Turkish Journal of Zoology Turk J Zool (2018) 42: 422-431 http://journals.tubitak.gov.tr/zoology/ © TÜBİTAK Research Article doi:10.3906/zoo-1801-14

The role of a novel Wolbachia (Rickettsiales: Anaplasmataceae) synthetic peptide, WolFar, in regulating prostaglandin levels in the hemolymph of Acheta domesticus (Orthoptera: Gryllidae)

1 1 Muhamad Azmi MOHAMMED , Ameyra AMAN-ZUKI , 1 2 1, Nurul Othman WAHIDA , Yohsuke TAGAMI , Salmah YAAKOP * 1 School of Environmental and Natural Resource Sciences, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, Bangi, Malaysia 2 Laboratory of Applied Entomology, Faculty of Agriculture, Shizuoka University, Shizuoka, Japan

Received: 09.01.2018 Accepted/Published Online: 26.06.2018 Final Version: 26.07.2018

Abstract: Prostaglandins and other eicosanoids are known as regulating agents for cellular immune responses to pathogen threats in an insect’s hemolymph. A novel synthetic peptide, WolFar, was customized from a conserved region of the Wolbachia surface protein (WSP), isolated from an economically important endoparasitoid species, Fopius arisanus. WolFar consists of nine amino acids (SYY VRL QYN) and was tested on the house cricket, Acheta domesticus. Three concentrations of peptides, 0.83 mmol/L (100%), 0.63 mmol/L

(75%), and 0.42 mmol/L (50%), were injected and observed for 72 h. The regulation of prostaglandin 2E (PGE2) in the hemolymph of A. domesticus was determined using ELISA and by observation of nodules in the internal system of A. domesticus. The results showed that there were significant increases in PGE2 in response to peptide injection at 24, 48, and 72 h after treatment. Furthermore, higher concentrations of peptide were directly proportional to the level of PGE2 activity. These findings were supported by the abundance of nodules that formed in the internal system and fat body of A. domesticus, detectable at 72 h into the treatment. This indicates that WolFar is able to stimulate the immune system of insects and can be further developed as a potential biopesticide.

Key words: Prostaglandin, hemolymph, Acheta domesticus, WSP, peptide

1. Introduction and hemocytes, which initiates the release of the PSP that Insect immunology consists of three main components: binds to the hemocyte receptor. The binding induces the physical barriers, humoral immune response, and cellular activation of phospholipase A2 to hydrolyze arachidonic immune response. Physical barriers include structures acids (AA) from phospholipid substrates. The AA is then such as cuticle-protecting epidermis, peritrophic matrix- oxygenated by cyclooxygenase or lipoxygenase enzymes to covering midgut lumen, and chitin lining of the tracheal form PGs and other eicosanoids, which later mediate the system (Wigglesworth, 1972; Billingsley and Lehane, 1996), hemocyte-spreading behavior (Strand, 2008; Stanley and which prevent unwanted pathogens from entering the Kim, 2011, 2014; Stanley et al., 2012). A study by Jurenka et hemolymph system of insects. Humoral immune response al. (1999) showed that levels of PG in the true armyworm, involves activities of biosynthesis to produce antimicrobial Pseudaletia unipuncta, were produced fourfold in relation peptide (AMP), enzyme lysozyme, and discharge of to injections of heat-killed bacteria (Serratia marcescens) prophenoloxidase (PPO), which results in melanization compared with saline-injected larvae. of the affected areas (Lemaitre and Hoffmann, 2007). As The endosymbiont Wolbachia (Rickettsiales: for cellular immune response, cellular actions—such as Anaplasmataceae) is considered to be among the most microaggregation of pathogens, followed by encapsulation significant and successful endosymbionts and has been and then nodulation—are controlled and signaled by estimated to infect 70% of arthropod hosts, such as prostaglandins (PGs) (Stanley and Kim, 2011) and other insects, mites, crustaceans, and nematodes (Saridaki eicosanoids (Büyükgüzel et al., 2007). One of the possible and Bourtzis, 2009). However, most of the discussions signaling pathways for cellular immune response is the regarding the roles played by this endosymbiont have mechanism of plasmatocyte-spreading peptide (Srikanth focused primarily on reproductive effects, such as et al., 2011). First, pathogens are detected by the fat body cytoplasmic incompatibility (Bordenstein and Werren, * Correspondence: [email protected] 422 MOHAMMED et al. / Turk J Zool

2007), male feminization (Asgharian et al., 2014), peptide was made using mild 9-fluorenylmethoxycarbonyl induction of parthenogenesis (Watanabe et al., 2013), and (Fmoc) chemistry methods (Sheppard, 2003). The peptide male mortality (Rasgon, 2012). The reproductive effects assembly and purification were computer-controlled to exerted by Wolbachia on their hosts make it possible to ensure the authenticity of the sequences. The synthesized implement biological control measures, thereby reducing peptides were then purified in an automated preparative populations of pests or increasing populations of beneficial HPLC system, and the best fractions were selected as the insects such as predators and parasitoids (Zabalou et al., final product, with a final mass of 509.7 mg at 93% of 2004; Brelsfoard and Dobson, 2009). Recent studies have purity by HPLC. The customized peptide was 1205.34 g/ shown that Wolbachia proteins are also able to interfere mol. The purified peptides were consistently estimated in the immune systems of their hosts. Pinto et al. (2012) by analytical reverse-phase HPLC (RP-HPLC), and they reported that recombinant Wolbachia surface protein were checked for correct identity using mass spectrometry (WSP) was shown to be a stimulator that increases the (MS). The peptide treatments were prepared by diluting transcription of immune genes (TEP1 and APL1), known the peptide in solid form with deionized distilled water at to be crucial in the killing of Plasmodium in the mosquito a ratio of 1:1 (100%) to produce molarity of 0.83 mmol/L. Anopheles gambiae, which is naturally uninfected with The 100% peptide then underwent a series of dilutions to Wolbachia. When tested on the mosquito Aedes albopictus, produce concentrations of 75% (0.63 mmol/L) and 50% which is naturally Wolbachia-infected, the upregulations (0.42 mmol/L). of immune genes were lower than in the A. gambiae cells. 2.2. Insect rearing In another study, Wolbachia lipoproteins were proven to Fifth instar females of Acheta domesticus, weighing 200 ± stimulate innate and adaptive immunity in the filarial 10 mg, were used as a model species. The cricket nymphs nematode, Brugia malayi, through toll-like receptors 2 and were bought from a pet shop and underwent a selective 6 (TLR2/6) (Turner et al., 2009). procedure for the fifth instar stage: the nymphs must Wolbachia has been proven to be effective when wholly not yet have formed the protruding ovipositor structure. introduced as a functional unit, a process that involves According to Patton (1978), female A. domesticus reach artificial infection or transinfection into novel hosts. A their fifth instar stage when the ovipositor structure begins number of case studies have reported on the intentional to show. Accordingly, all nymphs without ovipositors introduction of this endosymbiont into new hosts. were brought to the lab, placed in transparent plastic Most of these studies have focused on the properties of containers (24.5 cm × 13.5 cm × 13.0 cm), and reared reproduction manipulators in controlling vector diseases, under lab conditions until the emergence of the ovipositor for example in the mosquitoes Aedes albopictus (Xi et structure. The plastic containers were provided with al., 2006; Fu et al., 2010) and Ae. aegypti (Johnson, 2015; pieces of cotton wool soaked in water as a drinking supply Joubert and O’Neill, 2017). However, no study has yet and a few dry twigs for the nymphs to jump on. Rabbit been carried out on novel peptide synthesis isolated from pellets (Harringtons, UK) and larvae of the mealworm Wolbachia DNA sequences, and its potential for other Tenebrio molitor were supplied as food. The female useful effects is unknown. The objectives of the present crickets with newly formed ovipositor structures were study are first to investigate the effects of Wolbachia selected and relocated into new plastic containers prior peptide synthesized from the WSP gene on the regulations to the biochemical assay. All the rearing procedures were of PGs in the hemolymph of A. domesticus as a cellular conducted at 28 °C with 68% relative humidity. immune response and second to visualize the effects of the 2.3. Peptide treatment assay peptide on the internal system of A. domesticus. A total of 10 fifth instar nymphs of female A. domesticus, chilled to 2 °C for 10 min, were injected dorsoventrally 2. Materials and methods between the third and fourth abdominal segments with 2.1. Designation of Wolbachia synthesis peptide (WolFar) 15 µL of Wolbachia synthetic peptide. As a negative A total of 17 sequences of WSP were obtained from control, 10 samples were injected with 15 µL of deionized Mohammed et al. (2017). These sequences were amplified distilled water. The synthetic peptide injection assays from an endoparasitoid species, Fopius arisanus, which were set to 24 h, 48 h, and 72 h, in line with previous was collected from five carambola cultivation sites in biochemical assay studies on insect immune response (Bali Peninsular Malaysia. The WSP sequences were aligned and Kaur, 2013; Prisco et al., 2013), and the hemolymph to search for the conserved region that comprises 29 was collected with micropipettes 24, 48, and 72 h after nucleotides (5’-AGC TAC TAC GTT CGT TTG CAA injection. The hemolymph was collected by piercing the TAC AAC GG-3’) translated into nine amino acids (SYY prothoracic legs with a fine and sterile needle and pooled VRL QYN), and they were sent to Mimotopes Pty. Ltd. into Eppendorf tubes from all 10 samples. The average (Australia) for peptide synthesis. The custom-synthesized quantity of hemolymph collected was about 30.0 µL

423 MOHAMMED et al. / Turk J Zool per insect. The tubes were then centrifuged (12,000 × g, plate was covered with the plate sealer provided to avoid 27 °C, 10 min) to remove cell wastes. Phosphate buffered spillage. The contents of the wells were then discarded and saline (PBS), pH 7.4, was added to the hemolymph to washed by adding 400 µL of 1X wash buffer to each well. achieve the different concentrations (50%, 75%, and The washing procedure was repeated twice, resulting in a 100%). The mixtures of hemolymph and PBS were total of three washes. After the final wash, the wells were transferred to new Eppendorf tubes, labeled with their aspirated, and the plate was firmly tapped on a lint-free treatment concentrations, in preparation for ELISA. Three paper towel to remove any remaining wash buffer. PGE2 replications were taken for each treatment as well as for the alkaline phosphatase conjugate (5 µL) was added to the control, and the experiments were repeated twice. TA (total activity) wells only. Subsequently, 200 µL of the 2.4. Observation of nodules pNPP substrate solution was added to the wells, which were Samples of hemolymph, consisting of negative control then incubated at room temperature for 45 min without samples and samples treated with 100% WolFar shaking. The reaction was stopped by adding 50 µL of stop concentration, were successfully extracted according to solution to each well and the plate was read immediately at postinjection time (at 24 h, 48 h, and 72 h). The bodies 405 nm, preferably with correction between 570 and 590 of the nymphs were then dissected horizontally from the nm using microplate reader (Infinite 200, Tecan Trading, thorax to the anus using a microdissecting kit. The dissected Switzerland) with reader software Tecan i-control version bodies were observed for any physical abnormalities of the 1.7.1.12. internal system related to immune response. The internal 2.6. Statistical analysis system and fat body of A. domesticus were checked using Experimental analysis for PGE2 activity was carried out a surgical stereo microscope (Carl Zeiss Stemi 2000) and using one-way analysis of variance (ANOVA), and the photographed using an image analyzer connected to an significance among means of the different concentrations AxioCam ICc 3 microscope camera. (100%, 75%, and 50%) and times postinjection (0 h, 24 h, 2.5. Enzyme-linked immunosorbent assay (ELISA) 48 h, and 72 h) was determined at P ≤ 0.05 using Tukey’s The 96-well plate strips precoated with mouse IgG antibody range test. and reagents (ab133021 – prostaglandin E2 ELISA Kit, Abcam, UK) were equilibrated to room temperature prior 3. Results to use. A total of 100 µL of assay buffer provided by the The application of Wolbachia synthetic peptide generated manufacturer was added into the nonspecific binding differential PGE2 activity in the treated crickets compared

(NSB) and B0 (0 pg/mL standards) wells. Next, 100 µL of to the untreated negative control crickets (Table; Figure 1). prepared standard, provided by the manufacturer, and the The mean of PGE2 activity in the treated cricket nymphs diluted hemolymph samples were added to the appropriate was greater than in the control crickets, regardless of the wells. Assay buffer (50 µL) was added to the NSB wells only. number of hours postinjection. At 24 h, the PGE2 activity

A total quantity of PGE2 alkaline phosphatase conjugate had increased for all three concentrations. The 100%

(50 µL) was added to the NSB, B0, standard, and sample concentration had the highest level of PGE2 at 132.11 wells. Next, 50 µL of PGE2 antibody was added to the B0, pg/mL, followed by the 75% concentration. The 50% standard, and sample wells. The plate was incubated at concentration had the lowest level of PGE2 activity, at 72.76 room temperature on a plate shaker for 2 h at 500 rpm. The pg/mL (Table). At 48 h, all three peptide concentrations

Table. Regulation of prostaglandin E2 activity at different postinjection times and different concentrations of peptide treatment.

Prostaglandin E activity (pg/mL) (mean ± SE) Synthetic 2 F-value peptide (%) 0 h 24 h 48 h 72 h Control 29.7 ± 0.64a 29.43 ± 0.62a 30.87 ± 0.51a 28.92 ± 0.84a NS 100 30.3 ± 19.3a 132.11 ± 16.1b 78.16 ± 12.01a 107.96 ± 15.03b 76.3367 75 31.1 ± 12.5c 112.33 ± 18.41a 51.41 ± 16.12c 88.13 ± 15.11b 54.21667 50 28.9 ± 8.4c 72.76 ± 9.59a 39.80 ± 6.77bc 53.49 ± 8.71b 25.61

Significant at 5%, NS = nonsignificant. Values in each row followed by different letters are significantly different at P ≤ 0.05.

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180 Negat ve control 100% 75% 50% 160 ) L 140 / m g p (

120 t y

t v 100 a c 2 E 80 n d

la n 60 ta g s o

r 40 P 20

0 0 24 48 72 T me post nject on (h)

Figure 1. Prostaglandin E2 activity of Acheta domesticus at different postinjection times relative to the concentrations of WolFar applied. Each point represents the mean ± SE.

showed a significant decrease in PGE2 activity. The 100% species can be regarded as one of the best model study WolFar concentration recorded a drop of 40.84% from species because of several characteristics, such as absence the activity at 24 h; the drop was 45.3% for the 50% of diapause, gradual metamorphosis, hardiness, ease of concentration and the biggest decrease was recorded for handling, and the availability of an inexpensive and easily the 75% concentration, with a drop of 54.23%. However, administered diet (Clifford et al., 1977). A number of at 72 h, significant increases were recorded for all three studies regarding the establishment and rearing process synthetic peptide concentrations. The 100% concentration have been conducted in the past due to the potential of showed an increase of 38.13% in the concentration at 48 h, this species as nutrition and as a staple food for many pet and the 50% concentration had an increase of 34.4%. The species (Clifford et al., 1977; McFarlane and Distler, 1982; 75% concentration had the highest percentage increase in Clifford and Woodring, 1990; Nakagaki and Defoliart,

PGE2, with 71.43%. All the WolFar concentrations tested 1991). This species has also been widely used as a model in this study showed significant differences over time for studying the immune responses of insects when (P ≤ 0.05), with P = 0.0162 for the 100% concentration, under attack by pathogens, including nematodes (Wang P = 0.0003 for the 75% concentration, and P = 0.0007 for et al., 1994; Hallem et al., 2011), bacteria (Adamo, 1999; the 50% concentration. Simpson et al., 2000), fungi (Wang and Leger, 2006), and The application of the WolFar synthetic peptide resulted viruses (Liu et al., 2011; Szelei et al., 2011; Fauce and in the formation of nodules as an immune response to an Owens, 2013). For example, the bacterium Bacillus subtilis unidentified substance entering the systems of the crickets. was found to induce the activation of prophenoloxidase The negative control clearly shows no formation of nodules (humoral immune response) and to increase the level of (Figure 2A). At 24 h and 48 h (Figures 2B and 2C), nodules phenoloxidase activity when injected into the hemocoel of had formed, as shown by the blackish-red dots scattered A. domesticus nymphs (Silva, 2002). In another example, over the internal organs and system, but not as abundantly Wang and Leger (2006) successfully demonstrated that a as at 72 h (Figure 2D). The abundance of nodules is clearly particular gene, Mcl1, in fungus Metarhizium anisopliae, visible as blackish-red dots attached to the internal system was able to avoid detection and destruction by the and fat body of the crickets after 72 h of treatment with hemocyte of the hosts (A. domesticus and six other insect WolFar. species). Further study needs to be conducted to determine 4. Discussion the status of Wolbachia infection in A. domesticus as a 4.1. Sample selection Wolbachia prevalence study was not conducted in the The house cricket, Acheta domesticus (Orthoptera: present study. The status ofWolbachia in A. domesticus Gryllidae) is omnivorous, cosmopolitan, and easily is unknown because no previous studies regarding this reared under constricted conditions (Patton, 1978). This matter have been reported. In addition, there were also no

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Figure 2. Formation of nodules in the internal system and fat body of Acheta domesticus following injections of 100% concentration of WolFar. The red triangles indicate the positions of the nodules. A: Negative control; B: at 24 h; C: at 48 h; D: at 72 h. available data or sequences on A. domesticus as a possible isolated and purified from Galleria mellonella were able host for Wolbachia provided in the GenBank database, to induce antimicrobial activity when tested against strengthening the claim that A. domesticus has not yet gram-negative and gram-positive bacteria, yeast, and been infected with Wolbachia. Hence, the selection of A. filamentous fungi. However, a limited number of studies domesticus as a subject in this study can be considered as have focused on the introduction of different foreign accordant and appropriate to avoid bias due to the factor substances, such as peptides, into the insect’s system with of Wolbachia infection status. the aim of properly understanding the mechanisms of 4.2. Wolbachia synthetic peptide the immune responses of different insect species. Several Our study also considered the potential for WolFar, a related studies, including those of Windley et al. (2012) synthetic peptide isolated from the conserved region of and Fruttero et al. (2016), developed novel bioinsecticides, WSP, to be isolated from the economically important as in the present study. For example, Hardy et al. (2013) endoparasitoid Fopius arisanus and used as a biopesticide. successfully developed an oral insecticidal toxin, OAIP- Previous studies, including those of Bulet et al. (1991), 1, from the Australian tarantula, Selenotypus plumipes, to Cytryńska et al. (2006), Eleftherianos et al. (2009), and combat the agronomically important cotton bollworm, Ishii et al. (2010), have been heavily focused on specific Helicoverpa armigera. In another study, a recombinant AMPs extracted from various insects species and then peptide, Jaburetox, derived from the isoforms of Canavalia tested on pathogenic agents in order to prove their ensiformis (the jack bean) was tested on the Chagas disease significance for the regulation of antimicrobial activity. vector Rhodnius prolixus. The results of that study showed Cytryńska et al. (2006) reported that eight peptides that R. prolixus treated with the peptide had increased PO

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(phenoloxidase) activity compared to the control sample, injury-induced inflammation in rats. The levels of which had been treated with a sodium phosphate buffer. PGE2 were found to be decreased in parallel with the Based on these results, Frutterro et al. (2016) suggested expression of the lipopolysaccharide-induced related that the Jaburetox peptide triggers an immune response proteins as determined by using the ELISA approach. in R. prolixus. In the present study, the PGE2 activity was found to be Even though there has been no direct evidence significantly increased after 24 h of treatment for all regarding the toxicology effects of WSP, several studies peptide concentrations, proving that treatment with the were able to give indirect evidence of the pathogenicity peptide triggers an immune response in A. domesticus. The of WSP. Studies conducted on Wolbachia infection in level of PGE2 in the hemolymph significantly increased in nematodes proved that WSP played an antigenic role in the treated samples compared to the untreated samples, stirring the immune response of vertebrate hosts infected indicating that a series of immune responses against the with filarial nematodes (Bazzocchi et al., 2000; Brattig et peptide had been activated in the treated samples. The al., 2000, 2004). WSP also surprisingly showed analogies same situation was recorded in a study carried out by to the antigenic protein of pathogens despite the fact that Srygley and Jaronski (2011), which showed elevated PO this endosymbiont is not found in vertebrates (Baldo et activity in the Mormon cricket, Anabrus simplex, when al., 2005). According to Braig et al. (1998), WSP shows treated with the entomopathogenic fungus Beauveria sequence similarity with genes coding for major surface bassiana and compared to untreated samples. The PGE2 proteins of endosymbionts of Rickettsiales, which cause activity at the higher concentrations of treatment (100%) lethality and illness worldwide, and arthropods are was also slightly higher than at the lower concentrations known to be vectors of these pathogens (Azad and Beard, (75% and 50%). 1998). Moreover, the results of BLAST found significant The findings of the present study are also in line with homology between the WSP protein and products that a case study carried out by Bali and Kaur (2013). The PO arbitrate various pathogen–host cell interactions from activity in the hemolymph of Spodoptera litura, when treated pathogenic proteobacteria such as Neisseria sp. (Braig with a higher concentration of B. bassiana, was found to be et al., 1998). Hence, based on this evidence, this study significantly higher than at lower concentrations. However, provides additional data on the pathogenicity of WSP that the defensive responses toward the peptide treatments led can be further developed into a potential biopesticide. to elevated levels of PGE2 activity in the hemolymph at 48 h for all peptide concentrations. This result is congruent 4.3. Regulations of prostaglandin E2 The results of the present study show that treatment with with other studies that showed PO levels declining with the Wolbachia synthetic peptide regulates the immune an increase in the duration of B. bassiana treatments in S. litura. Dorrah (2009) found that when the flesh fly response of the subject, as shown by the PGE2 activity in the hemolymph of the cricket Acheta domesticus (Table; Figure Parasarcophaga hirtipes was infected with bacteria the 1). The present study focuses mainly on the regulation of the level of PO activity declined after 24 h, reaching a baseline immune response to a novel foreign substance (Wolbachia- after 72 h. In addition, the downregulation of the immune based peptide) that has the potential to be developed into response after treatments was possibly due to a reduction a biopesticide. It does not focus on toxicology tests for in expression of the genes that act in immunity (Fang the mortality rate of the target subject because such tests et al., 2010). Significant decreases were found in the have been carried out abundantly in previous studies (Lee expression of immunity-related genes in the small white et al., 2003; Park et al., 2003; Kreutzweiser et al., 2008; butterflyPieris rapae after treatment with the venom of the Yokoyama et al., 2009). Several studies were carried out endoparasitoid Pteromalus puparum (Fang et al., 2010). on the regulations of prostaglandins by treatments with However, the immune responses of insects differ from synthetic peptides. For example, wogonin (5,7-dihydroxy- species to species (Bali and Kaur, 2013).

8-methoxyflavone), a flavonoid originating from the root In the present study, the level of PGE2 activity was of Scutellaria baicalensis, was proven to induce preventive starting to reemerge, and it increased after 48 h for all effects against ethanol-induced gastric mucosal damage peptide concentrations. However, the increments were in rats. These results were supported by the regulations still below the peak activity level of PGE2 at 24 h, and of prostaglandins in the gastric mucosa of the rats (Park they did not exceed that level. The zigzag pattern of the et al., 2004). Another example was recorded by Cherng immune response during treatments was also common in et al. (2010), who studied the effects of the Chlorella-11 previous studies, as no specific patterns were introduced peptide (Val-Glu-CysTyr-Gly-Pro-Asn-Arg-Pro-Gln-Phe) to explain this situation. For example, a study by Ebrahimi customized from a type of fresh water-grown unicellular et al. (2014) showed that the level of PO activity in the green algae, Chlorella, on blocking lipopolysaccharide- Colorado potato beetle, Leptinotarsa decemlineata, when induced macrophage activation and alleviating thermal treated against entomopathogenic nematode, Steinernema

427 MOHAMMED et al. / Turk J Zool carpocapsae, also took the form of a zigzag pattern, with h, the nodules were hardly seen on the internal organs or several upregulations and downregulations during the the internal system of A. domesticus (Figure 2C). The delay experiments. The same situation was also shown in a in the formation of nodules may be due to the Wolbachia study by Flores-Villegas (2016), who recorded a zigzag synthetic peptide not being considered at first as a threat pattern in proPO activity over time in the Chagas vector to the immune system, but over time the peptide was Meccus pallidipennis against two entomopathogenic fungi, recognized as a foreign substance that had entered the Metarhizium anisopliae and Isaria fumosorosea. system of A. domesticus. The accumulation of the peptide 4.4. Observation of nodules after being injected into the system of A. domesticus every According to previous studies on nodulation as an 24 h triggers the immune response, and hence the nodule observable immune response, nodule formations vary structure was formed to combat the unknown substance across species and treatments. For example, a study by Zhou (i.e. WolFar). Our results showed that the nodules were et al. (2009) found that nodules in the larvae of the blowfly most abundant at 72 h (Figure 2D). In addition, the Chrysomya megacephala were observable (5–6 nodules) abundance of nodulation becoming visible at 72 h into the as early as 1 h after being injected with the bacterium treatment may be due to the smaller capsule size during Ureaplasma urealyticum. According to Büyükgüzel (2012), the encapsulation event in the first 48 h. However, over a nodulation (11 nodules) in the ichneumonid wasp Pimpla period of 72 h, the capsule became bigger and observable turionellae was clearly visible by as soon as 1 h after being to the naked eye. According to Ryder (2007), the capsule treated with bovine herpes simplex virus-1 (BHSV-1). size at 72 h in A. domesticus treated with synthetic nylon In another case, a novel protein, Noduler, known to be implants was more than double (≈0.7 mm3) the size during involved in nodulation, was extracted from the Indian the first 6 h of treatment. Despite being formed from the saturniid silkmoth, Antheraea mylitta, and detected in the conserved region of the Wolbachia surface protein, the hemolymph system as early as 30 min after being treated effects of this peptide have to be further investigated. In with the bacteria Escherichia coli, Staphylococcus aureus, particular, it is necessary to establish whether this peptide and Micrococcus luteus (Gandhe et al., 2007). leads to toxicity or lethality when applied to a target There are no specific methods for identifying the nodule species. structure in the previous studies as most of the studies The regulations of PG and other eicosanoids is one of related to nodule formation confirmed this matter along the early indications of an immune response toward threats with the quantification of immune response such as levels (pathogens, viruses, nematodes, and fungi). The present of phospholipase A2 in black cutworms, Agrotis ipsilon, study suggests that WolFar, a synthetic peptide originating and true armyworms, Pseudaletia unipuncta (Jurenka et from WSP sequences, triggers an immune response in al., 1997); cicadas, Magicicada septendecim and M. cassini A. domesticus by regulating the PGE2 in the hemolymph. (Tunaz et al., 1999); and levels of phenoloxidase in moth, Moreover, these results are supported by the observation Galleria mellonella (Mandato et al., 1996). However, the of nodule formations attached to the internal system structure of the nodules in this study that were attached and fat body of A. domesticus. A better understanding to the internal system and fat bodies were morphologically of the mechanism underlying the regulation of PG identified based on several characteristics of nodule-like levels triggering this immune response could lead to the structure that have been described in previous studies development of Wolbachia-based strategies for controlling such as melanotic (Mandato et al., 1997), small and insect pests. discrete (Jurenka et al., 1997), and melanized, darkened nodules, about 0.1 mm in typical diameter and attached to Acknowledgments body walls and various organs (Tunaz et al., 1999). Hence, The authors would like to thank the Malaysian Genome we were able to confirm that the nodule-like structures in Institute (MGI) for providing the workbench for the this study were indeed nodules due to immune response ELISA lab works. This research was funded by research as they matched the descriptions stated above. Up to 48 grant GUP-2016-022.

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