Leeches and their microbiota:
naturally simple symbiosis models
Joerg Graf, Yoshitomo Kikuchi and Rita V.M. Rio
Department of Molecular and Cell Biology, University of Connecticut, 91 North Eagleville Rd, Storrs, CT 06269-3125, USA
Strictly blood-feeding leeches and their limited
microbiota provide natural and powerful model systems
to examine symbiosis. Blood is devoid of essential nutrients
and it is thought that symbiotic bacteria synthesize
these for the host. In this review, three distinct leech–
microbe associations are described: (i) the mycetome,
which is the large symbiont-containing organ associated
with the esophagus; (ii) the nephridia and bladders that
form the excretory system; and (iii) the digestive tract,
where two bacterial species dominate the microbiota.
The current knowledge and features of leech biology
that promote the investigation of interspecific interactions
(host–microbe and microbe–microbe) and their
evolution are highlighted.
Model systems for bacteria–animal symbioses
Symbiosis forms a pivotal component in the existence of
many animals and plants by providing a multitude of indispensable
biological functions . The complexity and intimacy
of the majority of these relationships present
difficulties in examining how symbiosis arises, identifying
the mechanisms that contribute to specificity and elucidating
the functional roles of each partner .Many established
model systems are either monospecific (in which the host
maintains relations with a single microbial species), artificially
reconstituted from far more complex associations, or
toomultifaceted to reveal underlyingmechanisms [3,4]. The
study of these pioneering systems has resulted in exciting
discoveries but to assess how widely applicable these findings
are, a comparative approach using a wide range of
model systems is required. The medicinal leech is a promising,
bona fide model for symbiotic associations and has a
microbial community of limited complexity to facilitate
examination of fundamental aspects of interspecific relations
(Box 1). In this review, we describe three distinct
microbe–leech associations found in different leech species:
mycetome, nephridia and bladders, and digestive-tract symbioses.
Aspects of leech biology that promote its application
as a powerful model system for the study of host–microbe
and microbe–microbe interactions will also be highlighted.
Hirudinids: taxonomy, natural history and medicinal
Leeches are fascinating animals that can evoke contradictory
responses. One can observe with amazement the
leech undulating elegantly while swimming or with horror
as its whole body contracts rhythmically while pumping
the blood from an unsuspecting victim. Strictly blood-feeding
leeches are found in the orders Rhynchobdellida, species
of which feed using a tubular proboscis and have a
bacterial-symbiont-containing organ (the mycetome) associated
with the esophagus, and Arhynchobdellida, which
feed using toothed jaws and lack a mycetome . Recent
molecular studies have shown that the medicinal leech,
although usually marketed as Hirudo medicinalis
(Hirudinea: Arhynchobdellida: Hirudinidae), probably
consists of a complex of at least three species: Hirudo
orientalis, the commonly sold Hirudo verbana and the
rare H. medicinalis [6–9]. Hirudinids are hermaphrodites
that deposit cocoons containing multiple eggs at the
land–water interface . Juvenile leeches reportedly consume
their first blood meal from amphibians whereas
successive meals can be obtained from amphibians, fish
or mammals [5,10]. The ingested blood is quickly modified
in the crop by the discharge of water and osmolytes
through the multiple pairs of bladders that lie near the
lateral ceca of the crop (Figure 1). The erythrocytes are
stored apparently physically intact within the crop for up
to six months. The actual digestion of the blood meal and
absorption of nutrients is thought to occur in the much
smaller intestinum (located between the last pair of crop
ceca), which combines some functions of the intestine and
rectum (Figure 1).
The remarkable abilities of the medicinal leech to consume
five to six times its body weight in a single blood meal
and to release an array of potent chemicals with its saliva
has led to an unexpected resurgence of the use of leeches in
modern medicine . Recently, the medicinal leech was
approved as a medical device for its bloodletting capabilities
by the Food and Drug Administration of the USA
In a manner that has yet to be reproduced by pharmaceuticals,
the direct application of H. medicinalis to areas of
acute venous congestion provides a cost-effective and reliable
treatment to ameliorate the postoperative effects
associated with reconstructive surgery [11–13]. Powerful
vasodilators and anti-inflammatory and anticoagulation
molecules have been isolated, characterized and patented
from leech saliva [14,15]. From the microbiological perspective,
an interesting observation originally made in the
1980s was the diagnosis of wound infections caused by
Aeromonas in patients receiving leech therapy [12,16]. The
use of antibiotics before bloodletting usually prevents
these infections. Earlier studies had identified Aeromonas
as the sole digestive-tract symbiont of H. medicinalis. The
Review TRENDS in Microbiology Vol.14 No.8
Corresponding author: Graf, J. (email@example.com).
www.sciencedirect.com 0966-842X/$ – see front matter 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.tim.2006.06.009
detection of one culturable symbiont led us to pursue the
feasibility of using the medicinal leech as a naturally
occurring simple model for digestive-tract associations
Symbiotic associations of leeches
The most extreme and intimate examples of interspecific
relationships are intracellular symbioses. In these specialized
associations, leech symbionts are usually harbored
in the cytoplasm of mycetocytes. These are large
specialized cells that typically aggregate into a large
symbiotic organ, which, although it houses bacteria, is
called a mycetome for historical reasons . Important
physiological functions occur within mycetomes, such as
the provision of essential host nutrients (e.g. vitamins
and amino acids). Obligate insect symbionts such as
Wigglesworthia spp. in tsetse flies and Buchnera spp.
in aphids are well characterized by genome sequencing
and elegant functional assays, and currently represent
366 Review TRENDS in Microbiology Vol.14 No.8
Box 1. Advantages of using the leech as a symbiosis model
(i) Inexpensive and easily bred invertebrate host.
(ii) Simple host morphology.
(iii) Limited dietary intake (blood).
(iv) Simple trinary association in the crop.
(v) In the digestive tract, the dominant Aeromonas symbiont is
culturable and amenable to genetic manipulations and reintroduction.
(vi) Aeromonas is also a pathogen, which provides an opportunity to
compare symbiosis and virulence factors in one organism.
Figure 1. Leech internal morphology depicting the structural variety of mycetomes. Drawing of the digestive tract and excretory organs based on the medicinal leech. The
inset shows schematic illustrations of mycetome morphological variations: (i) basic structure lacking mycetocytes in Hirudo verbana; (ii) large mycetocytes form a tube-like
structure in Placobdelloides spp.; (iii) a pair of pear-shaped mycetomes join the esophagus at their narrowed ends in Placobdella spp.; (iv) two pairs of large bulbous sacs
connected to the esophagus by narrow ducts comprise the mycetomes in Haementeria species. Figure redrawn, with permission, from Refs  and . (2006)
American Society for Microbiology.
model systems for intracellular symbiosis in invertebrates
In leeches, mycetomes occur in the order Rhynchobdellida
(mainly in the family Glossiphoniidae [1,5]) but not in
the order Arhynchobdellida, which includes the medicinal
leech. Three distinct mycetome morphotypes have been
described in glossiphoniid leeches : large mycetocytes
that surround the esophagus lumen in Placobdelloides spp.
[Figure 1, part (ii)]; a pair of pear-shaped blind sacs in
Placobdella spp. [Figure 1, part (iii)]; and two pairs of large
bulbous sacs connected to the esophagus by narrow ducts
in Haementeria spp. [Figure 1, part (iv)].
Recent molecular phylogenetic analyses based on 16S
rRNA gene sequences have revealed that the symbionts of
Placobdelloides spp. and Haementeria spp. belong to the g-
3 subdivision of the Proteobacteria, whereas the symbionts
of Placobdella spp. belong to the Rhizobiaceae family in the
a-Proteobacteria [19–21]. The g-proteobacterial leech symbionts
cluster with the insect symbionts Buchnera spp. and
Wigglesworthia spp., although the symbionts of Placobdelloides
spp. and Haementeria spp. do not form a monophyletic
group (Figure 2) . These phylogenetic
relationships suggest that the evolution of mycetome symbioses
in glossiphoniid leeches has occurred multiple
times, which is also supported by the morphological diversity
of this organ or, alternatively, suggests that the current
symbionts replaced ancestral ones.
In addition to the mycetome symbionts, intracellular symbionts
that belong to the family Rickettsiaceae (a-Proteobacteria)
were discovered in three Japanese glossiphoniid
species [22,23]. The Rickettsiaceae are well-described parasitic
and/or commensalistic bacteria that can be isolated
from a wide range of animals . Unlike the mycetome
symbionts, rickettsial symbionts exhibit wider tropism:they
are detected in various leech tissues such as the epidermis,
esophagus and salivary glands and exhibit a heterogeneous
distribution within host populations .
Intracellular symbiont transmission
Although it remains unclear whether the leech intracellular
symbionts are transovarially transmitted, multiple
observations support a transmission from parent to
offspring through the egg. In Placobdelloides spp., the
same microbial species found in adult mycetomes were
also detected in 100% of examined eggs . Rickettsial
symbionts were also consistently detected in the eggs of
infected leeches . Finally, Placobdella parasitica juveniles
that had never received a blood meal were shown to
harbor already large symbiont populations in their mycetomes
. The inability to culture the mycetome symbionts
suggests specialization towards an intracellular
lifestyle, possibly because of their stable inheritance
through host lineages and associated genome reduction
(reviewed in Ref. ). The stability of these associations
supports their indispensable roles in host biology.
Nephridia and bladder symbiosis
The seminal paper by Bu¨ sing et al.  described the presence
of two morphologically distinct bacteria associated
within the nephridia and bladders, the excretory organs of
H. medicinalis (Figure 1). The nephridia serve to remove
waste products from the haemocoelomic fluid and recover
salts from the primary urine. The urine and nitrogenous
waste in the form of ammonia are stored in the bladder
until released . 16S rRNA gene sequencing was used to
identify the symbionts residing in the nephridia and
bladders as the intracellular Ochrobactrum, an a-proteobacterium
related to Sinorhizobium (Figure 2), and
an extracellular Flavobacterium, a member of the
Bacteroidetes.*,y Furthermore, bacteria were detected
microscopically in the bladder of embryos, which strongly
supports the hypothesis of vertical transmission .
Although their functional roles remain uncertain, experimental
evidence suggests that the bacteria contribute to the
degradation of nitrogenous waste .
The digestive-tract microbiota of hirudinid leeches is of
particular interest because of the medicinal application of
these leeches and their lack of mycetomes (unlike the
glossiphoniid leeches, in which the hallmark mycetomes
are presumed to perform an essential function for the host).
The first microbiologists who examined the hirudinid
digestive-tract microbiota in the 1950s reported the surprising
presence of a single, b-hemolytic bacterial species
that released many proteolytic enzymes . Combined
with an apparent lack of host digestive enzymes in the
crop, these early investigators posed three possible functions
for the gut symbiont: (i) aiding in the digestion of
the blood meal; (ii) providing essential nutrients; and
(iii) ‘colonization resistance’, in which they function to
prevent colonization by other potentially harmful microorganisms.
More recent studies demonstrated the presence
of host-produced proteases in the intestinum, which cast
some doubt on the importance of the bacterial-produced
proteases in digestion . However, the lack of direct
experimental evidence has not ruled out any of these
hypotheses (reviewed in Ref. ).
The symbiont was identified as Aeromonas using current
taxonomybut the species identity differed between research
groups [30–33]. Although initially reported as Aeromonas
hydrophila by several investigators, our group identified the
symbionts as Aeromonas veronii biovar sobria using biochemical
tests and 16S rRNA gene sequences . It is
interesting to note that Aeromonas culicicola, which was
isolated from the midgut of female Culex quinquefasciatus
and Aedes aegyptii mosquitoes , has recently been
renamed A. veronii , suggesting a propensity of this
species to colonize the gastrointestinal tract of blood-feeding
organisms. The differing species identifications probably
reflect the dynamic and complex taxonomy of Aeromonas.
A limitation of the previous characterizations of digestivetract
microbiota was that the studies were purely culturebased.
It is widely recognized that 99% of microbes are
Review TRENDS in Microbiology Vol.14 No.8 367
* A. Schramm et al., abstract 505, 101st General Meeting of the American Society
for Microbiology, Orlando, USA, 2001.
y J. Graf et al., abstract 478, 100th General Meeting of the American Society for
Microbiology, Los Angeles, USA, 2000.
368 Review TRENDS in Microbiology Vol.14 No.8
Figure 2. Phylogenetic tree based on the sequence of the 16S rRNA gene (neighbor-joining analysis with a Kimura’s correction; aligned 1050 bp). Bacterial phyla are shown
on the right. Leech symbionts are represented in bold and red text and their location within the host is stated in parentheses. The mycetome types in red text correspond to
those in Figure 1. Bootstrap values higher than 50% are depicted at the nodes. The scale bar represents 0.1 changes per base.
presently unable to be cultivated [36,37]. Technological
developments in the culture-independent profiling of
microbial community complexity and diversity have
revealed a plethora of novel cohabiting microorganisms
that far outnumber the culturable organisms. These developments
have also greatly advanced our understanding of
the residential microbiota of digestive tracts, to which
essential roles in host biology have been attributed, such
as the provision of essential nutrients (reviewed in Ref.
), development , energy balance  and the priming
of immunity [40,41]. Interesting parallels in the digestivetract
microbial composition among various host species
have been described [39,42], which raises the question of
whether their functional roles are universal or tailored to
the different hosts.
A recent culture-independent study discovered the presence
of a second symbiont in the crop that we have been
unable to cultivate in the laboratory . The 16S rRNA
gene sequence indicates that it is a relative of Rikenella,
members of the Bacteroidetes that have been found in
several different digestive tracts. An exciting aspect about
the discovery of a second symbiont is the natural occurrence
of a restricted – but not monospecific – digestive-tract
microbiota, which will enable us not only to investigate
microbe–host interactions but also to investigate the interaction
between different microbial species. Relevant features
of the Aeromonas and Rikenella species that
comprise the basic two-member microbial community in
the crop are discussed here.
Aeromonas species are motile, Gram-negative rods that
belong to the family Aeromonadaceae . A widely noted
characteristic of Aeromonas spp. is the production of a
large number of exported hydrolytic enzymes that could
aid in the breakdown of nutrients inside the digestive tract
of animals. This family currently consists of 17 facultatively
anaerobic species that occupy a spectrum of niches
ranging from free-living occupants of freshwater to opportunistic
pathogens of fish, amphibians and humans
(reviewed in Ref. ), and to the digestive tract symbionts
of a variety of blood feeders including mosquitoes, the
medicinal leech and the vampire bat [17,31,34,46]. Three
Aeromonas species including A. veronii are associated with
a range of maladies including wound infection, septicemia
and diarrhea in humans . Therefore, A. veronii seems to
have an innate ability to infect the digestive tracts of
multiple host species where manifestations of infection
span from pathogenesis to cooperative.
The recurring identification of 16S rRNA gene sequences
that belong to the Rikenellaceae from a wide range of
digestive tracts is suggestive of both evolutionary adaptation
and physiological contributions towards digestivetract
ecosystems (Figure 2). All of the isolates or sequences
were obtained from a variety of gastrointestinal environments
including goat rumen, termite gut, murine cecum
and the human colon [39,47,48]. Knowledge of the
Rikenella genus is further obscured because of their
fastidious growth and obligate anaerobic requirements.
A novel Rikenella species, related to Rikenella microfusus
isolated from the cecal and fecal samples of Japanese fowl
, has been identified as one of two dominant residents
of the medicinal leech crop . An intriguing question is
whether the leech crop is sufficiently anaerobic to support
the growth of the Rikenella symbiont or if A. veronii
has to remove residual oxygen from the ingested blood
meal to prime the microenvironment for the Rikenella
The presence of a basic two-member microbial community
in the leech digestive crop provides an exciting and
unique opportunity to further extend knowledge of
Rikenella species, albeit indirectly. For example, differential
antibiotic regimens might be used selectively to clear
the Aeromonas or Rikenella symbiont. The reintroduction
of various concentrations of A. veronii and/or isogenic
mutants into the host can reveal whether spatial or quantitative
alterations of the Rikenella population occur by
employing techniques such as fluorescence in situ hybridization
and quantitative PCR. Host fitness assays after
differential antibiotic treatments to examine classical life
history traits such as reproductive output, growth rate and
viability could also prove valuable towards the elucidation
of microbial functional roles.
Factors that contribute to a limited microbial
Factors that contribute to the unusual simplicity of the
leech digestive symbiosis could be derived from three
sources: the ingested blood, the host and/or the symbiotic
bacteria [17,50,51]. The complement system of vertebrate
blood contains powerful antimicrobial properties . Two
lines of evidence suggest that the ingested complement
system remains active for some time inside the leech
and contributes to the specificity of the microbiota.
Heat-inactivation of the blood before feeding enables
colonization by some bacterial species that were unable to
colonize when fed to the leech in fresh blood . Furthermore,
the importance of the Aeromonas lipopolysaccharide
(LPS) layer in protecting against the antimicrobial properties
of the complement system has been demonstrated 
by observing that serum-sensitive Aeromonasmutants with
a defect in their LPS had a dramatically reduced ability to
colonize the leech .
Other bacteria such as Pseudomonas aeruginosa and
Staphylococcus aureus were tested for their ability to
colonize the leech digestive tract and were able to persist
inside it but had a dramatically reduced ability to grow,
independent of the activity of the complement system,
which suggests the presence of a second layer of defense
. The discovery of the Aeromonas symbiont led to
speculation that this symbiont might release antimicrobial
compounds . As part of a culture-independent characterization
of the leech digestive system, the microbiota of
the intestinum in which the actual digestion of the blood
occurs was also characterized. The intestinum harbored a
more diverse microbial community with an average of eight
species detected . The microbial community of the
intestinum, similar to the crop, was dominated by the
Rikenella and Aeromonas symbionts. The presence of a
more diverse microbiota despite the presence of the crop
Review TRENDS in Microbiology Vol.14 No.8 369
symbionts suggests that these two species are not responsible
for inhibiting the growth of microorganisms in the
crop unless this activity is specifically downregulated
within the intestinal environment.
Symbiosis is an important driving force of metazoan evolution.
The association of an animal with microorganisms
provides the host animal with new metabolic capabilities –
for example, enabling animals to feed exclusively on blood.
Whereas intracellular symbioses presumably require the
tightest coordination between microbe and host, extracellular
digestive-tract associations are more prevalent and
usually involve more complex microbial communities. This
complexity not only makes understanding the molecular
interactions between symbionts and host difficult but also
complicates the dissection of those interactions between
the bacterial symbionts. Although the general behavior of
bacteria belonging to one species is well understood, we are
still at the early beginnings of understanding how different
species of bacteria interact in a microbial community.
Digestive tracts are an important environment where
microbial interactions are likely to have an important role.
The digestive-tract symbiosis of the medicinal leech with
the two dominant extracellular A. veronii and Rikenellalike
symbionts provides a unique opportunity to investigate
not only microbe–host but also microbe–microbe
interactions in a naturally simple system (Box 2).
J.G. would like to thank K. Schopfer for introducing him to the leech
symbiosis. We would like to thank V. Kask for the artwork. The research
in our laboratory is supported by an NSF Career award MCB 0448052
and a grant from the Research Foundation of the University of
Connecticut to J.G. Y.K. is the recipient of a postdoctoral training
fellowship from the Japan Society for the Promotion of Science.
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Box 2. Future directions
Mycetome symbiosis: The possibility of three distinct origins of this
symbiosis in the Rhynchobdellid leeches makes for exciting
evolutionary developmental biology (‘evo-devo’) studies. The ability
to dissect out these organs and isolate large numbers of symbionts
enables the implementation of genomic and proteomic tools to gain
an insight into their functional significance.
Nephridia and bladders symbiosis: This symbiosis has potential for
being used in comparative physiological studies with the Acidovorax-
like symbionts that are harbored within the nephridia of earthworms,
a terrestrial annelid [54,55]. It would be interesting to
observe whether the functional roles of these phylogenetically
distinct symbionts are similar.
Digestive-tract symbiosis: The combination of molecular genetic
tools for Aeromonas, fluorescence in situ hybridization for the
localization of both Aeromonas and Rikenella and the ability to
remove symbionts from juvenile hosts will provide new understanding
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