Beneficial bacterial symbionts, which are ubiquitous in nature, are often
characterized by the extent to which they interact with the host. In the case of
mutualistic symbioses, both partners benefit so that each one can inhabit diverse
ecological niches where neither could survive on its own. Unfortunately, little is
known about the functional mechanisms that underlie mutualistic relationships.
Insects represent a group of advanced multi-cellular organisms that harbor
well-documented symbiotic associations. One such insect, the tsetse fly, harbors a
maternally transmitted bacterial mutualist called Wigglesworthia
that provides its host with essential metabolites missing from its vertebrate
blood-specific diet. In this study, we further examine the relationship between
tsetse and Wigglesworthia by investigating the interaction between
this bacterium and its host's immune system. We have found that when
Wigglesworthia is absent from tsetse during the maturation of
immature larval stages, subsequent adults are characterized by an underdeveloped
cellular immune system and thus highly susceptible to infection with a normally
non-pathogenic foreign microbe. These findings represent an additional adaptation
that further anchors the steadfast relationship shared between tsetse and its
obligate symbiont.
Beneficial microbial symbionts serve important functions within their hosts,
including dietary supplementation and maintenance of immune system homeostasis.
Little is known about the mechanisms that enable these bacteria to induce specific
host phenotypes during development and into adulthood. Here we used the tsetse fly,
Glossina morsitans, and its obligate mutualist,
Wigglesworthia glossinidia, to investigate the co-evolutionary
adaptations that influence the development of host physiological processes.
Wigglesworthia is maternally transmitted to tsetse's
intrauterine larvae through milk gland secretions. We can produce flies that lack
Wigglesworthia
(GmmWgm
−) yet retain their other
symbiotic microbes. Such offspring give rise to adults that exhibit a largely normal
phenotype, with the exception being that they are reproductively sterile. Our results
indicate that when reared under normal environmental conditions
GmmWgm
− adults are also
immuno-compromised and highly susceptible to hemocoelic E. coli
infections while age-matched wild-type individuals are refractory. Adults that lack
Wigglesworthia during larval development exhibit exceptionally
compromised cellular and humoral immune responses following microbial challenge,
including reduced expression of genes that encode antimicrobial peptides
(cecropin and attacin), hemocyte-mediated
processes (thioester-containing proteins 2 and 4
and prophenoloxidase), and signal-mediating molecules
(inducible nitric oxide synthase). Furthermore,
GmmWgm
− adults harbor a reduced
population of sessile and circulating hemocytes, a phenomenon that likely results
from a significant decrease in larval expression of serpent and
lozenge, both of which are associated with the process of early
hemocyte differentiation. Our results demonstrate that
Wigglesworthia must be present during the development of immature
progeny in order for the immune system to function properly in adult tsetse. This
phenomenon provides evidence of yet another important physiological adaptation that
further anchors the obligate symbiosis between tsetse and
Wigglesworthia.
IntroductionBacteria comprise the most abundant and diverse life form on earth. The ubiquity of
bacteria means they have colonized virtually every ecological niche, including
habitation within more evolutionarily sophisticated multi-cellular animals. Co-evolution
over millions of years has provided an opportunity for beneficial symbiotic associations
to develop between phylogenetically distant taxa. Such affiliations are often
mutualistic, meaning both partners benefit so that each can successfully inhabit diverse
environments that neither could survive in on its own [1],[2]. Deciphering the mutualistic
relationships between prokaryotic bacteria and multi-cellular eukaryotic animals is a
rapidly advancing field of research.Performing detailed investigations into the relationships between symbiotic bacteria and
higher eukaryotes often involves costly and complex procedures. However, insects have
well-documented symbioses that are attractive to study because they have relatively
short generation times and are easy and less costly to rear. One insect that harbors
multiple symbionts is the tsetse fly, Glossina morsitans. These
microbes include the commensal Sodalis, the parasite
Wolbachia, and the obligate mutualist Wigglesworthia
glossinidia
[3]. Molecular
phylogenetic analysis indicates that tsetse's symbiosis with
Wigglesworthia is ancient, dating back 50–80 million years
[4]. The concordant
nature of tsetse's obligate association with Wigglesworthia has
driven the co-evolution of biological adaptations that are beneficial to both partners.
For example, the localization of Wigglesworthia cells within host
bacteriocytes provides a protective and metabolically favorable niche for this bacterium
[5]. In return
tsetse derives benefit from Wigglesworthia in at least two distinct
ways. First, tsetse feeds exclusively on vertebrate blood, which is deficient in
vitamins essential for survival. In accordance, a large proportion of
Wigglesworthia's streamlined (700 kB) genome encodes vitamin
biosynthesis pathways that presumably supplement tsetse's restricted diet [5],[6]. Second, more recent studies indicate
that Wigglesworthia may serve an immunologic role in tsetse. These
flies are the sole vector of pathogenic African trypanosomes, the causative agent of
sleeping sickness in humans [7]. In laboratory experiments infection with immunogenic
trypanosomes results in a decrease in tsetse fecundity [8]. Furthermore, parasite infection
prevalence is higher in flies that lack Wigglesworthia when compared to
age-matched wild-type (WT) individuals [9]. Wigglesworthia is thought to influence
tsetse's vectorial competence by modulating its host's humoral immune system
[10]. Thus, by
preventing energetically costly parasite infections, this obligate symbiont may
indirectly benefit the reproductive fitness of tsetse.Symbiotic bacteria are rapidly gaining recognition for their important contributions to
host development and immunity. The most well-known example of this type of interaction
involves the mammalian microbiome, which modulates gut development during early
postnatal life and subsequently shapes our mucosal and systemic immune systems [11],[12]. Symbionts also
serve similar functions in invertebrate hosts. For example, light organ morphogenesis in
juvenile bobtail squid (Euprymna scolopes) initiates only after
symbiotic Vibrio fischeri cells have stably colonized this tissue [13]. The pea aphid,
Acyrthosiphon pisum, harbors a secondary symbiont,
Hamiltonella defensa, which can be infected with a lysogenic
bacteriophage (A. pisum secondary endosymbiont; APSE). A.
pisum that harbor both H. defensa and APSE are protected
from being consumed by a parasitic wasp larvae through the action of phage-encoded
toxins [14].To the best of our knowledge no evidence exists that demonstrates insect symbionts can
confer an impending protective phenotype in their adult host by directing immune system
development during immature stages. In the present study we investigate the mechanism by
which Wigglesworthia contributes to the development and function of
cellular and humoral immune responses in adult tsetse. Our results show that the
maturation and normal function of adult cellular immune responses in tsetse are severely
compromised when Wigglesworthia is absent during larval development.
This study reveals an important new facet that further anchors the obligate relationship
between tsetse and Wigglesworthia and may serve as a useful model to
understand the highly integrated and dynamic relationship between hosts and their
beneficial bacterial fauna.ResultsTsetse's Susceptibility to E. coli Infection Is Dependent
Upon Age and Symbiotic StatusInsects are normally capable of mounting an immune response that combats infection
with various groups of bacteria. Interestingly, in comparison to
Drosophila, tsetse flies are uniquely susceptible to septic
infection with 103 colony-forming units (CFU) of normally non-pathogenic
Escherichia coli (E. coli) K12 [15]. In the present
study we further investigated tsetse's unique susceptibility to E.
coli infection by subjecting wild-type
(Gmm
WT) and adults from two age groups to hemocoelic
infections with varying quantities of E. coli K12. Three-day-old
Gmm
WT individuals (flies from this age group are
hereafter referred to as “young”) were highly susceptible to this
treatment, as 103 CFU resulted in the death of all flies by 8 d
post-infection (dpi; Figure
1A, top graph). In contrast, 77% and 55% of 8-d-old WT
individuals (flies 8 d old and older are hereafter referred to as
“mature”) survived for 14 dpi with 103 and 106 CFU
of E. coli K12, respectively (Figure 1A, middle graph).10.1371/journal.pbio.1000619.g001Figure 1Host survival correlates with symbiont status following septic infection
with E. coli K12.(A) The effects of age and symbiont status on the survival of tsetse following
systemic infection with E. coli K12. Mature adult
GmmWgm- flies were significantly more
susceptible to infection with 106 CFU of E. coli
than were their wild-type counterparts (bottom and middle panels;
p<0.001). (B)
Gmm
WT/Wgm− flies
harbored Wigglesworthia during immature development but lacked
the bacteria as mature adults. (C)
Gmm
WT/Wgm− adults were
infected with tetracycline resistant E. coli 1 d after their
last antibiotic-supplemented blood meal. Unlike their counterparts that lacked
Wigglesworthia throughout immature development,
Gmm
WT/Wgm− was able to
survive infection with E. coli. No significant difference in
survival was observed between mature adult Gmm
WT
versus Gmm
WT/Wgm− adults
infected with 106 CFU of E. coli (Figure 1A middle panel and
Figure 1C;
p = 0.07). (D) Relative
Sodalis and Wolbachia densities in
40-d-old Gmm
WT and
GmmWgm
− adults
(n = 5 of each) were normalized
against host β-tubulin copy number. (E) Analysis of
bacterial 16s rRNA clone libraries indicates that
Gmm
WT larvae harbored
Wigglesworthia, Sodalis, and
Wolbachia, while their counterparts from ampicillin treated
females harbored only Sodalis and Wolbachia.
No other bacteria were identified from either fly line. (F) Average number
(±SEM) of recE. coli
pIL per tsetse strain
over time (n = 3 individuals per strain
per time point) following septic infection with 103 CFU of bacteria.
Values shown in red represent lethal infections.We demonstrated previously that feeding pregnant female tsetse the antibiotic
ampicillin results in the generation offspring that lack
Wigglesworthia
(GmmWgm
−) but still harbor
Sodalis
[9] and presumably
Wolbachia. In the present study we determined the survival
outcome of mature GmmWgm
− inoculated
with 106 CFU of E. coli K12 (the dose required to kill
∼50% of mature WT flies; Figure 1A, middle graph). In comparison to age-matched WT tsetse,
GmmWgm
− flies were highly
susceptible to this infection. In fact, at 14 dpi, only 1% of these
individuals remained (Figure 1A,
bottom graph). We reasoned that the dramatic susceptibility of mature
GmmWgm
− flies to infection with
E. coli could result from one of two scenarios. In the first
scenario Wigglesworthia may directly contribute to the adult immune
response to a foreign micro-organism. In the second scenario, the absence of
Wigglesworthia during larvagensis may give rise to
GmmWgm
− adults with compromised
immune functions. To determine if the first scenario is correct we treated mature WT
tsetse with the antibiotic tetracycline to eliminate all of their microbiota,
including bacteriome-associated Wigglesworthia (Figure 1B), and subsequently challenged these adult
flies (Gmm
WT/Wgm−) with
106 CFU of tetracycline resistant E. coli K12. We
found that, similar to their WT counterparts (Gmm
WT;
Figure 1A, middle panel),
about 70% of Gmm
WT/Wgm−
individuals survived this infection (Figure 1C). Conversely,
GmmWgm
− adults were highly
susceptible (Figure 1A, bottom
graph). This result suggests that when Wigglesworthia is absent from
tsetse during larval development subsequent adults are severely
immuno-compromised.We demonstrated that when larval tsetse lacks exposure to
Wigglesworthia in utero their immune system is highly compromised
during adulthood. This finding does not exclude the possibility that treatment of
female flies with ampicillin to produce
GmmWgm
− offspring induces
perturbations in other constituents of tsetse's microbiota. Because
tsetse's other two known endosymbionts, Sodalis and
Wolbachia, are present at similar densities in
Gmm
WT and
GmmWgm
− adults (Figure 1D), we looked for the presence of
uncharacterized endosymbionts or digestive tract-associated microbes that are
potentially passed on to developing intra-uterine larvae where they subsequently
impact immunity. We generated a clone library containing 16s rRNA gene sequences PCR
amplified from 3rd instar
GmmWgm
− and
Gmm
WT larvae and then sequenced multiple clones from
each tsetse line. Our results indicate that the proportion of
Wigglesworthia, Sodalis, and
Wolbachia in 3rd instar
Gmm
WT larvae is 19%, 75%, and 6%,
respectively. As expected no Wigglesworthia 16s rRNA sequences were
present in GmmWgm
− larvae, and the
proportion of Sodalis and Wolbachia sequences
present was 71% and 29%, respectively (Figure 1E). No uncharacterized endosymbionts or
gut-associated environmental microbes were present in any sample. Taken together,
these results suggest that the presence of Wigglesworthia
specifically is responsible for enabling immune system maturation in WT tsetse.Our host survival curves indicate that mature Gmm
WT and
Gmm
WT/Wgm− can survive
infection with E. coli while young Gmm
WT
and mature GmmWgm
− perish. To determine
a cause for the variation in survival we observed between the four groups following
infection with E. coli, we monitored bacterial growth dynamics over
the course of the experiment in each group. Bacterial number within mature
Gmm
WT and
Gmm
WT/Wgm− peaked at
3.8×104 and 1.9×104
E. coli, respectively, over the 2-wk period, indicating that these
groups appeared able to control their infections. In contrast, E.
coli increased exponentially in young Gmm
WT
and mature GmmWgm
− and reached a maximum
density at 6 dpi of 4.2×106 and 8.8×106,
respectively (Figure 1F). These
results implicate bacterial sepsis as the cause of high mortality observed in young
Gmm
WT and mature
GmmWgm
−. All of the above-mentioned
results taken together indicate that mature Gmm
WT are
considerably more resistant to infection with a foreign microbe than are their
younger counterparts. Furthermore, tsetse's obligate mutualist,
Wigglesworthia, must be present during the development of
immature stages so that mature adults are able to overcome infection with E.
coli.Expression of Immunity-Related Genes in Adult Tsetse Varies Depending on Symbiont
StatusTo understand the basis of the compromised immunity we observed in mature
GmmWgm
− flies, we evaluated the
expression profile of a set of immunity-related genes from age-matched mature adult
Gmm
WT and
GmmWgm
− flies that were either
uninfected or 3 dpi with E. coli K12. We included the antimicrobial
peptides (AMPs) attacin, cecropin, and
defensin, which distinctly target gram-negative
(attacin and cecropin) and gram-positive
bacteria (defensin) [16]. Our analysis also included
thioester-containing proteins (tep2 and tep4),
prophenoloxidase (PPO), and inducible nitric oxide synthase
(iNOS). In insects TEPs likely function as pathogen-specific
opsonins that bind to bacteria or parasites and promote their
phagocytosis/encapsulation [17], while PPO initiates a proteolytic cascade that results in
melanin deposition [18]. iNOS catalyzes synthesis of the signaling molecule nitric
oxide (NO), which plays a role in humoral and cellular immunity in Anopheline
mosquitoes [19], reduvid bugs [20], and Drosophila
[21],[22] by inducing AMP
expression and recruiting hemocytes to the site of infection.Our expression results indicate that symbiont status plays little or no role in the
expression of immunity-related genes in uninfected adults. In fact, with the
exception of the AMP defensin, no significant differences in
immunity-related gene expression between mature uninfected
Gmm
WT and
GmmWgm
− adults (Figure 2A). However, we observed a considerably
different profile of immunity-related gene expression when these different fly
strains were infected with E. coli K12. Under these circumstances,
all of the genes evaluated (with the exception of defensin) were expressed at
significantly higher levels in Gmm
WT compared to
GmmWgm
− individuals (Figure 2B). Particular striking was
the fact that the induction of pathways associated with cellular immunity, such as
pathogen recognition (tep2 and tep4) and
melanization (PPO), were significantly compromised in mature
GmmWgm
− adults. The absence of a
robust cellular immune response is likely the cause of high mortality among these
individuals following E. coli infection. This analysis indicates
that Wigglesworthia must be present during the development of
immature tsetse in order for immune-related genes to subsequently be expressed in
mature E. coli–infected adults.10.1371/journal.pbio.1000619.g002Figure 2The effect of symbiont status on the expression of immunity-related genes
in adult tsetse.(A) Target gene expression in uninfected Gmm
WT and
GmmWgm
− adults is indicated
relative to the constitutively expressed tsetse β-tubulin gene. (B)
Fold-change in the expression of immunity-related genes in
Gmm
WT and
GmmWgm
− tsetse 3 dpi with
E. coli K12. All values for both tsetse strains are
represented as a fraction of average normalized gene expression levels in
bacteria-infected flies relative to expression levels in PBS-injected controls.
In (A) and (B), quantitative measurements were performed on three biological
samples in duplicate. Values are represented as means (±SEM). *
p<0.05, ** p<0.005
(Student's t-test).Hemocytes Play an Integral Role in Controlling E. coli Infection
Outcomes in TsetseOur analysis of immunity-related gene expression in tsetse suggests that cellular
immune pathway functions in adult tsetse are particularly compromised when
Wigglesworthia is absent during immature development. The most
prominent cellular immune mechanisms include melanization and phagocytosis. These
processes, which ultimately result in the removal of foreign invaders in
Drosophila
[23] and A.
aegypti
[24], both arise
from distinct crystal cell and plasmatocyte hemocyte lineages, respectively [25].We investigated the role hemocytes might play in determining the susceptible
phenotype we observed in tsetse following infection with E. coli. We
infected mature Gmm
WT individuals with GFP-expressing
E. coli and were able to observe that hemocytes had engulfed a
large number of the introduced cells by 12 hpi (Figure 3A). We next inhibited phagocytosis by
introducing blue fluorescent microspheres directly into tsetse's hemocoel and 12
h later infected the bead-treated individuals with GFP-expressing E.
coli. Microscopic inspection of hemocytes harvested 12 hpi with
E. coli revealed the presence of internalized microspheres and
the absence of engulfed E. coli. This observation indicated that we
were successful in blocking hemocyte phagocytosis (Figure 3B). We subsequently maintained our
microsphere-injected tsetse for 2 wk with the intention of determining the impact of
impaired phagocytosis on host survival outcome. Mature
Gmm
WT flies exhibiting impaired phagocytosis were
highly susceptible to infection with both 1×103 and
1×106
E. coli K12. In fact, by day 12 post-infection, all of these flies
had perished regardless of the initial dose used for infection (Figure 3C). This observation contrasts starkly with
infection outcome in mature Gmm
WT that exhibit normally
functioning hemocytes (Figure 1A,
middle graph). Our results suggest that defects in phagocytosis severely compromise
the ability of these tsetse flies to overcome bacterial infection.10.1371/journal.pbio.1000619.g003Figure 3Hemocyte-mediated phagocytosis is a critical component of tsetse's
immune response.(A) 8-d-old Gmm
WT were subjected to septic
infection with GFP-expressing E. coli K12. Twelve hours
post-infection hemolymph was collected, fixed on glass slides using 2%
paraformaldehyde, and microscopically examined for the presence of
hemocyte-engulfed bacterial cells. Scale bar = 10
µm. (B) The process of hemocyte-mediated phagocytosis in tsetse was
blocked by micro-injecting polystyrene beads into the hemocoel of 8-d-old WT
individuals. In consecutive 12 h intervals following bead injection, flies were
infected with GFP-expressing E. coli K12 and then hemolymph
was collected and fixed as described above. Hemocytes appear to have engulfed
the beads, thus prohibiting the subsequent uptake of bacterial cells. The inset
in each panel shows a higher magnification image of one hemocyte, which is
identified by a white triangle in the left-most panel. Scale bar = 20 μm. (C) Tsetse flies that harbor hemocytes
incapable of engulfing E. coli are susceptible to septic
infection with this bacterium while their wild-type counterparts are not. The
susceptible phenotype is exhibited regardless of whether phagocytosis-inhibited
tsetse were inoculated with 103 or 106 CFU of E.
coli. Beads alone had no effect on tsetse mortality. No significant
difference existed in survival outcome between mature
Gmm
WT phagocytosis inhibited flies infected with
103 versus 106 CFU of E. coli
(p = 0.47, log-rank analysis).
Furthermore, no significant difference was present between mature
GmmWgm
− flies with
uninhibited hemocytes (Figure
1A, bottom panel) and mature Gmm
WT
phagocytosis inhibited flies (p = 0.11)
infected with 106 CFU of E. coli.A notable result of our immunity-related gene expression analysis was a 37-fold
decrease in PPO levels in
GmmWgm
− flies. This enzyme is an
essential component of the melanization pathway, and its expression ultimately
results in host wound healing and the melanization, encapsulation, and subsequent
removal of foreign microorganisms [26]–[28]. In conjunction with the remarkable variation in
PPO expression observed between Gmm
WT
and GmmWgm
−, we were also able to
visually observe the absence of a melanization response to E. coli
infection in flies lacking Wigglesworthia. In fact, 30 min
post-injection with E. coli, hemolymph was still actively exuding
from the inoculation wound of GmmWgm
−
flies. Conversely, in WT individuals no hemolymph was detectable and melanin was
deposited at the wound site (Figure
4). These results further suggest that hemocyte-mediated cellular immunity
provides an imperative defense against the establishment of bacterial infections in
tsetse's hemocoel. Furthermore, the absence of this response in
GmmWgm
− individuals was likely
responsible for the compromised host survival phenotype we observed following
infection with E. coli.10.1371/journal.pbio.1000619.g004Figure 4The effect of symbiont status on melanization in tsetse.Mature Gmm
WT and
GmmWgm
− tsetse
(n = 10 of each strain) were
intra-thoracically inoculated with 1×103
E. coli K12. Thirty minutes post-inoculation the wound site on
individuals from each strain was inspected microscopically for the presence of
hemolymph clotting and melanin deposition. Thirty minutes post-inoculation,
neither hemolymph clotting nor melanin were observed at the wound site of
GmmWgm
− individuals
(indicated by a red arrow). In contrast, within the same amount of time,
hemolymph no longer exuded from the wound of WT flies and melanin was present
surrounding the site (indicted by a white arrow).Symbiotic Bacteria Impact the Maturation of Tsetse's Cellular Immune
Response During DevelopmentWe observed that young Gmm
WT were markedly more
susceptible to infection with E. coli K12 than were their mature
counterparts. Furthermore, symbiont status also altered infection outcome, as mature
GmmWgm
− perished following
E. coli infection while age-matched WT individuals survived.
These differential infection outcomes appeared to result from disparities in cellular
immune system function between the different tsetse lines we examined. Based on these
observations we hypothesized that the obligate mutualist
Wigglesworthia plays a crucial role in regulating the development
of cellular immunity in tsetse during immature stages. To test this hypothesis we
quantified the number of circulating and sessile hemocytes present in young and
mature adult Gmm
WT and mature adult
GmmWgm
−. Our results indicate that
a 1.4-fold increase in circulating hemocyte number occurs between day 3 and day 8 in
WT tsetse, while no significant change in circulating hemocyte number was observed
between young and mature GmmWgm
− (Figure 5A). Interestingly, mature
Gmm
WT adults harbored 3.4× more circulating
hemocytes than did mature GmmWgm
− adults
(Figure 5A). We also looked at
sessile hemocyte abundance as a further indicator of
Wigglesworthia's impact on the development of cellular
immunity in tsetse. In Drosophila, this hemocyte subtype
concentrates in large quantities around the anterior end of the fly's dorsal
vessel [29].
Thus, we indirectly quantified sessile hemocyte abundance immediately adjacent to the
anterior-most chamber of tsetse's dorsal vessel by measuring the fluorescent
emission of microspheres that were found engulfed in this region. Young
Gmm
WT adults engulfed 1.2× more microspheres
than their mature counterparts and 15.7× more than mature
GmmWgm
− adults. Furthermore,
mature WT adults engulfed 13.2× more microspheres than did age-matched
GmmWgm
− adults (Figure 5B).10.1371/journal.pbio.1000619.g005Figure 5The effect of age and symbiont status on the development of tsetse's
cellular immune response.(A) Number of hemocytes per µl of hemolymph in young (3 d) and mature (8
d) Gmm
WT and
GmmWgm
- tsetse (n = 5 individuals from each tsetse strain at both time
points). (B) Quantitative analysis of sessile hemocyte abundance in young and
mature Gmm
WT and mature
GmmWgm
- tsetse (n = 4 individuals from each tsetse strain and age point).
All tsetse strains tested were subjected to hemocoelic injection with blue
fluorescent microspheres. Twelve hours post-injection, flies were dissected to
reveal tsetse's heart. The left-most panel is a Brightfield image of the
three chambers that make up the dorsal vessel (DV; scale bar = 350 µm). The anterior-most chamber is indicated
within a white circle. The three remaining panels are close-ups of the anterior
chamber (scale bar for all 3 panels = 80 µm),
visualized by excitation with UV light (365/415 nm). Relative fluorescence per
tsetse group was determined using ImageJ software. (C) The presence of
Wigglesworthia affects the expression of genes involved in
hemocyte differentiation in immature larval tsetse. Target gene expression in
Gmm
WT and
GmmWgm
− larval instars
1–3 is indicated relative to the constitutively expressed tsetse
β-tubulin gene. Quantitative measurements were performed on three
biological samples in duplicate. All values in this figure are represented as
means (±SEM). * p<0.05, **
p<0.005 (Student' t-test).Our results demonstrate that Wigglesworthia must be present during
the development of immature tsetse in order for cellular immunity to develop and
function properly in adults. Thus, we speculate that the absence of hemocytes in
adult tsetse should reflect a lack of blood cell differentiation during the
development of immature stages. In Drosophila the process of blood
cell differentiation, or hematopoiesis, begins in the embryo and proceeds through all
larval stages [30].
During Drosophila embryogenesis early hematopoiesis can be
distinguished by the expression of the zinc finger transcription factor
“Serpent.” Subsequently, another transcription factor,
“Lozenge,” directs the differentiation of
serpent-expressing precursor cells into a specific lineage of
hemocytes called crystal cells. To address the relationship between the presence of
Wigglesworthia and early hematopoiesis in tsetse, we used qPCR to
evaluate the relative number of serpent and lozenge
transcripts present in 1st, 2nd, and 3rd instar
larvae dissected from pregnant Gmm
WT and
GmmWgm
− females. Larval instars
L1, L2, and L3 from WT females expressed 1.7, 2.1, and 1.9 times more
serpent transcripts, and 4, 4.4, and 3.9 times more
lozenge transcripts, respectively, than did their counterparts
from females that lacked Wigglesworthia (Figure 5C). This observed attenuated expression of
both serpent and lozenge may account for the
depleted hemocyte population, and compromised cellular immune function, we observed
in adult GmmWgm
− individuals.DiscussionIn the present study we demonstrate that Wigglesworthia is intimately
involved in regulating the maturation and function of tsetse's cellular immune
system during immature larval development. We present a model that links the presence of
Wigglesworthia in larval progeny with host immune system maturation
during development and the subsequent ability of adult tsetse to overcome infection with
foreign microbes (Figure 6).
Obligate symbioses between intracellular bacteria and multi-cellular eukaryotes
represent millions of years of co-evolution during which time both partners have adapted
to increase each other's overall fitness. The association between tsetse and
Wigglesworthia is an example of this reciprocal relationship in that
neither organism can survive in the absence of the other.10.1371/journal.pbio.1000619.g006Figure 6
Wigglesworthia modulates the development and function of
tsetse's immune system.Through a currently unknown mechanism, the presence of
Wigglesworthia in Gmm
WT larvae
stimulates hemocyte differentiation in a specialized organ that is homologous to
Drosophila's lymph gland. Upon metamorphosis, specialized
hemocyte subtypes are released from the lymph gland and carried over in a
functional state to the adult. In the absence of Wigglesworthia,
GmmWgm
− larvae produce
significantly less hemocytes than their WT counterparts. Several innate immunity
pathways are activated upon inoculation of E. coli into the
hemocoel of mature adult Gmm
WT. Tsetse's
preliminary line of defense against E. coli infection likely
involves melanization at the wound site. This process is initiated by localized
crystal cells, which instigate the melanization cascade by secreting
prophenoloxidase (PPO) into the hemolymph. Pathogens that circumvent the wound
site then encounter phagocyte-mediated cellular and humoral immune responses.
Soluble TEPs likely opsonize bacterial cells, thus tagging them for engulfment by
phagocytes. Lysis of engulfed bacteria causes the release of bacterial
peptidoglycan PGN that subsequently stimulates the production of AMPs by the fat
body. AMPs, which are also generated by hemocytes, are then secreted into the
hemolymph where they further abrogate microbial proliferation. Hemocytes also
produce reactive oxygen intermediates, such as iNOS, that exhibit direct bacterial
toxicity and further stimulate humoral immunity. In the case of
GmmWgm
− adults, incapacitated
hematopoiesis during larval stages results in severely compromised immunity that
renders these flies highly susceptible as adults to bacterial infection.We present several lines of evidence indicating that tsetse's resistance to
E. coli positively correlates with fly age and symbiont infection
status during juvenile stages. Specifically, our results signify that mature
Gmm
WT adults are resistant to E. coli
infection, while young Gmm
WT adults are susceptible. In
contrast, both young and old adult flies that lack Wigglesworthia
throughout all developmental stages are killed by E. coli infections.
Like their WT counterparts, GmmWgm
− larvae
acquire both Sodalis and Wolbachia while in utero
[31]. Although the
intrauterine larval environment is otherwise aseptic, adult
GmmWgm
− can be exposed to a wide range
of environmental microbes during adulthood. However, neither the presence of other
symbiotic bacteria (Sodalis and Wolbachia) in larvae,
nor environmental microbes acquired during adulthood, appear to be sufficient to induce
immunity in GmmWgm
− adults. Additionally,
we show that mature Gmm
WT adults treated with antibiotics to
eliminate Wigglesworthia
(Gmm
WT/Wgm−) remain resistant
to E. coli infections. The resistant phenotype of
Gmm
WT/Wgm− further signifies
that obligate Wigglesworthia is not directly responsible for the
ability of mature adult Gmm
WT to overcome septic bacterial
infection. Instead, it appears that Wigglesworthia's presence
during the maturation of larval progeny stimulates the development of host immunity in
adults.Our quantitative analysis of gene expression indicates that
Gmm
WT and
GmmWgm
− adults exhibit no significant
difference in the expression of genes that serve as hallmarks of humoral and cellular
immunity in the absence of microbial challenge. However, following infection with
E. coli, all pathways were significantly compromised in
GmmWgm
− versus
Gmm
WT adults. The most notable discrepancy observed
between the two tsetse lines involved the expression of
prophenoloxidase. Following E. coli infection, the
expression of this gene increased 37-fold in WT tsetse but remained virtually unchanged
in individuals that lacked Wigglesworthia. In WT insects, PPO, which is
an inactive zymogen, is proteolytically cleaved to produce phenoloxidase upon mechanical
injury or the presence of foreign pathogens. Phenoloxidase then facilitates the process
of melanin deposition [18]. A functional melanotic pathway would likely increase
tsetse's resistance to E. coli infection in several ways. First,
sequestration of E. coli in a melanotic capsule at the site of
inoculation would likely help prevent their dissemination into adjacent host tissues.
This phenomenon has been observed in both the hawk moth, Manduca sexta,
and Drosophila following infection with pathogenic bacteria
(Photorhabdus luminescens) and parasitic wasp eggs, respectively
[32],[33]. In both cases the
lack of melanization at the wound site severely compromised the host's ability to
subsequently overcome the foreign invader. Second, the melanization cascade results in
the production of reactive oxygen intermediates that are directly toxic to foreign
pathogens. In the flesh fly Sarcophaga peregrina and M.
sexta, melanization intermediates such as DOPA exhibit direct antimicrobial
activity [34],[35]. Finally, melanin at
the site of inoculation expedites wound healing that could prevent the spread of
secondary infections [26].The E. coli–susceptible phenotype of mature
GmmWgm
− adults is likely reflective
of their blood cell (hemocyte) deficit in comparison to E.
coli–resistant WT flies. In Drosophila
90%–95% of the total hemocyte population is composed of sessile and
circulating plasmatocytes, which are a distinct hemocyte lineage predominantly
responsible for engulfing and digesting foreign pathogens [36]. The inability of mature
GmmWgm
− tsetse to survive infection
with E. coli may result from their significantly reduced population of
phagocytic hemocytes available to engulf bacteria injected into the hemocoel. In fact,
by injecting polystyrene beads as a means of blocking this physiological process, we
demonstrate that phagocytosis is a critical component of tsetse's ability to manage
septic infection with E. coli in WT flies. Similarly, mutant
Drosophila that contain a depleted plasmatocyte population, or have
been injected with beads to prohibit phagocytosis, exhibit a remarkable susceptibility
to a variety of gram positive and negative bacteria [37],[38]. Interestingly,
Drosophila that lack functional plasmatocytes also exhibit a reduced
capacity to activate humoral immune responses, further inhibiting their ability to fight
bacterial infection [29].In Drosophila the crystal cell hemocyte lineage controls the humoral
melanization cascade via the release of PPO stored in large cytoplasmic inclusion bodies
[30],[39]. In addition to the
dramatic reduction in PPO expression in
GmmWgm
− tsetse, two further lines of
evidence indicate that this fly strain harbors a significantly reduced population of
hemocytes that function in a homologous manner to Drosophila crystal
cells. First, lozenge expression in all three larval instars from
GmmWgm
− females is significantly
lower than in their Gmm
WT counterparts. In
Drosophila larval crystal cells fail to form in the absence of
lozenge expression, yet the differentiation of other hemocyte types
proceeds normally [40].
Second, we observed that hemocyte-deficient
GmmWgm
− flies were unable to produce a
viable clot at the site of bacterial inoculation. In mutant Drosophila
strains that lack crystal cells, PPO is absent from the hemolymph. Consequently, the
melanization cascade fails to initiate and hard clots do not form at wound sites [41].We discovered that young Gmm
WT adults harbor significantly
less circulating hemocytes than do mature WT adults. This observation implies that
circulating hemocyte number in adult WT tsetse increases as a function of age, although
the specific mechanism underlying this process in tsetse is currently unknown. In adult
Drosophila intact lymph glands are absent and no evidence exists for
the de novo synthesis of hemocytes following metamorphosis [30],[40]. Interestingly, we did observe more
subepidermal sessile hemocytes in young compared to mature
Gmm
WT adults, although the number was not statistically
significant between the two groups. We speculate that the increased abundance of
circulating hemocytes we observed in mature compared to young
Gmm
WT may reflect a shift in the proportion of sessile to
circulating cells instead of de novo production of new hemocytes. A comparable process
occurs in Drosophila larvae and adults, where the proportion of sessile
to circulating hemocytes changes following immune stimulation [42],[43]. Furthermore, after larval
Drosophila receive an epidermal wound, circulating hemocytes are
rapidly recruited to the site of injury. These circulating cells are phagocytically
active and likely function as a front-line surveillance system against tissue damage and
microbial infection [44]. In the mosquito malaria vector Anopheles
gambiae, exposure to Plasmodium parasites stimulated an
increase in the number of granulocytes circulating in the hemolymph. These primed
mosquitoes were subsequently more resistant to infection with pathogenic bacteria than
their wild-type counterparts [45]. We propose that young Gmm
WT
adults and mature GmmWig
− adults may be
susceptible to E. coli infection in part because they harbor
significantly less circulating hemocytes than do older WT individuals that are
resistant. Our previous studies indicated that
GmmWig
− adults are highly susceptible to
trypanosome infection [9],[10] and that this phenotype may be modulated by tsetse's
humoral immune system [46]–[48]. Studies are ongoing to determine if cellular immunity also
modulates tsetse's trypanosome vectorial competence.Neonatal humans (and presumably other mammals as well) acquire probiotic gut microbes
through the act of breast feeding [49]. These bacteria subsequently stably colonize the naïve
intestine where they promote immune system maturation and enhance defense against
infection with pathogenic microbes [50]. Most lower eukaryotes,
including insects, hatch from an egg deposited into the environment and thus rely mainly
on environmental microbes to stimulate their innate immune systems during the
development of immature stages. For example, Anopheline mosquitoes and
Drosophila are exposed to environmental microbes throughout all
stages of their development. Under these natural conditions subsequent adults exhibit
potent innate immunity [51],[52]. However, when these insects are reared under germ-free
conditions, they exhibit a severely compromised humoral immune response [51],[53]. Tsetse flies are unique among other
insects because they lead a relatively aseptic existence. Not only do they feed
exclusively on sterile vertebrate blood, but they also exhibit a unique viviparous
reproductive strategy. During this type of reproduction, all embryonic and larval stages
develop within the female's uterus. The protected in utero environment in which
viviparous offspring develop limits their exposure to environmental microbes. However,
throughout tsetse larvagenesis maternal milk gland secretions provide the developing
offspring with nourishment as well as Wigglesworthia,
Sodalis, and Wolbachia
[31]. Interestingly,
recently established Sodalis and Wolbachia appear
unable to influence their host's physiology to the same extent as mutualist
Wigglesworthia despite their presence throughout larval development.
Future studies on this system will focus on determining the chemical and/or metabolic
elements provided by Wigglesworthia that stimulate the maturation of
its host's immune system during larval development.The results of this study provide evidence of a novel functional role for obligate
insect symbionts–host immune system activation during immature developmental
stages to ensure robust function during adulthood. We demonstrate that the obligate
Wigglesworthia provides tsetse offspring the stimuli necessary for
immune system development, a process that exhibits functional parallels with the
mammalian system following the transfer of beneficial microbes from mothers to their
neonates during that act of breast feeding. This phenomenon represents an adaptation
that further anchors the steadfast relationship shared between tsetse and its obligate
mutualist. The essential nature of tsetse's dependence on
Wigglesworthia provides a potentially exploitable niche to
experimentally modulate host immunity, with the intention of diminishing this
insect's capacity to vector deadly trypanosomes.Materials and MethodsTsetse and Septic InfectionsThroughout the text, 3-d-old and 8-d-old tsetse are referred to as
“young” and “mature,” respectively. Wild-type G. m.
morsitans (Gmm
WT) were maintained in
Yale's insectary at 24°C with 50%–55% relative humidity.
These flies received defibrinated bovine blood every 48 h through an artificial
membrane feeding system [54]. Two Wigglesworthia-free tsetse lines were
generated for use in our experiments. The first,
GmmWgm
−, was generated by
supplementing the diet of pregnant females with 25 µg of ampicillin per ml of
blood. GmmWgm
− (offspring of
ampicillin-fed females) adult are devoid of Wigglesworthia
throughout all developmental stages [9]. PCR using Wigglesworthia thiamine
C-specific primers (forward, 5′-TGAAAACATTTGCAAAATTTG-3′; reverse,
5′-GGTGTTACATAGCATAACAT-3′) confirmed that this
tsetse strain lacked Wigglesworthia. The second
Wigglesworthia-free tsetse line,
Gmm
WT/Wgm−, was generated by
feeding mature WT adults three blood meals supplemented with 40 µg/ml
tetracycline. Gmm
WT/Wgm−
individuals thus harbored Wigglesworthia, Sodalis,
and Wolbachia throughout the development of all immature stages and
early adulthood, but lacked these symbionts thereafter. The absence of
Wigglesworthia from mature
Gmm
WT/Wgm− adults was
confirmed microscopically by comparing the bacteriome
(Wigglesworthia-harboring organ) contents of mature WT and
Gmm
WT/Wgm− adults.Septic infection of tsetse was achieved by anesthetizing flies with CO2
and subsequently injecting individuals with live bacterial cells using glass needles
and a Narashige IM300 micro-injector. The methods used to produce
luciferase-expressing E. coli K12 (recE.
coli
pIL), and the assay used to quantify luciferase expression
in vivo, were described previously [15]. GFP-expressing and tetracycline-resistant E.
coli K12 were produced via electroporation with pGFP-UV (Clontech,
Mountain View, CA) and pBR322 (Promega, Madison, WI) plasmid DNA, respectively. All
flies treated with tetracycline were subsequently infected with tetracycline
resistant E. coli K12. The number of bacterial cells injected and
control group designations for all infection experiments are indicated in the
corresponding figures and their legends. For all survival experiments, treatments
were performed in triplicate, using 25 flies per E. coli treatment
replicate. LB media controls were performed once using 25 flies.Real-Time Quantitative PCR (qPCR)For analysis of immunity-related gene expression, sample preparation and qPCR were
performed as described previously [15]. Quantitative measurements were performed on three
biological samples in duplicate and results were normalized relative to tsetse's
constitutively expressed β-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.For symbiont quantification, total RNA was prepared from 40-d-old adult
Gmm
WT and
GmmWgm
− flies. Symbiont genome
numbers were quantified using single-copy Sodalis fliC and
Wolbachia groEL. Relative symbiont densities were normalized to
tsetse β-tubulin. All qPCR was performed with an icycler iQ real
time PCR detection system (Bio-Rad). Values are represented as the mean
(±SEM). qPCR primer sequences are shown in Table S1.Bacterial 16s rRNA Clone LibrariesUniversal bacterial 16S rRNA gene primers 27F (5′-AGAGTTTGATCCTGGCTCA
G-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′) [55] were used to produce a clone
library of 16s rRNA gene sequences found in 3rd instar
Gmm
WT and
GmmWgm
− larvae. Five individual
larvae from each tsetse line were dissecting under sterile conditions and washed in
DNase (Ambion, Austin, TX) to remove any surface contamination prior to DNA
extraction. Genomic DNA was isolated using Holmes-Bonner buffer (0.1 mol/L Tris-HCl,
pH 7.5; 0.35 mol/L NaCl; 10 mmol/L EDTA, pH 8.0; 2% SDS; 7
mol/L Urea), purified via phenol-chloroform extraction and precipitated in
100% EtOH. PCR was performed under standard reaction conditions [15], and the resulting
products were cloned in the pGEM-T vector (Promega, Madison, WI). Twenty clone
inserts from each larvae (100 in total from each tsetse line) were sequenced using
the T7 vector specific primer, and homology to previously described 16s rRNAs was
determined using the blastn database.Hemolymph Collection and Hemocyte QuantificationDepending on the subsequent experiment, tsetse hemolymph was collected using one of
two methods. For hemocyte quantification, 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 a glass micro-pipette and placed into a microfuge tube on ice.
Hemocytes were quantified microscopically using a Bright-Line hemocytometer, and
hemocyte numbers are represented as cells per µl of hemolymph.When hemocytes were required for microscopic visualization, hemolymph was collected
by employing a modified version of the high injection/recovery method previously
developed for use in mosquitoes [56]. In brief, tsetse flies were sedated on ice and injected
with 25 µl of chilled anticoagulant buffer [70% MM medium,
30% anticoagulant citrate buffer (98 mM NaOH, 186 mM NaCl, 1.7 mM EDTA, and 41
mM citric acid, buffer pH 4.5), vol/vol] between the last two abdominal
schlerites using a glass needle and a Narashige IM300 micro-injector. Following a 30
min incubation on ice, a front leg was removed at the joint most proximal to the
thorax. At this point internal pressure forced hemolymph diluted with anticoagulant
buffer to be expelled from the wound site (more liquid could be recovered by applying
gentle pressure to the distal end of the abdomen). Liquid was collected using a
pipette and either placed into a chilled microfuge tube or directly into a 24-well
cell culture plate. In the latter case, cells were allowed to adhere to the plate
bottom, after which anticoagulant buffer was replaced with MM media.Sessile hemocytes were observed by intra-thoracically injecting young and mature
Gmm
WT, and mature
GmmWgm
−
(n = 5 of each strain), with 2 µl of blue
fluorescent (365/415 nm) 0.2 µm carboxylate-modified beads (Invitrogen corp.).
Prior to use, beads were washed once in PBS and resuspended in 100% of their
original volume. Flies were dissected 12 h post-injection to reveal their dorsal
vessel and surrounding tissue, which was gently washed 3 times with PBS to remove any
potentially contaminating circulating (non-adherent) hemocytes. Engulfed microspheres
were visualized using a Zeiss steriomicroscope (Discovery v8) equipped with a coaxial
fluorescence module. Semi-quantitative comparison of sessile hemocyte number between
young and mature Gmm
WT adults, and mature
Gmm
WT and
GmmWgm
− adults, was performed by
quantifying fluorescent signal intensity (n = 4
individuals from each group) using ImageJ software (http://rsbweb.nih.gov/ij/).Phagocytosis and Melanization AssaysPhagocytosis by circulating tsetse hemocytes was observed by intra-thoracically
infecting mature Gmm
WT flies (n = 10) with 1×106 GFP-expressing E.
coli K12. Twelve hours post-infection, hemolymph was collected and
hemocytes monitored to determine if they had engulfed the GFP-expressing bacterial
cells. Hemolymph samples were fixed on glass microscope slides via a 2 min incubation
in 2% PFA. Prior to visualization using a Zeiss Axioscope microscope, slides
were overlayed with VectaShield hard set mounting medium containing DAPI (Vector
Laboratories, Burlingame, CA).Phagocytosis by tsetse hemocytes was inhibited with blue fluorescent (365/415 nm) 0.2
µm carboxylate-modified beads (Invitrogen Corp.). Prior to use, beads were
washed once in PBS and resuspended in 100% of their original volume.
Inhibition assays were performed by inoculating 8-d-old
Gmm
WT with 2 µl of beads via their thoracic
compartment. Twelve hours later, these flies were similarly infected with
1×103 and 1×106 GFP-expressing E.
coli K12 (experiment was performed in triplicate; n = 25 flies per replicate). Finally, 12 hours post-infection
with E. coli, hemolymph was collected and processed as described
above (these samples were overlayed with VectaShield hard set mounting medium that
lacked DAPI).Melanization assays were performed by intra-thoracically inoculating mature
Gmm
WT and
GmmWgm
− (n = 10 of each strain) with 1×103
E. coli K12. Subsequently, three individuals from each group were
monitored microscopically every 10 min for the presence of melanin at the wound site.
The remaining seven flies from each group were maintained for 2 wk in order to
observe infection outcome.StatisticsStatistical significance of survival curves was determined by log-rank analysis using
JMP (v8.02) software (www.jmp.com). Statistical analysis of qPCR data was performed by
Student's t test using JMP (v8.02) software. Statistical
significance between various treatments, and treatments and controls, is indicated in
corresponding figure legends.Supporting InformationTable S1Primers used for quantitative real-time PCR.(0.02 MB XLS)Click here for additional data file.
Microbiology/Immunity to Infections Microbiology/Innate Immunity