A mucin protein predominantly expressed in the female-specific symbiotic organ of the stinkbug Plautia stali

Diverse insects are obligatorily associated with microbial symbionts, wherein the host often develops special symbiotic organs and vertically transmits the symbiont to the next generation. What molecular factors underpin the host-symbiont relationship is of great interest but poorly understood. Here we report a novel protein preferentially produced in a female-specific symbiotic organ of the stinkbug Plautia stali, whose posterior midgut develops numerous crypts to host a Pantoea-allied bacterial mutualist. In adult females, several posteriormost crypts are conspicuously enlarged, presumably specialized for vertical symbiont transmission. We detected conspicuous protein bands specific to the female’s swollen crypts by gel electrophoresis, and identified them as representing a novel mucin-like glycoprotein. Histological inspections confirmed that the mucin protein is localized to the female’s swollen crypts, coexisting with a substantial population of the symbiotic bacteria, and excreted from the swollen crypts to the midgut main tract together with the symbiotic bacteria. Using RNA interference, we successfully suppressed production of the mucin protein in adult females of P. stali. However, although the mucin protein was depleted, the symbiont population persisted in the swollen crypts, and vertical symbiont transmission to the next generation occurred. Possible biological roles and evolutionary trajectory of the symbiosis-related mucin protein are discussed.


Results
Identification of swollen crypt-specific proteins. In an attempt to identify proteins that are specifically expressed in the females-specific swollen crypts, we first compared the protein profiles of the normal crypts and the swollen crypts dissected from adult females of P. stali by SDS-PAGE. Coomassie Brilliant Blue (CBB) staining of the SDS-PAGE gels revealed that the banding patterns were generally similar between the normal crypts and the swollen crypts, but two prominent protein bands were specifically detected in the swollen crypts, which were located at low mobility positions of apparent molecular mass around 200 kDa and 400 kDa (Fig. 1d). The edges of these bands were strongly stained and smiling upward. We found that these bands were preferentially detected by Periodic acid Schiff (PAS) staining (Fig. 1e), suggesting that they are sugar-conjugated proteins.
We performed LC-MS/MS-based identification of the PAS-positive proteins. Prior to the mass examination, we conducted RNA-sequencing and de novo gene assembly to establish a reference catalogue of proteins representing the symbiotic midgut. We analyzed each of two specific proteins that exhibited different mobility on SDS-PAGE gels (Fig. 1d) and found that, unexpectedly, both proteins were mapped to C-terminal region of the same protein record (TRINITY_DN2761_c1_g1_i1.p1) ( Table 1). We determined the full-length mRNA sequences by PCR-amplification using several primer sets ( Supplementary Fig. S1), and identified two sequences that shared the common 5′-and 3′-regions but differed in size. The shorter variant was 1138 bp long, while the longer variant contained a 237 bp additional sequence in the middle region. Note that several single nucleotide substitutions were found in the shared regions. These genes were deduced to produce 32.7 kDa and 39.9 kDa polypeptides that have a secretory signal motif at the N-terminus and a chitin-binding domain at the C-terminus (Fig. 2a). No similar proteins were found in the protein database by Blastp similarity search against non-redundant protein sequences of the National Center for Biotechnology Information (e-value cutoff = 1e −10 ). Notably, the major part of these proteins, including the inserted region of the longer variant, was occupied by a large number of potential O-glycosylated residues consisting of proline, threonine and serine (= PTS) (Fig. 2a,  Supplementary Fig. S2). These three amino acid residues accounted for 68.5% (161/235) and 73.9% (232/314) of the PTS-rich domains for the shorter and longer variants, respectively. Such PTS-rich domains are known to be typical of mucin family glycoproteins 50 . When monosaccharide compositions of these proteins were analyzed by LC-MS after acid hydrolysis, canonical sugar components of mucin-type O-glycosylation, including N-acetylgalactosamine, N-acetylglucosamine and galactose 51,52 , were abundantly detected from both the variants (Fig. 2b). These features indicate that these proteins are heavily glycosylated mucin-like proteins. Hereafter, we designate this swollen crypt-specific mucin-like protein as SC mucin. On the ground that each insect contained either one or both of the short-and long-type SC mucins ( Supplementary Fig. S3), it seemed likely that the two length forms represent allelic variants that arose from the same gene locus.
Expression and localization of SC mucin. We examined expression levels of the SC mucin gene in the symbiotic midgut by quantitative RT-PCR using the primers that were designed to amplify both the SC mucin isoforms ( Supplementary Fig. S1). In early fifth instar nymphs whose swollen crypts were still undeveloped, the expression levels did not remarkably differ between males and females and between middle and posterior regions of the symbiotic midgut, except that there was a significant difference between the regions in female nymphs (Fig. 3). In mature adults whose swollen crypts were fully developed, the expression levels were significantly higher in the posterior region of female's symbiotic midgut where the swollen crypts form (Fig. 3). Histochemical inspection of the SC mucin protein by PAS staining visualized strong PAS signals in the cavity of the swollen crypts at the posterior end region of the symbiotic midgut (Fig. 4), verifying the specific expression and localization of the polysaccharide-rich SC mucin protein in the female-specific swollen crypts.   www.nature.com/scientificreports/ epithelium of the swollen crypts was thicker than that of the normal crypts (Figs. 4,5), presumably reflecting secretion activities there. Detailed histological observations revealed that the swollen crypts were connected to the midgut main cavity via a narrow duct (Fig. 5a). Note that, in adult insects of P. stali, the inner cavities of the normal crypts are isolated from the midgut main cavity without connection, plausibly for enabling stable symbiont retention and massive food flow simultaneously 49 . We observed that the SC mucin protein was released from the swollen crypts through the narrow duct into the midgut main cavity (Fig. 5b). FISH on the adjacent tissue sections visualized the localization patterns of the symbiotic bacteria identical to those of the SC mucin protein, being located within the swollen crypts, excreted via the narrow duct, and accumulated in the midgut main cavity (Fig. 5c). These results indicated that the SC mucin protein is specifically produced in female's swollen crypts, stored in the cavity of the swollen crypts to enclose the symbiotic bacteria, and excreted to the midgut main cavity through the narrow duct together with the symbiotic bacteria.  www.nature.com/scientificreports/ Suppression of SC mucin production by RNAi. Based on these observations, we suspected that the SC mucin may be involved in, and possibly essential for, vertical symbiont transmission via egg surface contamination in P. stali. In order to test this hypothesis, we synthesized a dsRNA targeting the 5′ region of the SC mucin gene (Supplementary Fig. S1) and attempted to suppress the SC mucin production by RNAi. Quantitative RT-PCR confirmed that the expression levels of the SC mucin gene were significantly suppressed by injection of the dsRNA into adult females (Fig. 6a). SDS-PAGE and PAS staining showed that the SC mucin protein also drastically decreased in the swollen crypts after the RNAi treatment (Fig. 6b). In the SC mucin RNAi females, enlargement of the terminal crypts was less obvious in comparison with the control females ( Fig. 7a,b). Histochemical inspection revealed that PAS stainability of the terminal crypts was clearly reduced in the SC mucin RNAi females (Fig. 7c,d). Meanwhile, FISH observation showed that the symbiont population persisted in the terminal crypts of the SC mucin RNAi females (Fig. 7e,f), indicating that depletion of the SC mucin protein did not conspicuously affect the localization and abundance of the symbiotic bacteria in the terminal crypts.
Effects of SC mucin depletion on symbiont transmission to offspring. Finally, we evaluated the effects of SC mucin depletion on vertical transmission efficiency of the symbiotic bacteria. Newly emerged adult females were injected with either control (= bla) dsRNA or SC mucin dsRNA. The number of egg masses laid within two weeks per female (5.47 vs 5.87 for control and SC mucin RNAi females respectively, N = 15 for each treatment, P > 0.05, Tukey T-test), the total number of eggs (89.2 vs 89.7, P > 0.05, Tukey T-test), and their hatching rates (80.7% vs 76.1%, P > 0.05, a likelihood ratio test for GLM) were not affected by the RNAi treatments.
We assessed the symbiont transmission efficiency by quantifying symbiont titers in the young progenies (2nd instar nymphs) derived from the second egg masses. Note that their first egg masses were discarded on account of possible carryover of remnant SC mucin protein. It turned out that the symbiotic bacteria were detected in all the investigated nymphs, and no significant difference was found in the symbiont titers between the two RNAi groups (Fig. 8a). We further assessed the offspring growth rate, because acquisition of the wholesome symbiont is indispensable for normal development in this insect 18 . In both the control group and the SC mucin RNAi group, most of the nymphs grew normally and attained adult emergence rates around 90% within the normal developmental period (Fig. 8b). These results indicate that the maternal SC mucin depletion by RNAi suppressed neither vertical symbiont transmission nor offspring growth.

Discussion
In this study, we discovered a secretory glycoprotein, SC mucin, as a predominant protein in the female-specific terminal crypts of the symbiotic midgut of the stinkbug P. stali. This female-specific organ is relatively small and morphologically not so conspicuous in comparison with the normal crypts ( Fig. 1a-c), but our in-depth histochemical observations uncovered not only its morphological and cytological architecture but also its biochemical and molecular specialization (Figs. 4,5). While previous histological studies have reported the presence of the female-specific swollen crypts in a variety of pentatomid stinkbugs [44][45][46][47] , this study is the first to report a molecular aspect of the unique organ found in stinkbug females. Initially, we found the very dense protein bands, 200 kDa and 400 kDa in estimated size, specific to the swollen crypts on SDS-PAGE gels (Fig. 1d). On the other hand, by molecular cloning and sequencing, we identified 1.1 kbp and 1.4 kbp variant genes encoding the proteins, which correspond to protein sizes of 32.7 kDa and 39.9 kDa, respectively (Fig. 2a, Supplementary Fig. S2a). The stark size discrepancies, 200 kDa vs. 32.7 kDa and 400 kDa vs. 39.9 kDa, are accounted for by massive glycosylation of the proteins, which were verified by www.nature.com/scientificreports/ strong PAS stainability of the proteins (Fig. 1e), presence of abundant potentially O-glycosylated PTS residues throughout the proteins (Fig. 2a, Supplementary Fig. S2a), and LC-MS analysis of sugars liberated from the hydrolysed proteins (Fig. 2b). We also experienced several difficulties in analyzing the proteins. For example, LC-MS/MS-based protein mapping identified peptides only to the C-terminal region. We attempted to obtain an antibody against the protein by immunizing a rabbit with a synthetic peptide representing a partial protein sequence, but the antibody recognized the synthetic peptide but did not react to the proteins. These difficulties may be attributable to steric hinderance due to bulky polysaccharides that prevent the access of reagents to the polypeptide backbone of the proteins. Mucins, which are characterized by heavily glycosylated PTS domains, are widespread proteins among animals that form extracellular mucus layers 50,52 . In vertebrates, their protective functions against pathogen infection, desiccation, and physical and chemical injuries have been documented 53 , whereas in insects and other invertebrates, their diversity and functions are still to be fully elucidated on account of their substantial variety in protein structures and expression patterns [54][55][56] . The SC mucin of P. stali possesses a single chitin-binding domain (or peritrophin-A domain) in addition to the PTS domain (Fig. 2a, Supplementary Fig. S2). This protein structure is somewhat reminiscent of the feature of invertebrate intestinal mucins, which are peritrophic  www.nature.com/scientificreports/ membrane-associated glycoproteins called peritrophins [57][58][59][60] . The insect peritrophic membrane is an extracellular film consisting of chitins and proteins that surrounds the food boluses in the gut cavity 58,61 . It has been reported that invertebrate intestinal mucins are bound to chitin matrix of the peritrophic membrane via the chitin-binding domains, thereby involved in digestive and protective roles [62][63][64][65] . Meanwhile, previous studies have noted that chitinous peritrophic membranes are not found in hemipteran insects including stinkbugs 58,59 . Actually, we observed no chitinous membranes in the symbiotic crypt cavities of P. stali (see Figs. 4,5), suggesting that the SC mucin is unlikely to play a peritrophin-like role, although the possibility that these proteins might share the common deep ancestry, though unrecognizable based on the sequence similarity, cannot be ruled out. In this study, by making use of histochemistry and FISH, we unequivocally demonstrated co-localization of the SC mucin and the symbiotic bacteria in the swollen crypts (Fig. 4), although this protein is also expressed at quite low but non-negligible levels in the normal crypts (Figs. 3, 6a). We also demonstrated that this glycoprotein is involved in the process of symbiont excretion to the main midgut tract through the special duct structures characteristic to the swollen crypts (Fig. 5). The symbiont-containing mucus released to the posteriormost part of the midgut lumen is expectedly transferred to the egg surface from the anus. Therefore, it is conceivable that the SC mucin may somehow interact with the symbiotic bacteria, and its overexpression in the swollen crypts may be associated with vertical transmission. In this context, it seems relevant that polysaccharide chains of intestinal mucins can be adhesion sites for bacteria that recognize cell-surface sugar chains 61,66 . In Drosophila, it was reported that a hemolymphal mucin is used for entrapment of bacteria 67 . In addition to the PTS domain heavily loaded with polysaccharide chains, the SC mucin contains a chitin-binding domain despite the apparent absence of chitinous structures in the midgut symbiotic organ. Notably, chitin-binding domains of some antimicrobial peptides are needed for interaction with bacteria via binding to their surface polysaccharides [68][69][70] . Hence, although speculative, the polysaccharide coat and/or the chitin-binding domain of the SC mucin might serve as possible recognition and attachment sites for the symbiotic bacteria. In general, mucin glycoproteins possess high water holding capacity 53 and high resistance against digestive enzymes 59 . These features of mucin proteins may be beneficial for the symbiotic bacteria that are smeared on the egg surface for vertical transmission where they suffer desiccation, UV irradiation and other environmental stresses. Another possible function of the SC mucin may be as an organic carbon and nitrogen source for proliferation of the symbiotic bacteria. Upon every oviposition, the symbiotic bacteria in the swollen crypts must be excreted and consumed for transfer to the egg surface. Highly glycosylated mucin proteins can be an ideal energy source for symbiotic and commensal gut bacteria 53,71 . The enhanced supply of the SC mucin may support replenishment of the symbiont population in the swollen crypts.
By injecting SC mucin dsRNA, we successfully generated adult females whose swollen crypts were depleted of the SC mucin by RNAi (Figs. 6, 7). Considering the specific localization, abundance, and co-localization with the symbiotic bacteria, we expected that the SC mucin depletion would negatively influence vertical transmission of the symbiotic bacteria. Contrary to the expectation, however, newborn nymphs from the egg masses laid by the SC mucin RNAi females acquired a substantial amount of the symbiotic bacteria and attained high adult emergence rates that are comparable to those of control newborn nymphs (Fig. 8). There are several possible reasons as to why the SC mucin depletion did not suppress the vertical symbiont transmission. Firstly, considering that vertical transmission is a pivotal process for sustaining the obligate symbiosis, multiple factors may be involved in vertical transmission of the symbiotic bacteria, and the role of the SC mucin may be complemented by other www.nature.com/scientificreports/ factors. We are now surveying such factors by RNA sequencing of the female-specific swollen crypts in combination with RNAi knockdown of candidate genes. Secondly, the RNAi treatment may have certainly suppressed the SC mucin production but not completely, and the remnant protein may be sufficient for ensuring vertical symbiont transmission. Thirdly, the SC mucin may certainly play some roles, but the roles are not essential for successful vertical transmission of the symbiotic bacteria. It should be noted that our experiments are conducted under an unnatural laboratory condition. In the field, eggs of P. stali are normally laid on host plant leaves, and symbiont-containing secretion on the eggs must experience a variety of environmental challenges, including solar radiation, washout by rain, predation, desiccation, and invasion of microbial contaminants. Considering the potential protective functions of mucin-type glycoproteins as discussed above, we suspect that the SC mucin may function by embedding the symbiotic bacteria and protecting them against environmental stresses. Alternatively, the substantial amount of SC mucin produced in the swollen crypts may contribute to constituting voluminous mucus to facilitate the excretion and smearing of the symbiotic bacteria, but it may be not essential for symbiont transmission and survival. So far, we could not detect SC mucin on the egg surface, primarily due to the difficulty to construct a specific antibody as mentioned above. To explore the full transmission route of symbiont-containing secretion from the swollen crypts to the progenies and to identify the functional component for vertical transmission are our future subjects. In intimate host-symbiont associations, both the host and the symbiont are integrated into an almost inseparable biological entity, where the symbiont cannot survive without the host and vice versa. Such symbiotic bacteria tend to exhibit genome reduction and gene losses, which lead to their incapability of independent survival and proliferation 9,10 . This particularly matters for symbiont-dependent stinkbugs, because their extracellular gut symbiotic bacteria have to be excreted and spend some time from oviposition to egg hatching outside the host www.nature.com/scientificreports/ encased in "capsules" covered with chitinous shell, and deposited beside the eggs, where the protein is essential for vertical symbiont transmission 34 . In stinkbugs of the family Urostylididae, the genome reduced symbiont Tachikawaea is implemented in voluminous galactose polymer gel, and the symbiont-containing "jelly" covers the eggs, protects them against desiccation and other environmental stresses, and serves as food source and symbiont inoculum for the nymphs 21 . In P. stali belonging to the family Pentatomidae, we identified a novel mucin protein that is preferentially produced by the female-specific swollen crypts and associated with the genome-eroded uncultivable symbiont Pantoea, although its biological role is still to be elucidated. In this way, different stinkbug groups have established intimate mutualistic associations with different bacteria, and have co-opted different molecular factors for sustaining the symbiotic associations, which highlight the dynamic aspect of host-symbiont co-evolution that can be embodied through a variety of evolutionary trajectories.

Methods
Insect materials and preparation of symbiotic midgut. We used a laboratory-maintained strain of P. stali, which was originally collected in Tsukuba, Ibaraki, Japan. This strain is associated with an obligatory uncultivable gut symbiont, Pantoea sp. A 18 , with no coexisting facultative symbionts. They were reared on raw peanuts and water supplemented with 0.05% ascorbic acid at 25 °C under a 16 h light and 8 h dark photoperiodic cycle. To obtain the symbiotic midgut used for the experiments, ice-anesthetized adult insects were dissected in a phosphate buffered saline (PBS: 137 mM NaCl, 8.10 mM Na 2 HPO 4 , 2.68 mM KCl, 1.47 mM KH 2 PO 4 , pH 7.4) using fine forceps. The dissected midgut was washed several times with sterilized PBS and divided into the normal crypt region and the swollen crypt region using an ophthalmic razor blade.

LC-MS for protein identification.
After CBB staining of SDS-PAGE gels, some protein bands were cut and stored at − 80 °C for protein identification using liquid chromatography and mass spectrometry (LC-MS) 72 . www.nature.com/scientificreports/ The gel pieces were subjected to disulfide reduction with 10 mM dithiothreitol in 25 mM ammonium bicarbonate (ABC) solution, and then to alkylation with 55 mM 2-iodoacetamide in 25 mM ABC solution. After drying, the gel pieces were infiltrated with a digesting solution containing 10 μg/mL sequence grade modified trypsin (Promega), and incubated at 36 °C for 16 h. The digested peptides were extracted with 5% (v/v) formic acidand 50% (v/v) acetonitrile-containing water, and desalted using a solid-phase extraction column (GL Tip-SDB, GL Science). Mass analyses were performed using a liquid chromatography system (Prominence, Shimadzu) coupled with an ion-trap mass spectrometer (LCQ-Fleet, Thermo) equipping an electrospray ionization source in a positive ion detection mode. Peptides were separated using an FC-ODS column (2 mm i.d. × 150 mm, Shimadzu) in a gradient elution of acetonitrile against 0.1% formic acid-containing water at a flow rate of 0.2 mL/ min. The obtained precursor/fragment mass data were subjected to protein identification using an MS/MS ion search algorism on Mascot Server (v2.7, Matrix Science). For reference, we built a protein sequence database of P. stali based on RNA sequencing data prepared from the whole midgut symbiotic region. The total RNA was extracted using RNAiso Plus (Takara) and purified with RNeasy columns (QIAGEN). The libraries were constructed using TruSeq stranded mRNA Kit (Illumina) and sequenced using Hiseq 3000 (Illumina). The sequence data were subjected to de-novo assembling using Trinity v2.11 73 , and converted to protein sequences by Trans-Decoder v5.5.0 74 . The RNA sequencing data were deposited in the DNA Data Bank of Japan under accession numbers DRX303863-5.

SC
cDNA cloning and sequencing. To determine full-length sequences of the genes encoding the swollen crypt-specific proteins, total RNA was extracted from the symbiotic midgut as described above. Full-length cDNA libraries were built using SMART cDNA Library Construction Kit (Clontech), and the 5′-and 3′-terminal fragments of the target genes were amplified using the primers (5RACE_R, 3RACE_F) shown in Supplementary  Fig. S1. The sequences of these cDNA fragments were determined using BigDye Terminator v3.1 Cycle Sequencing Kit (Life Technologies) and a DNA sequencer (ABI3130x, Applied Biosystems). To resolve the variants of the swollen crypt-specific proteins, we verified the length polymorphism of the gene by amplifying mid-sections using the primers (SC_F1, SC_F2, SC_R1, SC_R2) indicated in Supplementary Fig. S1, and sequenced them as described above. The sequencing data were deposited in the DNA Data Bank of Japan under accession numbers LC661891-2.

Sugar component analysis.
To determine sugar compositions associated with the target proteins, the protein bands were excised from SDS-PAGE gels after zinc-imidazole negative staining 75 . The gel pieces were ground in an extraction buffer (1% SDS in 20 mM Tris-HCl, pH 8.0) and incubated for 16 h to allow protein diffusion. The extracted proteins were retrieved using an ultrafiltration column (Amicon Ultra, 10 kDa cut off, Merck), precipitated by adding acetone, and lyophilized. The samples were hydrolysed by incubating at 110 °C for 4 h in 2 M trifluoroacetic acid. After drying, the hydrolysate was subjected to N-acetylation using acetic anhydride and derivatized with 1-phenyl-3-methyl-5-pyrazolone 76 . Identification and quantification of the derivatized monosaccharides were performed using the above-mentioned LC/MS system. InertSustain C18 column (2 mm i.d. × 150 mm, GL Science) was used at a flow rate of 0.2 mL/min under a gradient elution of 10 mM ammonium formate solution and acetonitrile.

Quantification of gene expression levels.
To quantify gene expression levels, we prepared mRNA samples from the dissected symbiotic midgut. The total RNAs were extracted and purified using RNAiso Plus (Takara) and RNeasy kit (QIAGEN), and reverse-transcribed using ReverTra Ace (TOYOBO). Expression levels of target genes were quantified by real-time quantitative PCR using KAPA SYBR Fast qPCR kit (Nippon Genetics) and MX3000P (Stratagene). We calculated the gene expression levels based on Ct values of standard plasmids (pT7Blue T-vector, Merck) carrying the target fragment sequences and normalized them to the constitutive gene Elongation Factor 1 alpha (EF1α). The gene-specific primer pairs (SC_qF, SC_qR, PsEF1a_qF, PsEF1a_qR) used for the quantitative PCR are shown in Supplementary Fig. S1. The gene expression levels were quantified and compared between a variety of combinations of developmental stages, sexes, and M4 regions (Fig. 3) or combinations of RNAi treatments and M4 regions (Fig. 6a). To ensure flat and simple comparisons of the gene expression levels among these treatment groups, we adopted single one-factor GLM rather than multi-factor GLM for statistics.
Histological observation. The dissected symbiotic midgut samples were fixed with 4% paraformaldehyde in PBS. After washing with PBST (0.1% Triton-X containing PBS), the samples were dehydrated with a graded alcohol series and embedded in Technovit 7100 or 8100 resin (Kulzer). The embedded samples were processed into 2 μm sections using a microtome (RM2255, Leica). The sections were fixed on glass slides and subjected to PAS staining or fluorescent in situ hybridization (FISH). For PAS staining, the sections were treated with 0.5% periodic acid for 10 min and then cold-Schiff reagent (Fujifilm) for 2 min. After counterstaining with hematoxylin (Fujifilm), the sections were sealed in a mounting agent (Entellan New, Merck) and observed under a light microscope. FISH was performed essentially as described previously 49 using an oligonucleotide probe EUB917 conjugated with Alexa Fluor 647 fluorescent dye that was designed for eubacterium detection 77 . Counter fluorescent staining was conducted with 4′,6-diamidino-2-phenylindole (DAPI) for nucleic acid visualization.
RNA interference. RNA interference (RNAi) was performed as described previously 78 . A 331 bp fragment located in the shared region of the two isoforms was amplified using the primers (SCdsR_F, SCdsR_R) shown in Supplementary Fig. S1 and cloned into pT7Blue T-vector (Merck www.nature.com/scientificreports/ injected with 1 μL of the 200 ng/μL dsRNA solution using a glass capillary from the intersegmental membrane between head and prothorax. As a control treatment, dsRNA of β-lactamase (bla) gene fragment was injected in the same manner. To assess the transmission rates of symbiotic bacteria, the treated adult females were allowed to lay egg masses for two weeks. Note that most females started to lay eggs after about a week. For direct detection of the transmitted symbiont, we performed qPCR using specific primers for the 16S rRNA gene of symbiont A 18 . The offspring DNA samples were extracted from the whole body of second instar nymphs at the day of ecdysis using DNA MINI Kit (QIAGEN). qPCR was performed as mentioned above, but the different reagent [KOD One (TOYOBO) supplemented with SYBR green I (BioWhittaker Molecular Applications) fluorescent dye 79 ] was used. It should be noted that, when uninfected nymphs emerging from formaldehyde-sterilized eggs 18,80 were subjected to qPCR, no detectable amplification was observed within 32 PCR cycles in this procedure (0/8 nymphs). We also assessed the offspring growth rate as another index of infection status. The progenies were aseptically reared in plastic dishes with sterilized water and raw peanuts 80 , and recorded the adult emergence rate within a normal growth period (27 days from hatching). We used second egg masses for the qPCR and second and third egg masses for the rearing experiment. Firstly-laid egg masses were not used in order to exclude the possibility of remnant effects of target proteins.