APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 2008, p. 6138–6140
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Vol. 74, No. 19
“Candidatus Midichloria” Endosymbionts Bloom after the Blood Meal
of the Host, the Hard Tick Ixodes ricinus?
Davide Sassera,1Nathan Lo,2* Edwin A. P. Bouman,3Sara Epis,1
Michele Mortarino,1and Claudio Bandi1
Dipartimento di Patologia Animale, Igiene e Sanita ` Pubblica Veterinaria, Universita ` degli Studi di Milano, Milan, Italy1;
The Australian Museum, 6 College St., Sydney, New South Wales 2010, Australia2; and Biology Centre, Institute of Parasitology,
Academy of Sciences of the Czech Republic, Branisovska 31, 370 05 Ceske Budejovice, Czech Republic3
Received 28 January 2008/Accepted 4 August 2008
“Candidatus Midichloria mitochondrii,” an intracellular symbiont of the tick Ixodes ricinus, is the only
described organism able to invade the mitochondria of any multicellular organism. We used quantitative PCR
to examine cycles of bacterial growth and death throughout the host’s development and found that they
correspond with the phases of engorgement and molt, respectively.
The European hard tick Ixodes ricinus is a vector of numer-
ous pathogenic microorganisms (11). While these pathogens
have been extensively studied, less attention has been given to
the tick’s symbionts, which may have an important effect on
host biology. “Candidatus Midichloria mitochondrii” is an in-
tracellular alphaproteobacterial symbiont that inhabits the
germ line cells of its female host (3, 5, 13, 16). It has the unique
ability to invade and destroy mitochondria within ovarian cells
(12). “Ca. Midichloria mitochondrii” has been detected in
100% of the examined I. ricinus females across its geographical
distribution and in 44% of males (9). The basis of the symbiosis
is not well understood. I. ricinus tick lines raised in the labo-
ratory apparently lose the symbiont but continue to survive and
reproduce (9). Thus, the symbiosis does not appear to be
obligatory. We used real-time quantitative PCR (qPCR) to
examine the population dynamics of the symbiont during the
host’s life cycle, which involves a blood meal at each of the
larval, nymph, and adult stages. qPCR has successfully been
used to obtain insights into the basis of a number of other
intracellular symbiont-host interactions (1, 10, 15).
Adult ticks were collected in the woods surrounding Ceske
Budejovice (Czech Republic). All males and some of the fe-
males were preserved in 100% ethanol, while the other females
were fed on guinea pigs. Starting with these females, an entire
life cycle was completed. DNA was extracted from ethanol-
preserved ticks as described previously (3). Sybr green real-
time qPCR protocols were designed for the following: (i) the
gyrB gene of “Ca. Midichloria mitochondrii” (primers CTTG
AGAGCAGAACCACCTA [forward] and CAAGCTCTGCC
GAAATATCTT [reverse]; amplifying 125 bp); (ii) the I. rici-
nus nuclear gene cal (primers ATCTCCAATTTCGGTC
CGGT and TGAAAGTTCCCTGCTCGCTT; amplifying 109
bp); and (iii) the I. ricinus mitochondrial gene COII (primers
CCGACTTCTTGACGTAGACAAC and CTGATTAAGGC
GACCAGGAACG; amplifying 144 bp). PCR cycling for gyrB
and cal was as follows: 95°C for 2 min, 40 cycles at 95°C for 15 s
and at 60°C for 30 s, and melt curve from 55°C to 95°C with
increasing increments of 0.5°C per cycle. The cycling for COII
differed only in the annealing temperature, set at 58°C. All
reactions were performed in 25 ?l of Milli-Q water containing
400 nM of each primer, 12.5 ?l of iQ Sybr green supermix, and
1 ?l of DNA. PCR products were sequenced to confirm PCR
specificity and then ligated into the pGEM-T Easy vector and
cloned. Purified plasmids containing the desired fragments
were serially diluted to evaluate the efficiency and detection
limit of each PCR protocol (10 copies in each case). PCRs
were then performed on each tick DNA sample in triplicate.
PCR efficiency was assessed by serial dilution of samples from
each life stage subset. Using the software SPSS version 14.0,
the nonparametric Kruskal-Wallis H test and the Mann-Whit-
ney U test were used to compare genome copy numbers for
each life stage; P values of ?0.05 were considered to be sig-
nificant. A total of 156 I. ricinus samples from 12 different life
stages were examined. Threshold cycle values were found to be
highly reproducible for all three protocols, with mean intra-
and interassay coefficients of variation always less than 2% and
Estimates of the total number of symbiont, nuclear, and
mitochondrial genome copies were obtained via a comparison
of the qPCR results of each tick life stage with those of serial
dilutions of cloned fragments (containing known copy num-
bers). Although we did not determine how many genome cop-
ies each “Ca. Midichloria mitochondrii” cell contains, other
members of the Rickettsiales are known to have a single ge-
nome per cell (10) and a single copy of the gyrB gene (2). Thus,
the gyrB copy number can be assumed to be approximately
equivalent to bacterial numbers or at least directly correlated
with them. Since mitochondria can contain a variable number
of genome copies (3a), we can consider the COII gene copy
number as an approximation of the number of mitochondria
present in tick samples.
Life stages and qPCR results for each gene are shown in
Table 1. The copy numbers of both cal and COII rise during
the development of the tick from egg to adult, with notable
increases following each molt and constant numbers during
each stage. In adult females, 5 days after detachment, there
* Corresponding author. Mailing address: The Australian Museum,
6 College St., Sydney, NSW 2010, Australia. Phone: 61 2 9320 6346.
Fax: 61 2 9320 6486. E-mail: email@example.com.
?Published ahead of print on 8 August 2008.
is a drop in the copy number of cal. This is likely a result of
extensive apoptosis in the salivary glands and other tissues
(6, 7). Adult females have significantly higher COII copy
numbers than males, presumably due to the energy require-
ments of oogenesis. The gyrB copy number is relatively high
in eggs and drops markedly in newly hatched larvae. High
concentration in the eggs is typical for vertically inherited
symbionts (4) and may reflect competition among symbionts
for transmission to progeny (14). The drop in the copy
number of gyrB from the egg stage to the larval stage is
presumably due to bacteria being excluded from most tis-
sues of the embryo during development. The gyrB copy
number then increases following engorgement of larvae.
Following molting to the nymph stage, the gyrB copy number
drops again but increases again following engorgement of
nymphs. Following the molt to the adult female, the gyrB
copy number increases. This is probably due to the fact that
the ovaries, the primary niche of the symbiont, are fully
FIG. 1. The gyrB/cal (A) and COII/cal (B) ratios in the various I.
ricinus life stages, determined by qPCR. Abbreviations: Ed1, eggs day
1; Ed15, eggs day 15; Ld1, larvae day 1; Leng, engorged larvae; Nd1,
nymphs day 1; Neng, engorged nymphs; Fne, nonengorged females;
Fpe, partially engorged females; Fd5ad, engorged females 5 days after
detachment; Fd10ad, females 10 days after detachment; Fadep, fe-
males after egg deposition; M, males positive for “Candidatus Midi-
chloria mitochondrii.” The boxes represent the 25th and 75th percen-
tiles of the values, with the line inside the boxes marking the median.
The whiskers indicate the 10th and 90th percentiles. Outlying points
are represented by ● or ?.
TABLE 1. Median values and ranges of gyrB, cal, and COII copy numbers and of gyrB/cal and COII/cal ratiosa
Stage (no. of
gyrB copy no.
cal copy no.
COII copy no.
Eggs, day 1 (17)
2.0 ? 106
7.0 ? 105–4.2 ? 106
5.4 ? 102
9.3 ? 101–7.0 ? 103
6.4 ? 106
1.2 ? 105–2.4 ? 107
3.4 ? 103
1.4 ? 102–2.2 ? 104
8.3 ? 103
5.1 ? 102–1.3 ? 105
Eggs, day 15 (15)
2.9 ? 106
5.4 ? 105–6.7 ? 106
3.4 ? 103✽
1.8 ? 103–1.3 ? 104
2.8 ? 106✽
1.3 ? 105–6.0 ? 106
9.2 ? 102✽
4.3 ? 10–3,537.3
8.0 ? 102✽
6.0–32.1 ? 103
Larvae, day 1 (13)
2.5 ? 104✽
7.6 ? 103–6.6 ? 104
1.3 ? 105✽
1.9 ? 104–1.5 ? 105
1.9 ? 107✽
5.6 ? 106–2.9 ? 107
2.4 ? 102
6.2 ? 10–7.3 ? 102
Larvae, engorged (18)
1.8 ? 105✽
7.8 ? 104–4.3 ? 105
1.3 ? 105
2.4 ? 104–4.0 ? 105
4.2 ? 107✽
2.5 ? 107–4.5 ? 107
2.6 ? 102
1.1 ? 102–3.4 ? 102
N, day 1 (18)
1.9 ? 104✽
3.2 ? 103–1.6 ? 105
5.0 ? 105✽
3.4 ? 105–1.1 ? 106
8.2 ? 107
96.9 ? 103–24.5 ? 107
1.3 ? 102
0.2–7.1 ? 102
N, engorged (18)
2.9 ? 105✽
1.3 ? 104–1.0 ? 106
5.0 ? 105
1.7 ? 105–9.3 ? 105
6.8 ? 108✽
3.3 ? 108–1.2 ? 109
1.4 ? 103✽
9.2 ? 102–1.7 ? 103
F, unfed (13)
2.3 ? 106✽
7.2 ? 105–8.3 ? 106
3.4 ? 106✽
2.1 ? 106–6.1 ? 106
1.1 ? 109
2.2 ? 107–2.2 ? 109
2.4 ? 102✽
7.4–5.3 ? 102
F, partially engorged
7.0 ? 106
5.2 ? 105–2.5 ? 107
2.0 ? 106✽
4.6 ? 105–4.0 ? 106
2.0 ? 109
9.6 ? 107–5.2 ? 109
6.3 ? 102✽
2.1 ? 102–4.3 ? 103
F, 5 days after
3.3 ? 106
1.7 ? 106–2.4 ? 107
2.0 ? 105✽
9.3 ? 104–8.9 ? 105
10.0 ? 107✽
7.7 ? 106–2.5 ? 108
2.0 ? 10✽
2.5 ? 102
0.9–7.6 ? 102
F, 10 days after
1.4 ? 107✽
1.0 ? 106–1.1 ? 108
2.9 ? 106✽
3.1 ? 105–1.1 ? 107
9.4 ? 108✽
2.2 ? 108–2.2 ? 109
1.8 ? 102
7.8 ? 10–3.0 ? 102
F, after deposition (4)
7.9 ? 107✽
3.5 ? 107–1.5 ? 108
3.1 ? 106
7.0 ? 105–1.1 ? 107
1.6 ? 109
3.1 ? 108–5.6 ? 109
2.5 ? 10✽
1.4 ? 10–50.1
4.8 ? 102✽
4.1 ? 102–6.0 ? 102
Adult males (15)
3.2 ? 103✽
0–4.3 ? 105
1.3 ? 106
4.9 ? 105–2.1 ? 106
2.1 ? 108✽
6.5 ? 106–3.2 ? 108
1.3 ? 102
0.1–6.6 ? 102
aN, nymphs; F, females; ✽, statistically significant difference with respect to the previous stage.
VOL. 74, 2008MIDICHLORIA ENDOSYMBIONTS BLOOM AFTER HOST BLOOD MEAL6139
developed in adults. The gyrB copy number continues to
increase in females during engorgement and egg deposition.
We were not able to determine the sex of larvae and
nymphs. The available evidence indicates that the symbiont
does not cause sex ratio distortion (i.e., via male-killing, par-
thenogenesis, or feminization) (8). Based on the relatively low
variance of the gyrB copy number in larvae, it appears that the
symbiont is transferred to both male and female larvae. How-
ever, the higher variance in nymphs, combined with the large
difference in the gyrB copy numbers between adult females and
males, suggests a specialization toward females during the
nymph stage. Nine of 15 adult males were found positive for
the symbiont, but these had the lowest gyrB copy numbers of all
life stages examined.
The gyrB/cal and COII/cal ratios are shown in Fig. 1. The
gyrB/cal ratio was highest in day-old eggs and follows a
similar pattern to the gyrB copy number (Table 1). One
exception is the large jump in females 5 days postdetach-
ment, which is due to the large drop in the cal copy number
at this stage (see above). The COII/cal ratio is highest (104)
in day-old eggs and then drops to a stable level of 102to 103
in other stages. The fact that both the gyrB/cal and COII/cal
ratios are highest in day-old eggs is interesting, given the
tendency of “Ca. Midichloria mitochondrii” to invade mito-
chondria. The behavior of the symbiont in eggs has not yet
In conclusion, the increase in the gyrB copy numbers follow-
ing engorgement of each of the three stages indicates that
bacterial growth is linked to the blood meal. Whether this
coincides with the production of metabolites by “Ca. Midichlo-
ria mitochondrii” for its host remains to be determined. The
growth may also reflect competition among symbionts for
transmission to the next stage of the tick, although the increase
appears to occur in both female and male larvae.
We are grateful to Tiziana Beninati for helpful discussion.
N.L. is supported by an Australian Research Council Postdoctoral
Fellowship. C.B. is supported by the Ministero della Salute and
ISPESL grant RFPS-2006-4-336506.P3.
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6140SASSERA ET AL.APPL. ENVIRON. MICROBIOL.