The Journal of Immunology
Obligate Symbionts Activate Immune System Development in
the Tsetse Fly
Brian L. Weiss, Michele Maltz, and Serap Aksoy
Many insects rely on the presence of symbiotic bacteria 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 insect’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-
type exhibited by GmmApoadults 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 GmmApolarvae 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.
(1, 2). Insects represent a group of higher eukaryotes that harbors
a well-defined bacterial microbiota. Unlike their mammalian
counterparts, insects house less complex bacterial communities,
are relatively inexpensive to maintain, and produce large numbers
of offspring in a short period of time. Several studies demon-
strated the importance of symbiotic bacteria as they relate to the
proper function of their insect host’s immune system. For exam-
ple, Drosophila naturally infected with Wolbachia are protected
(through an unknown mechanism) from several otherwise harmful
RNA viruses (3). The malaria vector Anopheles gambiae is un-
usually susceptible to infection with Plasmodium parasites when
they lack their commensal microbiota. In this case, symbiotic
bacteria appear to mediate anti-Plasmodium immunity by acti-
vating basal expression of antimicrobial peptides (AMPs), in-
ll metazoan life forms interact with prokaryotic organ-
isms on a perpetual basis. These associations often result
in a fitness advantage for one or both partners involved
ducing the production of phagocytic granulocytes, and directly
generating antimalarial reactive oxygen species (4–6).
Tsetse flies (Glossina spp.) harbor three symbiotic bacteria that
regulate important aspects of their host’s physiology. Two of these
microbes, obligate Wigglesworthia and commensal Sodalis, are trans-
ferred to developing intrauterine progeny via maternal milk gland
secretions (7). Tsetse’s third symbiont, Wolbachia, is transferred via
the germline (8). Tsetse that undergo intrauterine larval development
in the absence of Wigglesworthia are immunocompromised during
adulthood. This phenotype is characterized by a significantly reduced
population of phagocytic sessile and circulating hemocytes, as well as
an unusual susceptibility to infection with pathogenic trypanosomes
and normally nonpathogenic Escherichia coli K12 (9–11). Further
studies on the tsetse/Wigglesworthia symbiosis, as it relates to host
immunity, have been obstructed by our inability to reconstitute sym-
biont-free flies with this bacterium.
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
microbes. We analyzed the immune system phenotype of apo-
symbiotic tsetse (Glossina morsitans morsitans; [GmmApo]) fol-
lowing microbial challenge, and investigated whether loss of
immunity in GmmApoflies could be rescued through either trans-
fer of immune cells from healthy individuals or symbiont provi-
sioning. We obtained results that reinforce the obligate nature
of tsetse’s relationship with Wigglesworthia and provide further
insights into the basic molecular mechanisms that underlie sym-
biont-induced maturation of host immunity.
Materials and Methods
Tsetse and bacteria
G. morsitans morsitans were maintained in Yale’s insectary at 24˚C with
50–55% relative humidity. These flies received defibrinated bovine blood
(Hemostat Laboratories) every 48 h through an artificial membrane feeding
system (12). Designations of all tsetse cohorts used in this study, the
composition of their symbiont populations, and the treatments that they
received are described in Table I.
Division of Epidemiology of Microbial Diseases, Department of Epidemiology and
Public Health, Yale University School of Medicine, New Haven, CT 06520
Received for publication December 21, 2011. Accepted for publication January 30,
This work was supported by grants from the National Institute of Allergy and Infec-
tious Diseases (AI051584), the National Institute of General Medical Science
(069449), and the Ambrose Monell Foundation (all to S.A.).
Address correspondence and reprint requests to Dr. Brian L. Weiss, Division of
Epidemiology of Microbial Diseases, Department of Epidemiology and Public
Health, Yale University School of Medicine, LEPH 607, 60 College Street, New
Haven, CT 06520. E-mail address: email@example.com
The online version of this article contains supplemental material.
Abbreviations used in this article: AMP, antimicrobial peptide; dpc, days postchal-
lenge; DV, dorsal vessel; GC, gonotrophic cycle; GmmApo, aposymbiotic Glossina
morsitans morsitans; GmmWT, wild-type Glossina morsitans morsitans; iNOS, induc-
ible NO synthase; PGN, peptidoglycan; PSA, polysaccharide A; qPCR, quantitative
real-time PCR; recE. coliGFP, GFP-expressing Escherichia coli K12; recE. colipIL,
luciferase-expressing Escherichia coli; tep, thioester-containing protein; WT, wild-
Luciferase-expressing E. coli (recE. colipIL) K12 were produced via
transformation with construct pIL, which encodes the firefly luciferase
gene under transcriptional control of Sodalis’ insulinase promoter (13).
The assay used to quantify recE. colipILcells in vivo was performed as de-
scribed previously (13). GFP-expressing E. coli K12 (recE. coliGFP) were
produced via electroporation with pGFP-UV plasmid DNA (Clontech).
Sodalis were isolated from surface-sterilized G. morsitans pupae and
cultured on Aedes albopictus C6/36 cells, as described previously (14).
Sodalis, which has a doubling time of ∼24 h, was subsequently maintained
in vitro, in the absence of C6/36 cells, at 25˚C in Mitsuhashi–Maramorosch
medium (1 mM CaCl2, 0.2 mM MgCl2, 2.7 mM KCl, 120 mM NaCl, 1.4
mM NaHCO3, 1.3 mM NaH2PO4, 22 mM D (+) glucose, 6.5 g/l lactal-
bumin hydrolysate, and 5.0 g/l yeast extract) supplemented with 5% heat-
inactivated FBS (14).
Systemic challenge of tsetse was achieved by anesthetizing flies with CO2
and subsequently injecting individuals with live bacterial cells using glass
needles and a Narashige IM300 microinjector. Per os bacterial challenges
were performed by adding 500 CFU E. coli per 20 ml (the approximate
amount consumed by a fly) of the total blood meal. The vertebrate host
complement system was heat inactivated (56˚C for 1 h) prior to inoculating
blood meals with bacterial cells. The number of bacterial cells injected or
fed, control group designations, and sample size for all infection experi-
ments are indicated in the corresponding figures and their legends.
Hemolymph collection and hemocyte quantification
Hemolymph collection from wild-type G. morsitans morsitans (GmmWT)
and GmmApoflies was performed using the high-injection/recovery
method, as described previously (15). Subsequent determination of cir-
culating hemocyte abundance was performed using a Bright-Line hemo-
cytometer (11). Sessile hemocyte abundance was quantified by subjecting
GmmWTand GmmApoflies (n = 3) to hemocoelic injection with blue
fluorescent microspheres. Twelve hours postinjection, flies were dissected
to reveal tsetse’s dorsal vessel (DV). Exposed tissue was rinsed three times
with PBS to remove contaminating circulating hemocytes or any beads not
engulfed by sessile hemocytes. Engulfed beads were visualized micro-
scopically by excitation with UV light (365/415 nm). Relative fluores-
cence, which was quantified using ImageJ software, represents the average
amount of light emitted from three GmmWTand GmmApoindividuals.
Quantitative analysis of immunity-related gene expression
For quantitative real-time PCR (qPCR) analysis of immunity-related gene
expression, whole flies were homogenized in liquid nitrogen, and total RNA
was extract using TRIzol reagent (Invitrogen). Randomly primed cDNAs
were generated with Superscript II reverse transcriptase (Invitrogen), and
qPCR analysis was performed using SYBR Green Supermix and a Bio-Rad
I. Quantitative measurements were performed on three biological samples
in duplicate, and results were normalized relative to tsetse’s constitutively
expressed b-tubulin gene (determined from each corresponding sample).
Fold-change data are represented as a fraction of average normalized gene
expression levels in bacteria-infected flies relative to expression levels in
corresponding uninfected controls. Values represent mean 6 SEM.
Undiluted hemolymph was collected by removing one front fly leg at the
joint nearest the thorax and then applying gentle pressure to the distal tip of
the abdomen. Hemolymph exuding from the wound was collected using
newly emerged aposymbiotic recipient flies were used, two of which were
designated GmmApo/WTor GmmApo/Apobased on whether they received
hemolymph transplanted from wild-type (WT) or aposymbiotic donors,
respectively. GmmApo/WTor GmmApo/Aporecipient flies received 1 ml donor
hemolymph (this volume represents approximately one third of the total
volume collected from donor flies). On day 8 posttransplantation, three of
these flies were sacrificed to quantify hemocyte number using a Bright-
Line hemocytometer. To separate GmmWTdonor hemolymph into soluble
and cellular fractions, samples were centrifuged at 3000 3 g for 5 min.
The cellular component was resuspended in a volume of chilled antico-
agulant buffer (70% Mitsuhashi–Maramorosch medium, 30% anticoagu-
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
from which they were collected. The remaining two cohorts of GmmApo
recipient flies were injected with either 1 ml cellular suspension (these flies
are designated GmmApo/Cell) or 1 ml the soluble hemolymph fraction (these
flies are designated GmmApo/Sol).
Allaposymbioticrecipient flies were challenged with either 103CFUlive
recE. colipILor recE. coliGFP. Injections were performed using glass needles
and a Narashige IM300 microinjector. Quantification of recE. colipILin
recipient tsetse was performed as described above. Phagocytic capacity of
transplanted hemocytes was determined by infecting GmmApo/Aporecipient
flies with 103CFU live recE. coliGFP. Twelve hours postchallenge, hemo-
lymph was collected from three individuals, and hemocytes were monitored
for the presence of engulfed GFP-expressing bacterial cells. Hemolymph
samples were fixed on glass microscope slides via a 2-min incubation in 2%
paraformaldehyde. Prior to visualization using a Zeiss Axioscope micro-
scope, slides were overlaid with VECTASHIELD HardSet Mounting Me-
dium containing DAPI (Vector Laboratories).
Bacterial complementation experiments
A cartoon illustrating how bacterial complement experiments were per-
formedis showninSupplementalFig.1. Threecohorts(n=120 individuals/
group) of pregnant female tsetse were fed a diet containing tetracycline (40
mg/ml blood) every other day for 10 d. Additionally, throughout the course
of the experiment, all blood meals (three/wk) also contained vitamin-rich
yeast extract (1% w/v) to restore fertility associated with the absence of
Wigglesworthia (16). Ten days postcopulation, two cohorts of symbiont-
cured females were regularly fed a diet supplemented with Wigglesworthia
and Sodalis cell extracts. By timing treatments in this manner, larvae from
the first gonotrophic cycle (GC) went through most of their development in
the absence of bacterial complement, whereas those from the second and
third GCs developed in the presence of bacterial complement. Offspring of
these females were designated GmmApo/Wgmand GmmApo/Sgm, respectively.
Wigglesworthia was obtained by dissecting tsetse bacteriomes (an organ
immediately adjacent to the midgut that houses this bacteria) from GmmWT
females, whereas Sodalis was maintained in culture, as described above.
GmmApo/Wgmfemales were fed one bacteriome equivalent per four females,
and GmmApo/Sgmfemales were fed 4 3 107Sodalis/ml blood (thus, these
flies ingested ∼1 3 106Sodalis each time they fed). A third control cohort
of symbiont-cured females received no bacterial complement (their off-
spring are designated GmmApo/NB), and a fourth cohort of WT offspring
(GmmWT) served as another control. To confirm the aposymbiotic status of
offspring from symbiont-cured mothers (Supplemental Fig. 2), genomic
DNA was extracted from larval offspring (third instar; n = 3) of all ex-
perimental cohorts using the Holmes–Bonner method (17). PCR (20-ml
reactions) was performed in an MJ Research Thermal Cycler using
Table I.Designation of tsetse cohorts used in this study, their symbiont status, and the treatment they received
Wgm, Sgm, Wol
Offspring of mothers treated with Amp, yeast extract
Offspring of mothers treated with Tet, yeast extract
Received hemolymph transplant from GmmWTdonors
Received hemolymph transplant from GmmApodonors
Received soluble fraction of GmmWTdonor hemolymph
Received cellular fraction of GmmWTdonor hemolymph
Offspring of symbiont-cured mothers complemented with Wgm cell extracts
Offspring of symbiont-cured mothers complemented with Sgm cell extracts
Offspring of symbiont-cured mothers that received no bacterial complement
Amp, ampicillin; Apo, aposymbiotic; Sgm, Sodalis; Tet, tetracycline; Wgm, Wigglesworthia; Wol, Wolbachia.
3396SYMBIONT-INDUCED IMMUNITY IN TSETSE
bacteria-specific primers (Supplemental Table I) and the following cycle
program: 95˚C for 5 min, followed by 30 cycles at 95, 55, and 72˚C, each
for 1 min, and a final 7-min elongation/extension at 72˚C.
To determine whether complementing symbiont-cured mothers with bac-
terial cell extracts impacted the immunesystem phenotype oftheir offspring,
individuals/group/GC). All remaining offspring were allowed to mature to
adulthood. At this time, three individuals from each cohort and GC were
taken to determine circulating hemocyte abundance (as described above).
Furthermore, qPCR was used to compare immunity-related gene expression
in E. coli-challenged GmmApo/Wgmand GmmApo/Sgmindividuals (n = 3) from
the second GC of symbiont-cured mothers. Finally, all remaining mature
adult offspring were challenged with 103CFU live recE. coliGFP. Twelve
hours postchallenge, hemolymph was collected and monitored to determine
whether hemocytes had engulfed GFP-expressing bacterial cells (n = 3
individuals/group/GC). Hemolymph samples were fixed and visualized as
Statistical significance among various treatments, as well as treatments and
controls,is indicatedin the figure legends. Survival curve comparisons were
made by log-rank analysis using JMP (v9.0) software (http://www.jmp.
com). Statistical analysis of qPCR data and hemocyte abundance was
performed by the Student t test using Microsoft Excel software.
Aposymbiotic tsetse exhibit atypical hallmarks of cellular and
A positive correlation exists between the proper function of an
insect’s immune system and the dynamics of its microbiome (18).
In an effort to better define the relationship between symbiosis and
immunity in tsetse, we fed pregnant females a diet supplemented
with tetracycline and yeast. This antibiotic treatment clears all
symbionts from the flies, whereas the vitamin-rich yeast extract
rescues the loss of fertility associated with the absence of obligate
Wigglesworthia (9, 16). We then investigated whether offspring
that underwent intrauterine development in the absence of all
symbiotic bacteria (GmmApo) exhibited an immune system phe-
notype during adulthood that was different from that of their WT
counterparts that developed in the presence of their complete
microbiome. To do so, we began by quantifying the number of
circulating and sessile hemocytes present in 8-d-old adult (here-
after referred to as “mature”) GmmWTand GmmApoflies. Our
results indicate that mature WT tsetse harbor 113-fold more cir-
culating hemocytes/ml of hemolymph than do their aposymbiotic
counterparts (GmmWT, 793 6 34 hemocytes/ml of hemolymph;
GmmApo, 7 6 1 hemocytes/ml of hemolymph; Fig. 1A). To de-
termine the functional relationship between symbiont status and
sessile hemocyte abundance, we thoracically microinjected WT
and aposymbiotic adults with fluorescent microspheres. In both
tsetse and Drosophila, sessile hemocytes concentrate in large
quantities around the anterior chamber of the fly’s DV (11, 19).
Thus, we indirectly quantified sessile hemocyte number by mea-
suring the fluorescent emission of injected microspheres that were
found engulfed in this region. We observed that mature GmmWT
flies engulfed 16-fold more microspheres than did age-matched
GmmApoindividuals (Fig. 1B, Supplemental Fig. 3).
Previously, we determined that several genes associated with
humoral, cellular,and epithelial immune pathways, includingthose
that encode the AMPs attacin and cecropin, as well as thioester-
containing proteins (teps) tep2 and tep 4, prophenoloxidase, and
inducible NO synthase (iNOS), were expressed at significantly
lower levels in GmmWgm2flies compared with GmmWTflies fol-
lowing infection with E. coli (11). In the current study, we mon-
itored expression of these same genes in age-matched GmmWTand
GmmApoflies that were either unchallenged or 3 d postchallenge
(dpc) with E. coli K12. Furthermore, we also evaluated the ex-
pression of PGRP-LB, caudal, domeless, and DUOX. In tsetse and
closely related Drosophila, PGRP-LB and caudal serve as nega-
tive regulators of NF-kB–dependent antimicrobial peptide ex-
pression (10, 20, 21), whereas domeless is a cytokine receptor that
mature GmmWTand GmmApoflies (n = 3 individuals from each tsetse line). (B) Quantitative analysis of sessile hemocyte abundance adjacent to the anterior
chamber of the DVof mature GmmWTand GmmApoflies (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 GmmApoand GmmWTindividuals is normalized relative to
constitutively expressed tsetse b-tubulin (left panel). Fold change in the expression of immunity-related genes in GmmApoand GmmWTtsetse 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.
Aposymbiotic tsetse display atypical hallmarks of cellular and humoral immunity. (A) Number of circulating hemocytes/ml of hemolymph in
The Journal of Immunology3397
regulates expression of tep4 through the JAK/STAT-signaling
pathway (22, 23). Finally, in Drosophila and mosquitoes, DUOX
is involved in generating infection-induced antimicrobial reactive
oxygen species (24–26).
Our expression analysis indicates that the presence of symbiotic
Specifically, we observed that DUOX, domeless, and caudal are
expressed at significantly lower levels in mature unchallenged
GmmApoflies compared with GmmWTflies (Fig. 1C, left panel).
Following per os challenge with E. coli, no significant difference
in immunity-related gene expression (with the exception of iNOS)
was observed between GmmWTand GmmApoflies (Fig. 1C, middle
panel). However, systemic challenge resulted in a significant dif-
ference in the expression of all of the genes that we analyzed.
Most notably, pathways associated with cellular immunity were
significantly downregulated in GmmApoindividuals compared
with GmmWTindividuals, whereas those associated with humoral
immune responses were significantly upregulated (Fig. 1C, right
panel). These findings indicate that tsetse’s symbiotic bacteria are
closely associated with the development of their host’s immune
system during larval maturation and its subsequent proper func-
tion in unchallenged and E. coli-challenged adults.
Aposymbiotic tsetse are highly susceptible to normally
nonpathogenic E. coli
We next determined whether GmmApoindividuals are more sus-
ceptible to challenge with E. coli than are WT tsetse or tsetse that
lack only Wigglesworthia (GmmWgm2). To do so, we compared
percent survival of mature adults from these three tsetse lines
following systemic challenge with E. coli K12. We determined
that 67% of mature GmmWTindividuals and 59% of mature
GmmWgm2individuals survived systemic challenge with 103CFU
of E. coli (Fig. 2A, top and middle panels). In contrast, all age-
matched GmmApoindividuals perished by 12 dpc (Fig. 2A, bottom
panel). We next challenged GmmWTand GmmApoflies per os with
103and 106CFU of E. coli and found that all individuals survived
this challenge (Fig. 2A, top and bottom panels). This finding
suggests that mature GmmApoflies are considerably more sus-
ceptible to systemic challenge with a foreign microbe than are
age-matched GmmWTand GmmWgm2individuals. Furthermore,
tsetse’s ability to overcome per os challenge with E. coli appears
to be independent of symbiont status.
To determine a cause for the variation in survival that we ob-
served among GmmWT, GmmWgm2, and GmmApoindividuals fol-
lowing challenge with E. coli, we monitored the dynamics of
bacterial growth in each of these fly groups over time. When fed
E. coli, both mature aposymbiotic and WT individuals cleared all
E. coli. Following systemic challenge with 103CFU of E. coli,
bacterial densities within mature GmmWTflies reached 8.3 3 103
cells before being cleared. Interestingly, GmmWgm2flies, which
perish following challenge with 106CFU of E. coli (11), were able
to clear all exogenous bacterial cells following challenge with this
lower dose. In contrast, bacterial density in GmmApoflies peaked
at 7.8 3 106on day 6 postchallenge, after which all flies soon
perished (Fig. 2B). This observation suggests that aposymbiotic
tsetse were unable to control systemic infection with E. coli and,
thus, likely perished as a result of their inability to tolerate high
densities of this bacterium in their hemolymph. Taken together,
these findings indicate that GmmApoflies are significantly more
susceptible to challenge with E. coli than are WT flies and flies
that lack only Wigglesworthia.
Hemocyte transfer from WT tsetse restores the ability of
GmmApoadults to overcome infection with E. coli
We next set out to provide a definitive correlation between tsetse
microbe. To do so, we transplanted hemolymph from mature
GmmWTand GmmApoindividuals (donor flies) into the hemocoel of
susceptible GmmApoflies (recipient flies are hereafter designated
GmmApo/WTand GmmApo/Apo, respectively). Five days after this
procedure, we determined that GmmApo/WTflies harbored 330 6
20.4 hemocytes/ml of hemolymph, whereas GmmApo/Apoflies har-
bored 5 6 3.8 hemocytes/ml of hemolymph (Fig. 3A). We next
able to rescue the E. coli-susceptible phenotype exhibited by
following systemic and per os challenge with E. coli K12. Mature adult GmmApoflies were significantly more susceptible to challenge with 103CFU of
E. coli than were age-matched GmmWTflies (bottom and top panels, p , 0.001) and GmmWgm2flies (bottom and middle panels, p , 0.001). Both GmmWT
and GmmApoflies survived per os challenge with E. coli. Infection experiments were performed in triplicate, using 25 flies/replicate. (B) Average number
(6 SEM) of recE. colipILper tsetse cohort over time (n = 3 individuals/cohort/time point) following systemic and per os challenge with 103CFU of bacteria.
Values shown in gray represent lethal infections. By 8 dpc, not enough E. coli-injected GmmApoflies remained to quantify bacterial density.
Symbiont status mediates tsetse’s ability to survive challenge with E. coli K12. (A) The effect of symbiont status on the survival of tsetse
3398SYMBIONT-INDUCED IMMUNITY IN TSETSE
individuals with 103CFU of E. coli 3 d posthemolymph trans-
plantation and subsequently monitored their survival over time. Our
results indicate that 72% of GmmApo/WTflies survived for 14 d
following challenge. In comparison, only 2% of GmmApo/Apoflies
survived their challenge (Fig. 3B, top panel). These results dem-
onstrate that GmmApoflies are able to clear a systemic challenge
with E. coli after they receive a transplant of hemolymph from WT
We next investigated whether hemocytes or a soluble antimi-
crobial or signaling molecule present in the transplanted hemo-
lymph was responsible for restoring the resistant phenotype
exhibited by recipient individuals. To address this issue, we col-
lected hemolymph from WT donors, separated it into soluble and
cellular fractions by centrifugation, and then transplanted the sep-
arate fractions into two distinct groups of GmmApoflies. Finally,
3 d later we systemically challenged both groups of recipient
flies with 103CFU of E. coli K12. All aposymbiotic flies that
received the soluble fraction of hemolymph from GmmWT
donors (GmmApo/Sol) perished by day 12 postchallenge. In com-
parison, 62% of GmmAporecipients that received the cellular
fraction of hemolymph from GmmWTdonors (GmmApo/Cell) sur-
vived for 14 d following bacterial challenge (Fig. 3B, bottom
panel). These host survival curves indicate that GmmApoflies
survived challenge with E. coli when they had previously received
a transplant of hemocytes, as opposed to soluble hemolymph mol-
ecules, from WT tsetse.
To determine a cause for the variation in survival that we ob-
served between these two groups, we monitored the dynamics of
bacterialgrowth in each groupover the course of the experiment. E.
coli within GmmApo/Apoflies replicated exponentially, until a peak
density of 2.1 3 107was reached at 6 dpc. This finding suggests
that bacterial sepsis was the cause of high mortality we observed in
this group of flies. In contrast, aposymbiotic recipients were able to
clear all E. coli by 8 dpc when they had previously received a
hemolymph transplant from GmmWTdonors (Fig. 3C). More so,
microscopic examination of hemolymph from GmmApo/WTflies
showed that transplanted hemocytes engulfed the introduced E.
coli (Fig. 3D). Our results demonstrate that immune resistance can
be restored in adult aposymbiotic tsetse if they harbor hemocytes
transplanted from their WT counterparts.
Supplementation of Wigglesworthia to symbiont-cured females
restores immune system development in aposymbiotic offspring
Previous experiments revealed that the milk gland population of
tsetse’s obligate symbiont, Wigglesworthia, must be present dur-
ing the development of immature stages for subsequent adults to
exhibit a functional cellular immune system (11). We have not
been able to culture Wigglesworthia and, thus, cannot recolonize
aposymbiotic flies with this bacterium. To circumvent this im-
pediment, we tested whether we could restore the process of im-
mune system development in GmmApooffspring by supplementing
the diet of pregnant, symbiont-cured females with Wigglesworthia-
containing extracts of bacteriome tissue collected from WT fe-
males. A detailed description of the experimental design that we
used to test this theory is provided in the Materials and Methods
and Supplemental Fig. 1.
In brief, two treatment cohorts of pregnant GmmWTfemales were
fed a diet supplemented with tetracycline and yeast extract (16).
Ten days postcopulation, these symbiont-cured females began re-
ceiving either Wigglesworthia or Sodalis cell extracts in every
blood meal. The immune system phenotype of offspring from these
females (GmmApo/Wgmand GmmApo/Sgm, respectively) was com-
flies and immediately transplanted into GmmAporecipients. Five days posthemolymph transplantation, hemocyte abundance in GmmApo/WTand GmmApo/Apo
recipient flies was quantified microscopically using a hemocytometer. GmmApo/WTflies housed significantly more circulating hemocytes than did GmmApo/Apo
flies. (B) GmmApo/WTand GmmApo/Aporecipient 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/Apocounterparts (top panel, p , 0.001). Donor hemolymph was then divided into cellular and
soluble fractions via centrifugation. Significantly more GmmApo/Cellindividuals survived E. coli challenge than did their GmmApo/Solcounterparts (bottom
panel, p , 0.001). (C) Average number (6 SEM) of recE. colipILper GmmApo/WTand GmmApo/Aporecipient fly over time (n = 3 individuals/treatment/time
point) following systemic challenge with 103CFU 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/WTrecipient flies harbor engulfed bacterial cells. Original magnification 3400. **p , 0.005.
Hemocytes modulate tsetse’s ability to overcome challengewith E. coli K12. (A) Hemolymph was collected from GmmApoand GmmWTdonor
The Journal of Immunology 3399
pared with that of control cohort offspring from symbiont-cured
mothers that received no bacterial supplement (GmmApo/NB) and
offspring from GmmWTmothers. We first evaluated the relative
abundance of transcripts that encode the transcription factors Ser-
pent and Lozenge. In Drosophila, these molecules direct hemocyte
differentiation, or hematopoiesis, during embryogenesis and early
larvagenesis (27). In tsetse, larvae that develop in the absence of
Wigglesworthia express significantly less serpent and lozenge than
do their WT counterparts (11). In the current study, we found that
GmmApo/Wgm, GmmApo/Sgm, and GmmApo/NBlarva from the first GC
expressed significantly less serpent and lozenge than did GmmWT
larva. However, after the onset of bacterial supplementation,
GmmApo/Wgmand GmmWTlarva from the second and third GCs
expressed comparable levels of serpent and lozenge, whereas
GmmApo/NBand GmmApo/Sgmlarva expressed less (Fig. 4A).
Because serpent and lozenge expression can be indicative of
hematopoiesis, we next compared the number of hemocytes pre-
sent in GmmApo/Wgmadults to that found in age-matched GmmWT,
GmmApo/NB, and GmmApo/Sgmflies. We found that the provi-
sioning of Wigglesworthia extracts to symbiont-cured females
resulted in an increase in the number of circulating hemo-
cytes present in their offspring. Specifically, hemocyte density
in GmmApo/Wgmadults from GCs 2 and 3 was significantly greater
(113 6 33 and 127 6 21 hemocytes/ml of hemolymph, respec-
tively) than that found in age-matched GmmApo/NB(7 6 3 and 9 6
4 hemocytes/ml of hemolymph, respectively) and GmmApo/Sgm
flies (10 6 4 and 4 6 1 hemocytes/ml hemolymph, respectively),
but it was significantly less than that of GmmWTadults (733 6 104
and 681 6 68 hemocytes/ml hemolymph, respectively; Fig. 4B).
Correspondingly, we observed that prophenoloxidase and tep4,
which are expressed predominantly by hemocytes (28, 29), are
found at significantly higher levels in adult GmmApo/Wgmflies
compared with adult GmmApo/Sgmflies (from GC2) following
systemic challenge with E. coli (Fig. 4C). A similar pattern was
observed with genes involved in the generation of reactive oxy-
gen species (DUOX and iNOS). Interestingly, humoral immunity-
associated genes (AMPs and their regulators) were expressed at
similar levels in E. coli-challenged GmmApo/Wgmand GmmApo/Sgm
Our results suggest that feeding symbiont-cured mothers a diet
supplemented with Wigglesworthia cell extracts induces a physi-
ological response that partially restores immune system develop-
ment in their aposymbiotic offspring. Specifically, GmmApo/Wgm
larvae exhibit increased expression of the hematopoietic tran-
scription factors serpent and lozenge, and, as adults, these flies
present a functional immune system characterized by the presence
of circulating phagocytic hemocytes. Furthermore, the expression
of genes involved in epithelial and cellular immunity is enhanced
GmmApo/Wgmflies are resistant to E. coli challenge
We observed that GmmApo/Wgmoffspring exhibit hallmarks of en-
hanced immunity. Thus, we next tested whether mature GmmApo/
Wgmadults would be resistant to systemic challenge with E. coli
K12, whereas age-matched GmmApo/Sgmand GmmApo/NBflies
would not. To this end, we observed that 38 and 43% of
GmmApo/Wgmadults from GCs 2 and 3, respectively, survived
challenge with 103E. coli (Fig. 5A). Correspondingly, microscopic
inspection of hemolymph from E. coli-resistant GmmApo/Wgmadults
revealed the presence of phagocytic hemocytes that harbored in-
ternalized E. coli cells (Fig. 5B). In contrast, GmmApo/NBand
GmmApo/Sgmflies were highly susceptible to E. coli challenge,
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/Wgmand GmmApo/Sgmmothers 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 significantdifference(p, 0.05)betweensamples.(C) Fold changeintheexpression ofimmunity-relatedgenes inGmmApo/WgmandGmmApo/Sgm
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.
Dietary supplementation of Wigglesworthia cell extracts to symbiont-cured female tsetse induces immune system development in their apo-
3400SYMBIONT-INDUCED IMMUNITY IN TSETSE
and, like their GmmApocounterparts, all perished within the 14-d
experimental period (Fig. 5A). This susceptible phenotype likely
resulted from the fact that GmmApo/NBand GmmApo/Sgmadults are
devoid of phagocytic hemocytes (Figs. 4B, 5B). These findings
suggest that aposymbiotic tsetse can survive infection with an
otherwise lethal dose of E. coli if they completed intrauterine
development while their mothers were fed a diet containing
Wigglesworthia cell extracts. This immunocompetent phenotype
exhibited by GmmApo/Wgmadults likely results from the presence
of phagocytic hemocytes in their hemolymph.
Symbiotic bacteria are gaining increased recognition as potent
modulators of insect immunity (18, 30). In the current study, we
provide evidence that tsetse’s symbiotic bacteria are intimately
associated with the maturation of their host’s immune system
during juvenile development and its subsequent proper function
during adulthood. We determined that aposymbiotic (GmmApo)
flies derived from symbiont-cured mothers present a severely
compromised cellular immune system and, as such, are highly
susceptible to systemic infection with normally nonpathogenic E.
coli. This immunocompromised phenotype can be reversed when
GmmApoadults receive hemocytes transplanted from WT indi-
viduals. Furthermore, the process of immune system development
in GmmApolarvae can be restored when their symbiont-cured
mothers are fed a diet supplemented with Wigglesworthia cell
extracts. Our results demonstrate that evolutionary time has stably
anchored the obligate association between tsetse and Wig-
glesworthia such that this bacterium directly engenders immunity
and, thus, ultimately the fecundity, of its host. In return, tsetse
provides Wigglesworthia with a protective and metabolite-rich
niche that has enabled this bacterium to survive in this environ-
ment for $50 million years (31).
Tsetse that undergo intrauterine larval development in the ab-
sence of only Wigglesworthia (GmmWgm2) exhibit a compromised
immune system that, when compared with WT flies (GmmWT), is
characterized by a 70% reduction in the number of phagocytic
hemocytes (11). In the current study, we found that eliminating all
symbiotic bacteria from female tsetse markedly enhances the im-
munocompromised phenotype of their offspring. In fact, GmmApo
adults harbor virtually no circulating (99% less than GmmWT
adults) or sessile hemocytes and are correspondingly more sus-
ceptible to systemic infection with E. coli than are WT tsetse and
tsetse that lack only Wigglesworthia. GmmWgm2flies, which un-
dergo intrauterine maturation in the presence of Sodalis and
Wolbachia, house ∼40-fold more circulating hemocytes than do
their aposymbiotic counterparts and are more tolerant to E. coli
challenge (11). The enhanced immunity exhibited by GmmWgm2
individuals in comparison with their aposymbiotic counterparts
suggests that the presence of Sodalis and Wolbachia during in-
trauterine development may induce a limited degree of immune
system maturation in their tsetse host. Although no experimental
evidence exists that demonstrates a functional role of this nature
for Sodalis, Wolbachia exhibits immunomodulatory properties in
GmmWTadults from three GCs following challenge with 103CFU of E. coli K12. Significantly more GmmApo/Wgmflies from the second GC survived this
challenge than did age-matched GmmApo/Sgmand GmmApo/NBindividuals (p , 0.01). However, significantly fewer GmmApo/Wgmflies 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 GmmWTand GmmApo/Wgmflies; n = 20 for GmmApo/Sgmand GmmApo/NBflies)
infection experiments were performed in triplicate for all tsetse groups. GC3 is denoted with an asterisk because not enough GmmApo/Sgmand 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.
Original magnification 3400. In (A) and (B), GC1 is indicated in gray to signify that bacterial complement of GmmApo/Wgmand GmmApo/Sgmmothers began
after their first intrauterine larval offspring were approximately midway through their third developmental instar.
GmmApo/Wgmflies exhibit resistance to challenge with E. coli. (A) Percent survival of mature GmmApo/Wgm, GmmApo/Sgm, GmmApo/NB, and
The Journal of Immunology3401
other insect models. For example, Drosophila treated with anti-
biotics to clear their Wolbachia infections are significantly more
susceptible to a range of RNA viruses (32, 33). Furthermore, the
mosquito Aedes aegypti can be stably transinfected with an ex-
ogenous strain of Wolbachia (wMelPop) (34). The presence of
wMelPop appears to activate the immune system of offspring from
transinfected females, which subsequently exhibit enhanced im-
munity against a range of pathogens (35, 36). Interestingly, unlike
our laboratory colony, many natural populations of tsetse do not
harbor Sodalis and/or Wolbachia but are apparently still immu-
nocompetent (37). It remains to be seen whether these symbionts
play a role in stimulating immune system development in natural
populations of tsetse.
Many insects, including Drosophila, Anopheles, and Manduca,
likely rely on their cellular immune systems as a potent first line of
defense against systemic infection with pathogenic bacteria (38–
41). Similarly, tsetse become susceptible to infection with E. coli
after their hemocyte function is abrogated via the uptake of poly-
styrene microspheres (11). In this study, we provide further evi-
dence that tsetse’s ability to overcome systemic infection with
E. coli also depends on the presence of a functional cellular im-
mune system. First, E. coli kills GmmApoadults despite the fact
that they express dramatically more of the AMPs cecropin and
attacin than do resistant WT flies. This finding suggests that
AMPs alone are insufficient for tsetse to overcome systemic in-
fection with E. coli. Secondly, GmmApoadults survived this same
infection if they previously received hemolymph transplanted
from WT donors. However, when WT donor hemolymph was
separated into cellular and soluble fractions prior to transplan-
tation, only GmmAporecipients that received the cellular frac-
tion (hemocytes) exhibited an E. coli-resistant phenotype. Thus,
hemolymph-soluble factors, such as an AMPs, hematopoietic
molecules, or reactive oxygen species, presumably do not induce
E. coli resistance when transplanted into GmmApoflies. Instead,
resistance appears fixed to the cellular immunity-related activity
of hemocyte-mediated phagocytosis.
Beneficial microbes in the human gut produce symbiosis factors
that, unlike disease-causing virulence factors produced by patho-
genic microbes, promote favorable health-related outcomes (42).
For example, the human commensal, Bacteroides fragilis, pro-
duces one such molecule called polysaccharide A (PSA). Colo-
nization of germ-free mice with this bacterium restores CD4+
T cell populations to levels conventionally found in mice that
house their native microbiota. This process is consistent with B.
fragilis PSA-induced development of secondary lymphoid tissues.
B. fragilis PSA mutants fail to induce these systemic responses in
germ-free animals (43). Similarly, mouse intestinal microbiota
serve as a source of peptidoglycan (PGN) that enhances the effi-
cacy of phagocytic neutrophils against pathogenic bacteria (44).
No immunomodulatory symbiosis factors have been characterized
from insect-associated microbes. In this study, we demonstrated
that immune system development in GmmApolarvae was activated
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-
tional priming response in the intrauterine larvae of symbiont-
cured females. This response restores the process of immune
system maturation in larvae in the absence of milk gland-asso-
ciated Wigglesworthia. Tsetse houses two distinct populations
of Wigglesworthia, the first of which is found extracellularly in
female milk gland secretions. These bacterial cells presumably
colonize developing intrauterine larvae, which receive maternal
milk for nourishment during tsetse’s unique mode of viviparous
reproduction (7). Tsetse’s second population of Wigglesworthia
resides within the cytosol of specialized bacteriocytes that col-
lectively compromise an organ located immediately adjacent to
midgut called the bacteriome (45). Interestingly, GmmWgm2adults,
which arise from female tsetse that house bacteriome-associated
Wigglesworthia but lack their milk gland population, are immu-
nocompromised (11). Thus, this population of Wigglesworthia is
insufficient to stimulate immune system development in intra-
uterine GmmWgm2larvae. However, Wigglesworthia-containing
bacteriome extracts supplemented in the diet of symbiont-cured
mothers can stimulate immune system development in GmmApo
larvae. Bacteriome-associated Wigglesworthia appear to produce
the molecule(s) required to actuate immune system development
in GmmApooffspring, but they are concealed within the cytosol of
The mechanism by which Wigglesworthia extracts induce
immune system development in GmmApolarvae is unknown. In
the mammalian system, symbiosis factors are translocated from
the gut lumen to peripheral target immune tissues. In the mouse
model B. fragilis cells, or B. fragilis PSA, is presumably taken
up by gut-associated dendritic cells, which subsequently migrate
to outlying lymphoid tissues where they signal for the differ-
entiation of T cell lineages (43). In a similar manner, PGN shed
by mouse intestinal microbiota is translocated from the luminal
side of the gut epithelia into the circulatory system. A positive
correlation exists between the concentration of PGN present in
host sera and neutrophil function (44). Further experiments are
required in the tsetse system to determine whether immunosti-
mulatory Wigglesworthia molecules are transported to the de-
veloping larvae where they exhibit direct activity, or whether
they act locally in the gut to induce a maternally derived sys-
temic response that subsequently induces larval immune system
Nutritional symbioses between bacteria and insects are well
documented (46, 47). The relationship between tsetse and Wig-
glesworthia presumably also has a nutritional component, because
flies that lack this bacterium are reproductively sterile (48, 9). In
fact, Wigglesworthia’s highly reduced genome encodes many
vitamins and cofactors that are missing from tsetse’s vertebrate
blood-specific diet (49). In this study, we demonstrate that the
tsetse–Wigglesworthia symbiosis is multidimensional in that this
microbe is also intimately involved in activating the development
of its host’s immune system. As such, tsetse may be exploitable as
a relatively simple and efficient model for deciphering the basic
molecular mechanisms that underlie symbiont-induced maturation
of host immunity.
We thank Dr. Yineng Wu for assistance with qPCR and members of the
Aksoy laboratory for critical review of the manuscript.
The authors have no financial conflicts of interest.
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