Critical review of the manuscript

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YMH: Conception, design and critical analysis of the manuscript

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Tables

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Table 1. In-vitro haemocytes reactions of Octodonta nipae larvae to Steinernema carpocapsae and Heterorhabditis bacteriophora

594 595

Percentage Nematode (% ± SEM) Free Moving Encapsulated Melanized Sc Hb Sc Hb Sc Hb 1 86 ± 6.67 33 ± 8.81 20 ± 6.33 66 ± 8.85 0 ± 0.00 0 ± 0.00 8 80 ± 5.77 30 ± 5.77 13 ± 8.60 70 ± 5.80 0 ± 0.00 0 ± 0.00 23 ± 3.33 66 ± 3.33 0 ± 0.00 13 ± 1.46 24 70 ± 10.00 20 ± 2.32 Hb: Heterorhabditis bacteriophora; Sc: Steinernema carpocapsae; SEM: standard error mean

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598 599 600 601 602 603

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Time Point (h)

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Figures and legends

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Fig. 1. Survival curve of O. nipae larvae infected with S. carpocapsae at different concentrations

5

are significantly different, (p < 0.0001, log-rank test)

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Fig. 2. In vitro immune responses of Octodonta nipae larvae hemocytes to nematodes A) At 1

12

hour: Steinernema carpocapsae are free moving B) At 1 hour: Heterorhabditis bacteriophora

13

are encapsulated C) At 8 hours: Steinernema carpocapsae are free moving D) At 8 hours:

14

Heterorhabditis bacteriophora are completely encapsulated E) At 24 hours: Steinernema

15

carpocapsae are free moving nematode F) At 24 hours: Heterorhabditis bacteriophora;

16

completely melanized nematode

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Fig. 3. In vitro host proPO system in the presence of Steinernema carpocapsae and

20

Heterorhabditis bacteriophora. Error Bars labeled with different letters are significantly

21

different (one-way ANOVA followed by LSD test, p < 0.05).The asterisks *** (P < 0.0001) * (P

22

< 0.01 0; indicates different significant levels between the control and S. carpocapsae treatments

23

at the indicated time period.

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Fig. 4. Steinernema carpocapsae cuticles isolated in 20 volumes of extraction buffer using a

29

TissueLyser.

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Fig. 5: In vitro encapsulation response of Octodonta nipae larvae to Steinernema carpocapsae

33

isolated cuticles A) At 2 hours: Free moving NCT and encapsulated microbeads. Arrow

34

indicated the encapsulated microbead B) At 2 hours: HKCT and microbeads encapsulated.

35

Arrow indicated encapsulated microbead. C) At 8 hours:

36

microbead. Arrow indicated the melanized microbead D) At 8 hours: HKCT and microbead

37

melanized. Arrow indicated the melanized HKCT.

Free moving NCT and melanized

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Fig. 6. Inhibition of O. nipae proPO system in the presence of S. carpocapsae NCT and HKCT

40

compared to the Control Treatment (CK). Error bars labeled with different letters are

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significantly different (one-way ANOVA followed by LSD test, p < 0.05) .The asterisks *** (P

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< 0.0001); indicates different significant levels between the control and S. carpocapsae

43

treatments at the indicated time period.

55 56 57 58 59 60 61 62

TE D EP

54

AC C

44 45 46 47 48 49 50 51 52 53

M AN U

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(A)

M AN U

SC

RI PT

63

64 (B)

66 67 68 69 70 71 72

AC C

EP

TE D

65

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(C)

M AN U

SC

RI PT

73

74 (D)

76 77 78 79 80 81

AC C

EP

TE D

75

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(E)

M AN U

SC

RI PT

82

83

Fig. 7. Transcription of antimicrobial peptide (AMP) genes in O. nipae larvae infected with S.

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carpocapsae. AMP transcription levels are shown for (a) Attacin C1, (b) Attacin C2, (c) Attacin

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C3, (d) Defensin 2A, (e) Defensin 2B. Error bars labeled with different letters are significantly

87

different (one-way ANOVA followed by LSD test, p < 0.05).The asterisks *** (P < 0.0001); **

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(P < 0.001); * (P < 0.01) indicates different significant levels between the control and S.

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carpocapsae treatments at the indicated time period; while “ns” indicates no significant

90

difference.

AC C

EP

TE D

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Highlights •

S. carpocapsae efficiently resists being encapsulated and melanized within the host’s hemolymph S. carpocapsae isolated cuticles too withstand encapsulation by host hemocytes at all

RI PT

•

time points •

Heat killed S. carpocapsae isolated cuticles was encapsulated and melanized by host

SC

hemocytes

Host’s phenoloxidase activity was suppressed by S. carpocapsae and its isolated cuticles

•

Some antimicrobial peptide genes were up-regulated in the S. carpocapsae-challenged O.

M AN U

•

AC C

EP

TE D

nipae