Obligate Symbionts Activate Immune System Development in the Brian L. Weiss, Michele Maltz and Serap Aksoy This information is current as J Immunol 2012; 188:3395-3403; Prepublished online 24 of September 27, 2021. February 2012; doi: 10.4049/jimmunol.1103691 http://www.jimmunol.org/content/188/7/3395 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Obligate Symbionts Activate Immune System Development in the Tsetse Fly

Brian L. Weiss, Michele Maltz, and Serap Aksoy

Many rely on the presence of symbiotic for proper immune system function. However, the molecular mechanisms that underlie this phenomenon are poorly understood. Adult tsetse flies (Glossina spp.) house three symbiotic bacteria that are vertically transmitted from mother to offspring during this ’s unique viviparous mode of reproduction. Larval tsetse that undergo intrauterine development in the absence of their obligate mutualist, Wigglesworthia, exhibit a compromised immune system during adulthood. In this study, we characterize the immune phenotype of tsetse that develop in the absence of all of their endogenous symbiotic microbes. Aposymbiotic tsetse (Glossina morsitans morsitans [GmmApo]) present a severely compromised immune system that is characterized by the absence of phagocytic hemocytes and atypical expression of immunity-related genes.

Correspondingly, these flies quickly succumb to infection with normally nonpathogenic Escherichia coli. The susceptible pheno- Downloaded from type exhibited by GmmApo adults can be reversed when they receive hemocytes transplanted from wild-type donor flies prior to infection. Furthermore, the process of immune system development can be restored in intrauterine GmmApo larvae when their mothers are fed a diet supplemented with Wigglesworthia cell extracts. Our finding that molecular components of Wigglesworthia exhibit immunostimulatory activity within tsetse is representative of a novel evolutionary adaptation that steadfastly links an obligate symbiont with its host. The Journal of Immunology, 2012, 188: 3395–3403. http://www.jimmunol.org/

ll metazoan life forms interact with prokaryotic organ- ducing the production of phagocytic granulocytes, and directly isms on a perpetual basis. These associations often result generating antimalarial (4–6). A in a fitness advantage for one or both partners involved Tsetse flies (Glossina spp.) harbor three symbiotic bacteria that (1, 2). Insects represent a group of higher eukaryotes that harbors regulate important aspects of their host’s physiology. Two of these a well-defined bacterial microbiota. Unlike their mammalian microbes, obligate Wigglesworthia and commensal Sodalis,aretrans- counterparts, insects house less complex bacterial communities, ferred to developing intrauterine progeny via maternal milk gland are relatively inexpensive to maintain, and produce large numbers secretions (7). Tsetse’s third symbiont, Wolbachia, is transferred via

of offspring in a short period of time. Several studies demon- the germline (8). Tsetse that undergo intrauterine larval development by guest on September 27, 2021 strated the importance of symbiotic bacteria as they relate to the in the absence of Wigglesworthia are immunocompromised during proper function of their insect host’s immune system. For exam- adulthood. This phenotype is characterized by a significantly reduced ple, Drosophila naturally infected with Wolbachia are protected population of phagocytic sessile and circulating hemocytes, as well as (through an unknown mechanism) from several otherwise harmful an unusual susceptibility to infection with pathogenic trypanosomes RNA viruses (3). The malaria vector Anopheles gambiae is un- and normally nonpathogenic Escherichia coli K12 (9–11). Further usually susceptible to infection with Plasmodium parasites when studies on the tsetse/Wigglesworthia symbiosis, as it relates to host they lack their commensal microbiota. In this case, symbiotic immunity, have been obstructed by our inability to reconstitute sym- bacteria appear to mediate anti-Plasmodium immunity by acti- biont-free flies with this bacterium. vating basal expression of antimicrobial peptides (AMPs), in- In the current study, we investigated the intimate relationship between immunity and symbiosis in tsetse by producing flies that underwent larval development in the absence of all endogenous Division of Epidemiology of Microbial Diseases, Department of Epidemiology and microbes. We analyzed the immune system phenotype of apo- Public Health, Yale University School of Medicine, New Haven, CT 06520 symbiotic tsetse (Glossina morsitans morsitans;[GmmApo]) fol- Received for publication December 21, 2011. Accepted for publication January 30, lowing microbial challenge, and investigated whether loss of 2012. immunity in GmmApo flies could be rescued through either trans- This work was supported by grants from the National Institute of Allergy and Infec- fer of immune cells from healthy individuals or symbiont provi- tious Diseases (AI051584), the National Institute of General Medical Science (069449), and the Ambrose Monell Foundation (all to S.A.). sioning. We obtained results that reinforce the obligate nature Address correspondence and reprint requests to Dr. Brian L. Weiss, Division of of tsetse’s relationship with Wigglesworthia and provide further Epidemiology of Microbial Diseases, Department of Epidemiology and Public insights into the basic molecular mechanisms that underlie sym- Health, Yale University School of Medicine, LEPH 607, 60 College Street, New biont-induced maturation of host immunity. Haven, CT 06520. E-mail address: [email protected] The online version of this article contains supplemental material. Materials and Methods Abbreviations used in this article: AMP, antimicrobial peptide; dpc, days postchal- lenge; DV, dorsal vessel; GC, gonotrophic cycle; GmmApo, aposymbiotic Glossina Tsetse and bacteria WT morsitans morsitans; Gmm , wild-type Glossina morsitans morsitans; iNOS, induc- G. morsitans morsitans were maintained in Yale’s insectary at 24˚C with ible NO synthase; PGN, peptidoglycan; PSA, polysaccharide A; qPCR, quantitative 50–55% relative humidity. These flies received defibrinated bovine blood real-time PCR; recE. coliGFP, GFP-expressing Escherichia coli K12; recE. colipIL, luciferase-expressing Escherichia coli; tep, thioester-containing ; WT, wild- (Hemostat Laboratories) every 48 h through an artificial membrane feeding type. system (12). Designations of all tsetse cohorts used in this study, the composition of their symbiont populations, and the treatments that they Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 received are described in Table I. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1103691 3396 SYMBIONT-INDUCED IMMUNITY IN TSETSE

Table I. Designation of tsetse cohorts used in this study, their symbiont status, and the treatment they received

Tsetse Designation Symbiont Status Origin/Treatment Reference/Source GmmWT Wgm, Sgm, Wol None None GmmWgm2 Sgm, Wol Offspring of mothers treated with Amp, yeast extract (9) GmmApo Apo Offspring of mothers treated with Tet, yeast extract (16) GmmApo/WT Apo Received transplant from GmmWT donors This study GmmApo/Apo Apo Received hemolymph transplant from GmmApo donors This study GmmApo/Sol Apo Received soluble fraction of GmmWT donor hemolymph This study GmmApo/Cell Apo Received cellular fraction of GmmWT donor hemolymph This study GmmApo/Wgm Apo Offspring of symbiont-cured mothers complemented with Wgm cell extracts This study GmmApo/Sgm Apo Offspring of symbiont-cured mothers complemented with Sgm cell extracts This study GmmApo/NB Apo Offspring of symbiont-cured mothers that received no bacterial complement This study Amp, ampicillin; Apo, aposymbiotic; Sgm, Sodalis; Tet, tetracycline; Wgm, Wigglesworthia; Wol, Wolbachia.

Luciferase-expressing E. coli (recE. colipIL) K12 were produced via the abdomen. Hemolymph exuding from the wound was collected using transformation with construct pIL, which encodes the firefly luciferase a glass micropipette and placed into a microfuge tube on ice. Four cohorts of gene under transcriptional control of Sodalis’ insulinase promoter (13). newly emerged aposymbiotic recipient flies were used, two of which were Apo/WT Apo/Apo

The assay used to quantify recE. colipIL cells in vivo was performed as de- designated Gmm or Gmm based on whether they received Downloaded from scribed previously (13). GFP-expressing E. coli K12 (recE. coliGFP) were hemolymph transplanted from wild-type (WT) or aposymbiotic donors, produced via electroporation with pGFP-UV plasmid DNA (Clontech). respectively. GmmApo/WT or GmmApo/Apo recipient flies received 1 ml donor Sodalis were isolated from surface-sterilized G. morsitans pupae and hemolymph (this volume represents approximately one third of the total cultured on Aedes albopictus C6/36 cells, as described previously (14). volume collected from donor flies). On day 8 posttransplantation, three of Sodalis, which has a doubling time of ∼24 h, was subsequently maintained these flies were sacrificed to quantify hemocyte number using a Bright- in vitro, in the absence of C6/36 cells, at 25˚C in Mitsuhashi–Maramorosch Line hemocytometer. To separate GmmWT donor hemolymph into soluble medium (1 mM CaCl2, 0.2 mM MgCl2, 2.7 mM KCl, 120 mM NaCl, 1.4 and cellular fractions, samples were centrifuged at 3000 3 g for 5 min. mM NaHCO3, 1.3 mM NaH2PO4, 22 mM D (+) glucose, 6.5 g/l lactal- The cellular component was resuspended in a volume of chilled antico- http://www.jimmunol.org/ bumin hydrolysate, and 5.0 g/l yeast extract) supplemented with 5% heat- agulant buffer (70% Mitsuhashi–Maramorosch medium, 30% anticoagu- inactivated FBS (14). lant citrate buffer [98 mM NaOH, 186 mM NaCl, 1.7 mM EDTA, and 41 mM citric acid (pH 4.5) v/v]) (15) equal to the total amount of hemolymph Tsetse infections from which they were collected. The remaining two cohorts of GmmApo m Systemic challenge of tsetse was achieved by anesthetizing flies with CO recipient flies were injected with either 1 l cellular suspension (these flies 2 GmmApo/Cell m and subsequently injecting individuals with live bacterial cells using glass are designated )or1 l the soluble hemolymph fraction (these flies are designated GmmApo/Sol). needles and a Narashige IM300 microinjector. Per os bacterial challenges 3 were performed by adding 500 CFU E. coli per 20 ml (the approximate All aposymbiotic recipient flies were challenged with either 10 CFU live recE. coli or recE. coli . Injections were performed using glass needles amount consumed by a fly) of the total blood meal. The vertebrate host pIL GFP and a Narashige IM300 microinjector. Quantification of recE. colipIL in complement system was heat inactivated (56˚C for 1 h) prior to inoculating by guest on September 27, 2021 blood meals with bacterial cells. The number of bacterial cells injected or recipient tsetse was performed as described above. Phagocytic capacity of GmmApo/Apo fed, control group designations, and sample size for all infection experi- transplanted hemocytes was determined by infecting recipient 3 E. coli ments are indicated in the corresponding figures and their legends. flies with 10 CFU live rec GFP. Twelve hours postchallenge, hemo- lymph was collected from three individuals, and hemocytes were monitored Hemolymph collection and hemocyte quantification for the presence of engulfed GFP-expressing bacterial cells. Hemolymph samples were fixed on glass microscope slides via a 2-min incubation in 2% Hemolymph collection from wild-type G. morsitans morsitans (GmmWT) Apo paraformaldehyde. Prior to visualization using a Zeiss Axioscope micro- and Gmm flies was performed using the high-injection/recovery scope, slides were overlaid with VECTASHIELD HardSet Mounting Me- method, as described previously (15). Subsequent determination of cir- dium containing DAPI (Vector Laboratories). culating hemocyte abundance was performed using a Bright-Line hemo- cytometer (11). Sessile hemocyte abundance was quantified by subjecting Bacterial complementation experiments GmmWT and GmmApo flies (n = 3) to hemocoelic injection with blue fluorescent microspheres. Twelve hours postinjection, flies were dissected A cartoon illustrating how bacterial complement experiments were per- to reveal tsetse’s dorsal vessel (DV). Exposed tissue was rinsed three times formed is shown in Supplemental Fig. 1. Three cohorts (n = 120 individuals/ with PBS to remove contaminating circulating hemocytes or any beads not group) of pregnant female tsetse were fed a diet containing tetracycline (40 m engulfed by sessile hemocytes. Engulfed beads were visualized micro- g/ml blood) every other day for 10 d. Additionally, throughout the course scopically by excitation with UV light (365/415 nm). Relative fluores- of the experiment, all blood meals (three/wk) also contained vitamin-rich cence, which was quantified using ImageJ software, represents the average yeast extract (1% w/v) to restore fertility associated with the absence of amount of light emitted from three GmmWT and GmmApo individuals. Wigglesworthia (16). Ten days postcopulation, two cohorts of symbiont- cured females were regularly fed a diet supplemented with Wigglesworthia Quantitative analysis of immunity-related gene expression and Sodalis cell extracts. By timing treatments in this manner, larvae from the first gonotrophic cycle (GC) went through most of their development in For quantitative real-time PCR (qPCR) analysis of immunity-related gene the absence of bacterial complement, whereas those from the second and expression, whole flies were homogenized in liquid nitrogen, and total RNA third GCs developed in the presence of bacterial complement. Offspring of was extract using TRIzol reagent (Invitrogen). Randomly primed cDNAs these females were designated GmmApo/Wgm and GmmApo/Sgm, respectively. were generated with Superscript II reverse transcriptase (Invitrogen), and Wigglesworthia was obtained by dissecting tsetse (an organ qPCR analysis was performed using SYBR Green Supermix and a Bio-Rad immediately adjacent to the midgut that houses this bacteria) from GmmWT C1000 thermal cycler. Amplification primers are listed in Supplemental Table females, whereas Sodalis was maintained in culture, as described above. I. Quantitative measurements were performed on three biological samples GmmApo/Wgm females were fed one equivalent per four females, in duplicate, and results were normalized relative to tsetse’s constitutively Apo/Sgm 3 7 b and Gmm females were fed 4 10 Sodalis/ml blood (thus, these expressed -tubulin gene (determined from each corresponding sample). flies ingested ∼1 3 106 Sodalis each time they fed). A third control cohort Fold-change data are represented as a fraction of average normalized gene of symbiont-cured females received no bacterial complement (their off- expression levels in bacteria-infected flies relative to expression levels in Apo/NB 6 spring are designated Gmm ), and a fourth cohort of WT offspring corresponding uninfected controls. Values represent mean SEM. (GmmWT) served as another control. To confirm the aposymbiotic status of Hemolymph transplantation offspring from symbiont-cured mothers (Supplemental Fig. 2), genomic DNA was extracted from larval offspring (third instar; n = 3) of all ex- Undiluted hemolymph was collected by removing one front fly leg at the perimental cohorts using the Holmes–Bonner method (17). PCR (20-ml joint nearest the thorax and then applying gentle pressure to the distal tip of reactions) was performed in an MJ Research Thermal Cycler using The Journal of Immunology 3397 bacteria-specific primers (Supplemental Table I) and the following cycle symbiotic bacteria (GmmApo) exhibited an immune system phe- program: 95˚C for 5 min, followed by 30 cycles at 95, 55, and 72˚C, each notype during adulthood that was different from that of their WT for 1 min, and a final 7-min elongation/extension at 72˚C. counterparts that developed in the presence of their complete To determine whether complementing symbiont-cured mothers with bac- terial cell extracts impacted the immune system phenotype of their offspring, microbiome. To do so, we began by quantifying the number of qPCR was used (as described above) to monitor the expression of serpent and circulating and sessile hemocytes present in 8-d-old adult (here- lozenge in larvae (first, second, and third instar) from each of three GCs (n =3 after referred to as “mature”) GmmWT and GmmApo flies. Our individuals/group/GC). All remaining offspring were allowed to mature to results indicate that mature WT tsetse harbor 113-fold more cir- adulthood. At this time, three individuals from each cohort and GC were m taken to determine circulating hemocyte abundance (as described above). culating hemocytes/ l of hemolymph than do their aposymbiotic WT Furthermore, qPCR was used to compare immunity-related gene expression counterparts (Gmm , 793 6 34 hemocytes/ml of hemolymph; in E. coli-challenged GmmApo/Wgm and GmmApo/Sgm individuals (n =3)from GmmApo,76 1 hemocytes/ml of hemolymph; Fig. 1A). To de- the second GC of symbiont-cured mothers. Finally, all remaining mature 3 termine the functional relationship between symbiont status and adult offspring were challenged with 10 CFU live recE. coliGFP.Twelve sessile hemocyte abundance, we thoracically microinjected WT hours postchallenge, hemolymph was collected and monitored to determine whether hemocytes had engulfed GFP-expressing bacterial cells (n =3 and aposymbiotic adults with fluorescent microspheres. In both individuals/group/GC). Hemolymph samples were fixed and visualized as tsetse and Drosophila, sessile hemocytes concentrate in large described above. quantities around the anterior chamber of the fly’s DV (11, 19). Statistics Thus, we indirectly quantified sessile hemocyte number by mea- suring the fluorescent emission of injected microspheres that were Statistical significance among various treatments, as well as treatments and WT controls, is indicated in the figure legends. Survival curve comparisons were found engulfed in this region. We observed that mature Gmm made by log-rank analysis using JMP (v9.0) software (http://www.jmp. flies engulfed 16-fold more microspheres than did age-matched Downloaded from com). Statistical analysis of qPCR data and hemocyte abundance was GmmApo individuals (Fig. 1B, Supplemental Fig. 3). performed by the Student t test using Microsoft Excel software. Previously, we determined that several genes associated with humoral, cellular, and epithelial immune pathways, including those Results that encode the AMPs attacin and cecropin, as well as thioester- Aposymbiotic tsetse exhibit atypical hallmarks of cellular and containing (teps) tep2 and tep 4, prophenoloxidase, and humoral immunity inducible NO synthase (iNOS), were expressed at significantly http://www.jimmunol.org/ 2 A positive correlation exists between the proper function of an lower levels in GmmWgm flies compared with GmmWT flies fol- insect’s immune system and the dynamics of its microbiome (18). lowing infection with E. coli (11). In the current study, we mon- In an effort to better define the relationship between symbiosis and itored expression of these same genes in age-matched GmmWT and immunity in tsetse, we fed pregnant females a diet supplemented GmmApo flies that were either unchallenged or 3 d postchallenge with tetracycline and yeast. This antibiotic treatment clears all (dpc) with E. coli K12. Furthermore, we also evaluated the ex- symbionts from the flies, whereas the vitamin-rich yeast extract pression of PGRP-LB, caudal, domeless, and DUOX. In tsetse and rescues the loss of fertility associated with the absence of obligate closely related Drosophila, PGRP-LB and caudal serve as nega- Wigglesworthia (9, 16). We then investigated whether offspring tive regulators of NF-kB–dependent antimicrobial peptide ex- that underwent intrauterine development in the absence of all pression (10, 20, 21), whereas domeless is a cytokine receptor that by guest on September 27, 2021

FIGURE 1. Aposymbiotic tsetse display atypical hallmarks of cellular and humoral immunity. (A) Number of circulating hemocytes/ml of hemolymph in mature GmmWT and GmmApo flies (n = 3 individuals from each tsetse line). (B) Quantitative analysis of sessile hemocyte abundance adjacent to the anterior chamber of the DV of mature GmmWT and GmmApo flies (n = 3 individuals from each tsetse line). Relative fluorescence is proportional to the number of microspheres engulfed by sessile hemocytes and, thus, the number of these cells present in the region examined. (C) The effect of symbiont status and route of infection on the expression of selected immunity-related genes. Gene expression in uninfected GmmApo and GmmWT individuals is normalized relative to constitutively expressed tsetse b-tubulin (left panel). Fold change in the expression of immunity-related genes in GmmApo and GmmWT tsetse 3 d after per os (middle panel) and intrathoracic (right panel) challenge with E. coli K12. All fold change values are represented as a fraction of average normalized gene expression levels in bacteria-challenged flies relative to expression levels in PBS-injected controls. All quantitative measurements were performed on three biological samples in duplicate. Genes without a corresponding bar did not exhibit a fold change in expression between samples compared or their ex- pression was undetectable via qPCR. Values are presented as means. *p , 0.05, **p , 0.005, Student t test. 3398 SYMBIONT-INDUCED IMMUNITY IN TSETSE regulates expression of tep4 through the JAK/STAT-signaling ceptible to systemic challenge with a foreign microbe than are pathway (22, 23). Finally, in Drosophila and mosquitoes, DUOX age-matched GmmWT and GmmWgm2 individuals. Furthermore, is involved in generating infection-induced antimicrobial reactive tsetse’s ability to overcome per os challenge with E. coli appears oxygen species (24–26). to be independent of symbiont status. Our expression analysis indicates that the presence of symbiotic To determine a cause for the variation in survival that we ob- bacteria during larval development induce basal immunity in tsetse. served among GmmWT, GmmWgm2, and GmmApo individuals fol- Specifically, we observed that DUOX, domeless, and caudal are lowing challenge with E. coli, we monitored the dynamics of expressed at significantly lower levels in mature unchallenged bacterial growth in each of these fly groups over time. When fed GmmApo flies compared with GmmWT flies (Fig. 1C, left panel). E. coli, both mature aposymbiotic and WT individuals cleared all Following per os challenge with E. coli, no significant difference E. coli. Following systemic challenge with 103 CFU of E. coli, in immunity-related gene expression (with the exception of iNOS) bacterial densities within mature GmmWT flies reached 8.3 3 103 was observed between GmmWT and GmmApo flies (Fig. 1C, middle cells before being cleared. Interestingly, GmmWgm2 flies, which panel). However, systemic challenge resulted in a significant dif- perish following challenge with 106 CFU of E. coli (11), were able ference in the expression of all of the genes that we analyzed. to clear all exogenous bacterial cells following challenge with this Most notably, pathways associated with cellular immunity were lower dose. In contrast, bacterial density in GmmApo flies peaked significantly downregulated in GmmApo individuals compared at 7.8 3 106 on day 6 postchallenge, after which all flies soon with GmmWT individuals, whereas those associated with humoral perished (Fig. 2B). This observation suggests that aposymbiotic immune responses were significantly upregulated (Fig. 1C, right tsetse were unable to control systemic infection with E. coli and, panel). These findings indicate that tsetse’s symbiotic bacteria are thus, likely perished as a result of their inability to tolerate high Downloaded from closely associated with the development of their host’s immune densities of this bacterium in their hemolymph. Taken together, system during larval maturation and its subsequent proper func- these findings indicate that GmmApo flies are significantly more tion in unchallenged and E. coli-challenged adults. susceptible to challenge with E. coli than are WT flies and flies that lack only Wigglesworthia. Aposymbiotic tsetse are highly susceptible to normally nonpathogenic E. coli Hemocyte transfer from WT tsetse restores the ability of

Apo http://www.jimmunol.org/ We next determined whether GmmApo individuals are more sus- Gmm adults to overcome infection with E. coli ceptible to challenge with E. coli than are WT tsetse or tsetse that We next set out to provide a definitive correlation between tsetse lack only Wigglesworthia (GmmWgm2). To do so, we compared hemocytes and the fly’s ability to overcome challenge with a foreign percent survival of mature adults from these three tsetse lines microbe. To do so, we transplanted hemolymph from mature following systemic challenge with E. coli K12. We determined GmmWT and GmmApo individuals (donor flies) into the hemocoel of that 67% of mature GmmWT individuals and 59% of mature susceptible GmmApo flies (recipient flies are hereafter designated GmmWgm2 individuals survived systemic challenge with 103 CFU GmmApo/WT and GmmApo/Apo, respectively). Five days after this of E. coli (Fig. 2A, top and middle panels). In contrast, all age- procedure, we determined that GmmApo/WT flies harbored 330 6 Apo Apo/Apo matched Gmm individuals perished by 12 dpc (Fig. 2A, bottom 20.4 hemocytes/ml of hemolymph, whereas Gmm flies har- by guest on September 27, 2021 panel). We next challenged GmmWT and GmmApo flies per os with bored 5 6 3.8 hemocytes/ml of hemolymph (Fig. 3A). We next 103 and 106 CFU of E. coli and found that all individuals survived investigated whether our hemolymph-transplantation procedure was this challenge (Fig. 2A, top and bottom panels). This finding able to rescue the E. coli-susceptible phenotype exhibited by suggests that mature GmmApo flies are considerably more sus- GmmApo flies. To do so, we challenged GmmApo/WT and GmmApo/Apo

FIGURE 2. Symbiont status mediates tsetse’s ability to survive challenge with E. coli K12. (A) The effect of symbiont status on the survival of tsetse following systemic and per os challenge with E. coli K12. Mature adult GmmApo flies were significantly more susceptible to challenge with 103 CFU of E. coli than were age-matched GmmWT flies (bottom and top panels, p , 0.001) and GmmWgm2 flies (bottom and middle panels, p , 0.001). Both GmmWT and GmmApo flies survived per os challenge with E. coli. Infection experiments were performed in triplicate, using 25 flies/replicate. (B) Average number 3 (6 SEM) of recE. colipIL per tsetse cohort over time (n = 3 individuals/cohort/time point) following systemic and per os challenge with 10 CFU of bacteria. Values shown in gray represent lethal infections. By 8 dpc, not enough E. coli-injected GmmApo flies remained to quantify bacterial density. The Journal of Immunology 3399 Downloaded from

FIGURE 3. Hemocytes modulate tsetse’s ability to overcome challenge with E. coli K12. (A) Hemolymph was collected from GmmApo and GmmWT donor flies and immediately transplanted into GmmApo recipients. Five days posthemolymph transplantation, hemocyte abundance in GmmApo/WT and GmmApo/Apo Apo/WT Apo/Apo

recipient flies was quantified microscopically using a hemocytometer. Gmm flies housed significantly more circulating hemocytes than did Gmm http://www.jimmunol.org/ flies. (B) GmmApo/WT and GmmApo/Apo recipient flies were challenged with E. coli 3 d after receiving a hemolymph transplant. Significantly more GmmApo/WT individuals survived E. coli challenge than did their GmmApo/Apo counterparts (top panel, p , 0.001). Donor hemolymph was then divided into cellular and soluble fractions via centrifugation. Significantly more GmmApo/Cell individuals survived E. coli challenge than did their GmmApo/Sol counterparts (bottom Apo/WT Apo/Apo panel, p , 0.001). (C) Average number (6 SEM) of recE. colipIL per Gmm and Gmm recipient fly over time (n = 3 individuals/treatment/time point) following systemic challenge with 103 CFU of bacteria. Values shown in gray represent lethal infections. (D) Twelve hours postchallenge with recE. coliGFP, hemolymph was collected, fixed on glass slides using 2% paraformaldehyde, and microscopically examined for the presence of hemocyte-engulfed bacterial cells. GmmApo/WT recipient flies harbor engulfed bacterial cells. Original magnification 3400. **p , 0.005.

individuals with 103 CFU of E. coli 3 d posthemolymph trans- that bacterial sepsis was the cause of high mortality we observed in by guest on September 27, 2021 plantation and subsequently monitored their survival over time. Our this group of flies. In contrast, aposymbiotic recipients were able to results indicate that 72% of GmmApo/WT flies survived for 14 d clear all E. coli by 8 dpc when they had previously received a following challenge. In comparison, only 2% of GmmApo/Apo flies hemolymph transplant from GmmWT donors (Fig. 3C). More so, survived their challenge (Fig. 3B, top panel). These results dem- microscopic examination of hemolymph from GmmApo/WT flies onstrate that GmmApo flies are able to clear a systemic challenge showed that transplanted hemocytes engulfed the introduced E. with E. coli after they receive a transplant of hemolymph from WT coli (Fig. 3D). Our results demonstrate that immune resistance can donors. be restored in adult aposymbiotic tsetse if they harbor hemocytes We next investigated whether hemocytes or a soluble antimi- transplanted from their WT counterparts. crobial or signaling molecule present in the transplanted hemo- lymph was responsible for restoring the resistant phenotype Supplementation of Wigglesworthia to symbiont-cured females exhibited by recipient individuals. To address this issue, we col- restores immune system development in aposymbiotic offspring lected hemolymph from WT donors, separated it into soluble and Previous experiments revealed that the milk gland population of cellular fractions by centrifugation, and then transplanted the sep- tsetse’s obligate symbiont, Wigglesworthia, must be present dur- arate fractions into two distinct groups of GmmApo flies. Finally, ing the development of immature stages for subsequent adults to 3 d later we systemically challenged both groups of recipient exhibit a functional cellular immune system (11). We have not flies with 103 CFU of E. coli K12. All aposymbiotic flies that been able to culture Wigglesworthia and, thus, cannot recolonize received the soluble fraction of hemolymph from GmmWT aposymbiotic flies with this bacterium. To circumvent this im- donors (GmmApo/Sol) perished by day 12 postchallenge. In com- pediment, we tested whether we could restore the process of im- parison, 62% of GmmApo recipients that received the cellular mune system development in GmmApo offspring by supplementing fraction of hemolymph from GmmWT donors (GmmApo/Cell) sur- the diet of pregnant, symbiont-cured females with Wigglesworthia- vived for 14 d following bacterial challenge (Fig. 3B, bottom containing extracts of bacteriome tissue collected from WT fe- panel). These host survival curves indicate that GmmApo flies males. A detailed description of the experimental design that we survived challenge with E. coli when they had previously received used to test this theory is provided in the Materials and Methods a transplant of hemocytes, as opposed to soluble hemolymph mol- and Supplemental Fig. 1. ecules, from WT tsetse. In brief, two treatment cohorts of pregnant GmmWT females were To determine a cause for the variation in survival that we ob- fed a diet supplemented with tetracycline and yeast extract (16). served between these two groups, we monitored the dynamics of Ten days postcopulation, these symbiont-cured females began re- bacterial growth in each group over the course of the experiment. E. ceiving either Wigglesworthia or Sodalis cell extracts in every coli within GmmApo/Apo flies replicated exponentially, until a peak blood meal. The immune system phenotype of offspring from these density of 2.1 3 107 was reached at 6 dpc. This finding suggests females (GmmApo/Wgm and GmmApo/Sgm, respectively) was com- 3400 SYMBIONT-INDUCED IMMUNITY IN TSETSE pared with that of control cohort offspring from symbiont-cured found at significantly higher levels in adult GmmApo/Wgm flies mothers that received no bacterial supplement (GmmApo/NB) and compared with adult GmmApo/Sgm flies (from GC2) following offspring from GmmWT mothers. We first evaluated the relative systemic challenge with E. coli (Fig. 4C). A similar pattern was abundance of transcripts that encode the transcription factors Ser- observed with genes involved in the generation of reactive oxy- pent and Lozenge. In Drosophila, these molecules direct hemocyte gen species (DUOX and iNOS). Interestingly, humoral immunity- differentiation, or hematopoiesis, during embryogenesis and early associated genes (AMPs and their regulators) were expressed at larvagenesis (27). In tsetse, larvae that develop in the absence of similar levels in E. coli-challenged GmmApo/Wgm and GmmApo/Sgm Wigglesworthia express significantly less serpent and lozenge than adults. do their WT counterparts (11). In the current study, we found that Our results suggest that feeding symbiont-cured mothers a diet GmmApo/Wgm, GmmApo/Sgm, and GmmApo/NB larva from the first GC supplemented with Wigglesworthia cell extracts induces a physi- expressed significantly less serpent and lozenge than did GmmWT ological response that partially restores immune system develop- larva. However, after the onset of bacterial supplementation, ment in their aposymbiotic offspring. Specifically, GmmApo/Wgm GmmApo/Wgm and GmmWT larva from the second and third GCs larvae exhibit increased expression of the hematopoietic tran- expressed comparable levels of serpent and lozenge, whereas scription factors serpent and lozenge, and, as adults, these flies GmmApo/NB and GmmApo/Sgm larva expressed less (Fig. 4A). present a functional immune system characterized by the presence Because serpent and lozenge expression can be indicative of of circulating phagocytic hemocytes. Furthermore, the expression hematopoiesis, we next compared the number of hemocytes pre- of genes involved in epithelial and cellular immunity is enhanced sent in GmmApo/Wgm adults to that found in age-matched GmmWT, in GmmApo/Wgm adults.

Apo/NB Apo/Sgm Downloaded from Gmm , and Gmm flies. We found that the provi- Apo/Wgm sioning of Wigglesworthia extracts to symbiont-cured females Gmm flies are resistant to E. coli challenge resulted in an increase in the number of circulating hemo- We observed that GmmApo/Wgm offspring exhibit hallmarks of en- cytes present in their offspring. Specifically, hemocyte density hanced immunity. Thus, we next tested whether mature GmmApo/ in GmmApo/Wgm adults from GCs 2 and 3 was significantly greater Wgm adults would be resistant to systemic challenge with E. coli (113 6 33 and 127 6 21 hemocytes/ml of hemolymph, respec- K12, whereas age-matched GmmApo/Sgm and GmmApo/NB flies Apo/NB tively) than that found in age-matched Gmm (7 6 3 and 9 6 wouldnot.Tothisend,weobservedthat38and43%of http://www.jimmunol.org/ 4 hemocytes/ml of hemolymph, respectively) and GmmApo/Sgm GmmApo/Wgm adults from GCs 2 and 3, respectively, survived flies (10 6 4 and 4 6 1 hemocytes/ml hemolymph, respectively), challenge with 103 E. coli (Fig. 5A). Correspondingly, microscopic but it was significantly less than that of GmmWT adults (733 6 104 inspection of hemolymph from E. coli-resistant GmmApo/Wgm adults and 681 6 68 hemocytes/ml hemolymph, respectively; Fig. 4B). revealed the presence of phagocytic hemocytes that harbored in- Correspondingly, we observed that prophenoloxidase and tep4, ternalized E. coli cells (Fig. 5B). In contrast, GmmApo/NB and which are expressed predominantly by hemocytes (28, 29), are GmmApo/Sgm flies were highly susceptible to E. coli challenge, by guest on September 27, 2021

FIGURE 4. Dietary supplementation of Wigglesworthia cell extracts to symbiont-cured female tsetse induces immune system development in their apo- symbiotic offspring. Three groups of pregnant female tsetse were provided four blood meals supplemented with the antibiotic tetracycline to clear all of their endogenous microbiota. Two cohorts of these symbiont-cured females then received diets supplemented with either Wigglesworthia or Sodalis cell extracts to complement the absence of these bacteria. The third group of symbiont-cured females received no bacterial complement. Finally, a fourth group of WT fe- males received no tetracycline or bacterial complementation. Offspring of these females, which are designated GmmApo/Wgm, GmmApo/Sgm, GmmApo/NB, and GmmWT, respectively, were collected from three GCs and subsequently monitored to determine their immune system phenotype. GC1 is indicated in gray to signify that bacterial complement of GmmApo/Wgm and GmmApo/Sgm mothers began after their first larval offspring were fully developed. (A) qPCR was per- formed on larval offspring (n = 3 larva/cohort/GC) to determine their levels of serpent and lozenge expression. (B) Circulating hemocyte abundance in adult offspring (n = 3 flies/cohort/GC) was quantified microscopically using a Bright-Line hemocytometer. In (A) and (B), bars with different letters indicate a statistically significant difference (p , 0.05) between samples. (C) Fold change in the expression of immunity-related genes in GmmApo/Wgm and GmmApo/Sgm adults challenged with E. coli. Adult flies used for this experiment were from the second GC of symbiont-cured mothers. All fold-change values are represented as a fraction of average normalized gene expression levels in bacteria-challenged flies relative to expression levels in PBS-injected controls. Genes without a corresponding bar did not exhibit a fold change in expression between samples compared, or their expression was undetectable via qPCR. All quantitative measurements were performed on three biological samples in duplicate. Values are presented as means. *p , 0.05, **p , 0.005. The Journal of Immunology 3401 Downloaded from http://www.jimmunol.org/

FIGURE 5. GmmApo/Wgm flies exhibit resistance to challenge with E. coli.(A) Percent survival of mature GmmApo/Wgm, GmmApo/Sgm, GmmApo/NB, and GmmWT adults from three GCs following challenge with 103 CFU of E. coli K12. Significantly more GmmApo/Wgm flies from the second GC survived this challenge than did age-matched GmmApo/Sgm and GmmApo/NB individuals (p , 0.01). However, significantly fewer GmmApo/Wgm flies from these GCs survived this challenge than did their WT counterparts (p , 0.01). Values shown in gray represent lethal infections. Sample sizes are as follows: GC1 (n = 25 flies/replicate for all tsetse cohorts) and GC2 (n = 25 flies/replicate for GmmWT and GmmApo/Wgm flies; n = 20 for GmmApo/Sgm and GmmApo/NB flies) infection experiments were performed in triplicate for all tsetse groups. GC3 is denoted with an asterisk because not enough GmmApo/Sgm and GmmApo/NB offspring were produced to perform the experiment in triplicate (even in the presence of yeast extract, the fecundity of symbiont-cured females decreases over time). Thus, statistical comparisons between these two groups were not performed. (B) Twelve hours postchallenge with recE. coliGFP, hemolymph was

collected from all individuals (n = 3 flies/group/GC) to monitor for the presence of phagocytic hemocytes. Samples were processed as previously described. by guest on September 27, 2021 Original magnification 3400. In (A) and (B), GC1 is indicated in gray to signify that bacterial complement of GmmApo/Wgm and GmmApo/Sgm mothers began after their first intrauterine larval offspring were approximately midway through their third developmental instar. and, like their GmmApo counterparts, all perished within the 14-d anchored the obligate association between tsetse and Wig- experimental period (Fig. 5A). This susceptible phenotype likely glesworthia such that this bacterium directly engenders immunity resulted from the fact that GmmApo/NB and GmmApo/Sgm adults are and, thus, ultimately the fecundity, of its host. In return, tsetse devoid of phagocytic hemocytes (Figs. 4B, 5B). These findings provides Wigglesworthia with a protective and metabolite-rich suggest that aposymbiotic tsetse can survive infection with an niche that has enabled this bacterium to survive in this environ- otherwise lethal dose of E. coli if they completed intrauterine ment for $50 million years (31). development while their mothers were fed a diet containing Tsetse that undergo intrauterine larval development in the ab- Wigglesworthia cell extracts. This immunocompetent phenotype sence of only Wigglesworthia (GmmWgm2) exhibit a compromised exhibited by GmmApo/Wgm adults likely results from the presence immune system that, when compared with WT flies (GmmWT), is of phagocytic hemocytes in their hemolymph. characterized by a 70% reduction in the number of phagocytic hemocytes (11). In the current study, we found that eliminating all Discussion symbiotic bacteria from female tsetse markedly enhances the im- Symbiotic bacteria are gaining increased recognition as potent munocompromised phenotype of their offspring. In fact, GmmApo modulators of insect immunity (18, 30). In the current study, we adults harbor virtually no circulating (99% less than GmmWT provide evidence that tsetse’s symbiotic bacteria are intimately adults) or sessile hemocytes and are correspondingly more sus- associated with the maturation of their host’s immune system ceptible to systemic infection with E. coli than are WT tsetse and during juvenile development and its subsequent proper function tsetse that lack only Wigglesworthia. GmmWgm2 flies, which un- during adulthood. We determined that aposymbiotic (GmmApo) dergo intrauterine maturation in the presence of Sodalis and flies derived from symbiont-cured mothers present a severely Wolbachia, house ∼40-fold more circulating hemocytes than do compromised cellular immune system and, as such, are highly their aposymbiotic counterparts and are more tolerant to E. coli susceptible to systemic infection with normally nonpathogenic E. challenge (11). The enhanced immunity exhibited by GmmWgm2 coli. This immunocompromised phenotype can be reversed when individuals in comparison with their aposymbiotic counterparts GmmApo adults receive hemocytes transplanted from WT indi- suggests that the presence of Sodalis and Wolbachia during in- viduals. Furthermore, the process of immune system development trauterine development may induce a limited degree of immune in GmmApo larvae can be restored when their symbiont-cured system maturation in their tsetse host. Although no experimental mothers are fed a diet supplemented with Wigglesworthia cell evidence exists that demonstrates a functional role of this nature extracts. Our results demonstrate that evolutionary time has stably for Sodalis, Wolbachia exhibits immunomodulatory properties in 3402 SYMBIONT-INDUCED IMMUNITY IN TSETSE other insect models. For example, Drosophila treated with anti- resides within the cytosol of specialized bacteriocytes that col- biotics to clear their Wolbachia infections are significantly more lectively compromise an organ located immediately adjacent to susceptible to a range of RNA viruses (32, 33). Furthermore, the midgut called the bacteriome (45). Interestingly, GmmWgm2 adults, mosquito Aedes aegypti can be stably transinfected with an ex- which arise from female tsetse that house bacteriome-associated ogenous strain of Wolbachia (wMelPop) (34). The presence of Wigglesworthia but lack their milk gland population, are immu- wMelPop appears to activate the immune system of offspring from nocompromised (11). Thus, this population of Wigglesworthia is transinfected females, which subsequently exhibit enhanced im- insufficient to stimulate immune system development in intra- munity against a range of pathogens (35, 36). Interestingly, unlike uterine GmmWgm2 larvae. However, Wigglesworthia-containing our laboratory colony, many natural populations of tsetse do not bacteriome extracts supplemented in the diet of symbiont-cured harbor Sodalis and/or Wolbachia but are apparently still immu- mothers can stimulate immune system development in GmmApo nocompetent (37). It remains to be seen whether these symbionts larvae. Bacteriome-associated Wigglesworthia appear to produce play a role in stimulating immune system development in natural the molecule(s) required to actuate immune system development populations of tsetse. in GmmApo offspring, but they are concealed within the cytosol of Many insects, including Drosophila, Anopheles, and Manduca, bacteriocytes. likely rely on their cellular immune systems as a potent first line of The mechanism by which Wigglesworthia extracts induce defense against systemic infection with pathogenic bacteria (38– immune system development in GmmApo larvae is unknown. In 41). Similarly, tsetse become susceptible to infection with E. coli the mammalian system, symbiosis factors are translocated from after their hemocyte function is abrogated via the uptake of poly- the gut lumen to peripheral target immune tissues. In the mouse styrene microspheres (11). In this study, we provide further evi- model B. fragilis cells, or B. fragilis PSA, is presumably taken Downloaded from dence that tsetse’s ability to overcome systemic infection with up by gut-associated dendritic cells, which subsequently migrate E. coli also depends on the presence of a functional cellular im- to outlying lymphoid tissues where they signal for the differ- mune system. First, E. coli kills GmmApo adults despite the fact entiation of T cell lineages (43). In a similar manner, PGN shed that they express dramatically more of the AMPs cecropin and by mouse intestinal microbiota is translocated from the luminal attacin than do resistant WT flies. This finding suggests that side of the gut epithelia into the . A positive

AMPs alone are insufficient for tsetse to overcome systemic in- correlation exists between the concentration of PGN present in http://www.jimmunol.org/ fection with E. coli. Secondly, GmmApo adults survived this same host sera and neutrophil function (44). Further experiments are infection if they previously received hemolymph transplanted required in the tsetse system to determine whether immunosti- from WT donors. However, when WT donor hemolymph was mulatory Wigglesworthia molecules are transported to the de- separated into cellular and soluble fractions prior to transplan- veloping larvae where they exhibit direct activity, or whether tation, only GmmApo recipients that received the cellular frac- they act locally in the gut to induce a maternally derived sys- tion (hemocytes) exhibited an E. coli-resistant phenotype. Thus, temic response that subsequently induces larval immune system hemolymph-soluble factors, such as an AMPs, hematopoietic development. molecules, or reactive oxygen species, presumably do not induce Nutritional symbioses between bacteria and insects are well E. coli resistance when transplanted into GmmApo flies. Instead, documented (46, 47). The relationship between tsetse and Wig- by guest on September 27, 2021 resistance appears fixed to the cellular immunity-related activity glesworthia presumably also has a nutritional component, because of hemocyte-mediated phagocytosis. flies that lack this bacterium are reproductively sterile (48, 9). In Beneficial microbes in the human gut produce symbiosis factors fact, Wigglesworthia’s highly reduced genome encodes many that, unlike disease-causing virulence factors produced by patho- vitamins and cofactors that are missing from tsetse’s vertebrate genic microbes, promote favorable health-related outcomes (42). blood-specific diet (49). In this study, we demonstrate that the For example, the human commensal, Bacteroides fragilis, pro- tsetse–Wigglesworthia symbiosis is multidimensional in that this duces one such molecule called polysaccharide A (PSA). Colo- microbe is also intimately involved in activating the development nization of germ-free mice with this bacterium restores CD4+ of its host’s immune system. As such, tsetse may be exploitable as T cell populations to levels conventionally found in mice that a relatively simple and efficient model for deciphering the basic house their native microbiota. This process is consistent with B. molecular mechanisms that underlie symbiont-induced maturation fragilis PSA-induced development of secondary lymphoid tissues. of host immunity. B. fragilis PSA mutants fail to induce these systemic responses in germ-free animals (43). Similarly, mouse intestinal microbiota Acknowledgments serve as a source of peptidoglycan (PGN) that enhances the effi- We thank Dr. Yineng Wu for assistance with qPCR and members of the cacy of phagocytic neutrophils against pathogenic bacteria (44). Aksoy laboratory for critical review of the manuscript. No immunomodulatory symbiosis factors have been characterized from insect-associated microbes. In this study, we demonstrated Disclosures that immune system development in GmmApo larvae was activated The authors have no financial conflicts of interest. when their mothers were fed a diet supplemented with extracts of Wigglesworthia cells. This finding suggests that a molecular component of this obligate bacterium can actuate a transgenera- References tional priming response in the intrauterine larvae of symbiont- 1. Wernegreen, J. J. 2002. Genome evolution in bacterial of insects. cured females. This response restores the process of immune Nat. Rev. Genet. 3: 850–861. 2. Moran, N. A. 2006. Symbiosis. Curr. Biol. 16: R866–R871. system maturation in larvae in the absence of milk gland-asso- 3. Teixeira, L., A. Ferreira, and M. Ashburner. 2008. The bacterial symbiont ciated Wigglesworthia. Tsetse houses two distinct populations Wolbachia induces resistance to RNA viral infections in Drosophila mela- of Wigglesworthia, the first of which is found extracellularly in nogaster. PLoS Biol. 6: e2. 4. Dong, Y., F. Manfredini, and G. Dimopoulos. 2009. Implication of the mosquito female milk gland secretions. These bacterial cells presumably midgut microbiota in the defense against malaria parasites. PLoS Pathog. 5: colonize developing intrauterine larvae, which receive maternal e1000423. 5. Rodrigues, J., F. A. Brayner, L. C. Alves, R. Dixit, and C. Barillas-Mury. 2010. milk for nourishment during tsetse’s unique mode of viviparous Hemocyte differentiation mediates innate immune memory in Anopheles gam- reproduction (7). Tsetse’s second population of Wigglesworthia biae mosquitoes. Science 329: 1353–1355. The Journal of Immunology 3403

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Obligate symbionts activate immune system development in the tsetse fly

Brian L. Weiss, Michele Maltz and Serap Aksoy

Supplemental Fig. 1. Cartoon illustrating the procedures used to perform bacterial compliment experiments.

Supplemental Fig. 2. Symbiont status of tsetse offspring used in bacterial compliment experiments.

Supplemental Fig. 3. Quantitative analysis of sessile hemocyte abundance in mature

GmmWT and mature GmmApo flies (n=3). The anterior-most chamber of tsetse’s dorsal vessel is indicated within a white circle (scale bar = 400 μm). The 2 remaining panels are close-ups of the anterior chamber (scale bar for both panels = 80 μm) visualized by excitation with UV light (365/415 nm).

Supplemental Table 1. PCR primers used in this study. Procedure Genea Primer sets Tm (°C) PCR thiC (Wgm) F-5’-TGAAAACATTTGCAAAATTTG-3’ 54 R-5’-GGTGTTACATAGCATAACAT-3’ exochitinase F-5’-ACCGACTGGG GACAGTACGATG-3’ 54 (Sgm) R-5’- CAAAGAAGTCATAG GTCATAAC-3’ bacterial 16s F-5’-AGAGTTTGATCCTGGCTCA G-3’ 54 rRNA gene R-5’-GGTTACCTTGTTACGACTT-3’ tubulin (tsetse) F-5'-ACGTATTCATTTCCCTTTGG-3' 54 F-5'-AATGGCTGTGGTGTTGGACAAC-3' qPCR attacin F-5'-ATGCCAACCTCTTCAACGAC-3' 56 R-5'-CGTAACCTAAGCCTCCACCA-3' cecropin F-5'-AGAGCGAAGCTGGTTGGTTA-3' 56 R-5'-GCAACATTTGCTGCTTGGTTG-3' defensin F-5'-GCACGAGGAAACAGTCAACA-3' 56 R-5'-CAGTAACGGCAACAGCACAT-3' tep2 F-5'-TGCTATCGTGGCAGACAAAG-3' 56 R-5'-ATACAGAGTGGCGGGAACTG-3' tep4 F-5'-CTGGGCATTACAGCAGACAA-3' 55 R-5'-CGGAATAAGGCAAACGGATA-3' PPO F-5'-ATGGTATGTGCGGGAACTTT-3' 54 R-5'-CCTTTCAGCCTGAGAACTGC-3' iNOS F-5'-GCTCTCAAGGCTCAGGATG-3' 55 R-5'-CTTGCCAGAAAGATCGGAATG-3' caudal F-5'-GCGAACTATTTGCCCCATCA-3' 54 R - 5'-GCGGATATTCACTCGGATGC-3' dual oxidase F - 5'-GATCGCAACGGAGTCATT-3' 55 R - 5'-CATTCCATCAATCAGTTCAGTT-3' domeless F-5'-GACGATCGCGTGTGGTATGC-3' 55 R-5'-TGTTGGGGCCGAATCATTTT-3' PGRP-LB F-5'-CACAACAAACCAAAAAGCAACA-3' 55 R-5'-AACAACACAAAACCGAAAGC-3' serpent F-5'-ATGGCTGCTTAACGCTATGC-3' 56 R-5'-GCCTGCTCAAATTGCTTAACG-3' lozenge F-5'-GAGGCAACTTGTAGAGGATATGG-3' 55 R-5'-CGCTATGGTCACCGTCAAAG-3' beta-tubulin F-5'-CCATTCCCACGTCTTCACTT-3' 55 R-5'-GACCATGACGTGGATCACAG-3' a Wgm, Wigglesworthia, Sgm, Sodalis