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Physiological Entomology (2006) 31, 164169

DOI: 10.1111/j.1365-3032.2006.00504.x

Oogenesis in the date stone beetle, Coccotrypes


dactyliperda, depends on symbiotic bacteria
EINAT ZCHORI-FEIN1, C H A N D R E S H B O R A D 2 and A L L Y R . H A R A R I 2
1

Department of Vegetable Crops, Newe Yaar Research Center, Agricultural Research Organization, Ramat Yishai, Israel
and 2Department of Entomology, Agricultural Research Organization, Beit Dagan, Israel
Abstract. It has been suggested that sex ratio distorting symbionts are involved in
the sex determination and female-biased sex ratios observed in strongly inbred
scolytid beetles. Coccotrypes dactyliperda (Coleoptera: Scolytinae) is a species in
which mother-son- and sib-mating occur inside the date seeds it inhabits, and the
sex ratios produced are highly skewed toward females. In the present study,
polymerase chain reaction (PCR) techniques and antibiotic treatments are applied
to determine the possible role of Bacteria in this system. PCR with primers
specifically designed to target the 16S rDNA gene in all Bacteria reveals the
presence of Wolbachia and Rickettsia in control beetles, but not in antibiotictreated individuals. Virgin females fed with antibiotics lay no eggs, and no sign of
oogenesis is detected compared with all-male progeny of virgin control females.
Mated females fed with antibiotics lay significantly fewer eggs than control
females, with a strong effect of female age at the time of antibiotic feeding on the
number of eggs laid. The study suggests that symbiotic bacteria are not involved in
female-biased sex ratios but are required for oogenesis in C. dactyliperda. The
specific role each of the bacteria (Wolbachia and Rickettsia) plays in the oogenesis
remains to be determined.
Key words. Coccotrypes dactyliperda, oogenesis, Rickettsia, Scolytinae, sym-

bionts, Wolbachia.

Introduction
It is now widely acknowledged that bacteria manipulating
the mode of reproduction play an essential role in the
biology of many arthropods. Among these symbionts are
a number of male-killing bacteria, such as Spiroplasma and
Rickettsia (Hurst & Jiggins, 2000), and two other bacteria,
Wolbachia and Cardinium, which induce a number of
reproduction-associated phenotypes (Stouthamer et al.,
1999; Zchori-Fein et al., 2004). Wolbachia, an alphasubdivision proteobacterium, has been the most extensively
studied genus among bacteria that take control of host
reproduction to increase their own numbers. This bacterium has been implicated in all types of reproductive manipulations discovered to date, including cytoplasmic
incompatibility (in which uninfected females that mate

Correspondence: Ally R. Harari, Department of Entomology,


Agricultural Research Organization, Beit Dagan, 50250, Israel.
Tel.: 972 3968 3910; fax: 972 3968 3445; e-mail: ally@int.gov.il

164

with infected males fail to reproduce), male killing, feminization (in which genetic males develop as females) and
thelytokous parthenogenesis induction (ONeill et al.,
1997; Werren, 1997; Stouthamer et al., 1999).
Recently, the symbiotic proteobacterium Wolbachia
was found in the coffee berry borer, Hypothenemus hampei
(Coleoptera: Curculionidae: Scolytinae) (Vega et al., 2002).
This species is characterized by strong female-biased sex
ratios, continuous inbreeding inside the berry and dwarfed
males (Baker, 1999). The presence of Wolbachia in most of
the H. hampei populations tested (11 out of 15) prompted
Vega et al. (2002) to suggest that the symbiont may be
involved in the skewed sex ratios exhibited by this species.
To establish the role that Wolbachia may be playing in the
sex determination of members of the Scolytinae, Vega et al.
(2002) suggested comparing the occurrence of Wolbachia in
other inbreeding and outbreeding members of this family.
The date stone beetle Coccotrypes dactyliperda F.
(Curculionidae: Scolytinae) is a strongly inbreeding species.
It is widespread in warm temperate regions, where it feeds
on either green dates (Kehat et al., 1976) or date seeds,

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2006 The Royal Entomological Society

Beetle oogenesis depends on symbiotic bacteria


which are also used as their breeding habitat (Hussein,
1990; Siverio & Montesdeoca, 1990). The newly emerged
female colonizes a new date stone and the entire life cycle
takes place inside that seed (Blumberg & Kehat, 1982). The
sex determination mechanism of C. dactyliperda is haplodiploidy, where the haploid dwarf males are produced from
unfertilized eggs and the diploid females result from sexual
reproduction. The offspring sex ratio of mated females is
strongly female-biased and inbreeding is the norm (Herfs,
1959).
The present study investigates whether C. dactyliperda
beetles serve as hosts to bacterial symbionts, to test
the hypothesis that symbionts are involved in the beetles
sex determination and to test the symbionts other effects
on the beetles. Polymerase chain reaction (PCR) is used
to detect the presence of Wolbachia and/or other
bacteria and antibiotic treatments are used to study the
influence of the symbionts on the reproduction of their
beetle host.

Materials and methods


Rearing Coccotrypes dactyliperda
Date seeds inhabited by C. dactyliperda were collected in
a date plantation in Ein-Gev, in the eastern part of the Sea
of Galilee, Northern Israel, in January, 2003. Voucher
specimens were deposited with Lawrence R. Kirkendall
(University of Bergen, Norway), who confirmed the identification using both morphology and DNA sequencing.
More than 200 seeds were cut open and pupae, as well as
adults, were collected and released on fresh date seeds
placed in plastic bowls (10  5 cm) covered with a perforated plastic lid and kept at 28  1  C and an average
relative humidity of 70  2%. One month later, the seeds
were cut open, young mated females were collected daily
and transferred into small Petri dishes with sliced date seeds
(< 2 mm thick) as food. To obtain virgin females, pupae
were collected from cut seeds and kept individually in small
tubes. Upon emergence, the beetles were transferred into a
small Petri dish with sliced date seeds as above. The beetles
do not oviposit on sliced-dates, but can be maintained on
this diet for long periods of time. Because preliminary
observations suggested that the beetles prefer moist seeds
both as food and oviposition substrate, all date seeds were
soaked in water for 5 days before they were presented to
the beetles.

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1992). Reactions contained 5 mL of the template DNA


lysate, 15 pmol of each primer, 1.5 mM dNTP and
0.5 units of REDTaq (Sigma, St Louis, Missouri) with a
final MgCl concentration of 1.5 mM in a total volume of
25 mL. PCR parameters were denaturation for 2 min at
95  C, followed by 30 cycles of 30 s at 92  C, 30 s at
57  C, 30 s at 72  C, and a 5-min final extension at 72  C.
Every reaction set included sterile water as a negative
control and Aphytis sp., which has been reported to carry
Wolbachia (Zchori-Fein & Perlman, 2004), as a positive
control. A primer set specifically designed to amplify a
gene encoding for the Wolbachia surface protein (wsp)
was used to obtain a better characterization of that
symbiont. The PCR was performed on two randomly
chosen females as described above, with primers and
conditions as previously reported (Zhou et al., 1998),
which yield an expected product of approximately 600 bp.
The DNA produced by the PCR was extracted from the gel
and cloned into Escherichia coli using pGEM-T Easy
plasmid (Promega, Madison, Wisconsin) according to the
manufacturers protocol. Clones were sequenced using an
ABI 3700 DNA analyser (Macrogen Inc., Korea), and the
sequence obtained was compared with sequences deposited
in GenBank.
Detection of other symbionts To determine whether
the Wolbachia found in C. dactyliperda is the causative
agent of the observed female-biased sex ratio, the 16S
rDNA gene fragment (approximately 1500 bp) was
amplified by PCR using primers and conditions
known to amplify this gene from all known Bacteria
(Weisburg et al., 1991). Reactions contained 5 mL of
the template DNA lysate, 15 pmol of each primer,
1.5 m M dNTP and 0.5 units of REDTaq (Sigma) with
a final MgCl concentration of 1.5 mM in a total
volume of 25 mL. The PCR products were cloned,
sequenced and analysed as described above.
The database search revealed the presence of an additional symbiont, other than Wolbachia, which showed
98% identity to various Rickettsia species. Based on this
rickettsial 16S rDNA gene sequence and similar sequences
found in the databases, primers were designed that are
capable of specifically amplifying this gene from the genus
Rickettsia (forward- Rb-F 5 -GCTCAGAACGAACGCT
ATC-3 and reverse- Rb-R 5 -GAAGGAAAGCATCTCT
GC-3 ). The combination of these primers was expected to
yield a product of approximately 900 bp.
Vertical transmission

Symbiont detection and identification


Wolbachia detection To test for the presence of
Wolbachia, the abdomens of 20 adult C. dactyliperda
from the laboratory colony were ground individually in a
lysis buffer as described by Frohlich et al. (1999). Each
sample was subjected to a PCR using a set of Wolbachiaspecific primers for the 16S rDNA gene (ONeill et al.,

To determine vertical transmission of the symbionts via


the eggs to the offspring, three 1-day-old eggs were ground
in a lysis buffer and subjected to a PCR analysis using both
the Wolbachia- and Rickettsia-specific primers, as described
above, with an annealing temperature of 57  C. The PCR
products were visualized by ethidium bromide on an agarose gel.

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166 E. Zchori-Fein, C. Borad and A. R. Harari


Oviposition pattern
To be able to assess the influence of antibiotic treatments
on C. dactyliperda reproduction, it was essential to determine the baseline reproductive potential of this species
under our experimental conditions. Mated females were
placed individually in a 50  15 mm Petri dish and offered
an intact date seed (n 170). During the next 17 days,
seeds were cut open, females were removed and the number
of oviposited eggs was counted (n 10 seeds each day).

soaked in either 3% tetracycline solution or sterile distilled water as described above were offered as food for 7
consecutive days to 1, 7 or 21-day-old mated females (10
females in each treatment). After the treatment, the food
was removed and the females were placed individually in
a 50  15 mm Petri dish and offered an intact,
untreated, date seed as an oviposition substrate. Ten
days later, the seeds were cut open and the number of
eggs of each female was counted and compared amongst
age groups and treatments using two-way factorial
ANOVA.

The effect of antibiotics on female survival


To determine the antibiotic concentration that affects the
target symbionts, but not the insect longevity, various concentrations were tested in a preliminary experiment. Date
seeds were chopped to pieces of approximately 60 mg each
(< 2 mm width), soaked in 25 mL of water (control) or 1, 3
and 5% tetracycline (Sigma) solution in water and placed
on a mechanical shaker for 24 h to ensure even absorption.
The chopped seeds were then air dried for 12 h, and offered
as food for 2-day-old mated females for 4 days. Groups of
10 females were then placed in a Petri dish (50  15 mm),
together with an intact water-soaked date seed to feed on
and oviposit in (n 10 for each treatment). After 30 days,
seeds were cut open and the number of live beetles was
recorded. The data were analysed using one-way analysis of
variance (ANOVA) followed by Tukey post hoc test.

The effect of antibiotics on female oviposition over time


Subsequent to the results of the preliminary experiment
above, date seeds, chopped to pieces as above, were soaked
in 25 mL of 3% tetracycline (Sigma) solution in water and
placed on a mechanical shaker for 24 h. After air drying for
12 h, the chopped seeds were offered as food for 2-day-old
mated females for 4 days (n 80). The females were then
placed individually in a Petri dish (50  15 mm), with half
of the females offered an intact, water-soaked date seed to
feed on and oviposit in, and the remaining half received an
intact, antibiotic-soaked seed treated as above. Control
females (n 40) were presented with water-soaked
chopped seeds for 4 days and then placed individually in
a Petri dish with an intact, water-soaked seed to feed on
and oviposit in. On days 1, 5, 9 and 13 after the females
penetration into the new seed, 10 females from each of the
three treatments were taken, the seeds were cut open, and
the number of eggs in each seed was counted. The data
were analysed using two-way factorial ANOVA.

The effect of female age on antibiotic treatment outcome


To test the hypothesis that symbionts are required in
the early stages of oogenesis, C. dactyliperda females
were treated at different ages. Chopped date seeds

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Parthenogenesis
The effect of antibiotics on eggs produced by virgin
females was also tested. One-day-old virgin females
were offered tetracycline (3%) (n 15) or water-treated
(n 20) chopped seeds, as described above. The food was
removed 10 days later and a whole seed was provided to
each female in a Petri dish for an oviposition period of
2 weeks. The number of offspring in each treatment group
was counted and compared using a t-test.

Results
Symbiont detection and identification
All samples of C. dactyliperda tested with the 16S rDNA
Wolbachia-specific primers by means of PCR gave a product
of the expected size. The use of wsp primers yielded two
identical PCR products of 595 bp, which exhibit over 99%
sequence similarity to the Wolbachia found in Drosophila
melanogaster (AF020065). Using universal Bacteria primers,
a second symbiont was detected in the beetle. The 16S rDNA
sequence of this bacterium showed homology to sequences of
various Rickettsia found in GenBank. This sequence is
1454 bp long, has a 52% GC content and exhibits over
98% homology to the Ixodes tick symbiont Rickettsia bellii
(U11014). The Rickettsia sequence was deposited in
GenBank under accession number AY961085. PCR analysis
of C. dactyliperda eggs with both Wolbachia and Rickettsia
primers showed that these two bacteria are present in the eggs
produced by the control beetles, but not in the very few eggs
produced by the antibiotic-treated ones.

Oviposition pattern
Mated females started to lay eggs 3 days after inhabiting
a new seed. An average number of 3.4  0.9 eggs
(mean  SE) was oviposited daily and these eggs were
accumulated in the seed (Fig. 1). The maximum number
of mature eggs found in the ovaries of females randomly
taken from the laboratory colony was 1, with up to 45% of
the tested females showing no mature eggs.

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2006 The Royal Entomological Society, Physiological Entomology, 31, 164169

167

No. of oviposited eggs

No. of oviposited eggs

Beetle oogenesis depends on symbiotic bacteria


60
50
40
30
20
10
0
1

25
20
15
10
5
0

9 10 14 16 17

9
13
Days after treatment

Days in the date


Fig. 1. Number of eggs oviposited by mated Coccotrypes dactyliperda females fed with water-soaked date seeds (mean  SE).

17

Fig. 2. Number of eggs oviposited by mated Coccotrypes dactyliperda females fed with date seeds treated with 3% tetracycline for
4 (striped columns) or 17 days (dark columns) or water (clear
columns) (mean  SE).

The effect of antibiotics on female survival

The effect of antibiotics on female oviposition over time


The effects of both treatment and day of oviposition were
highly significant (F2.108 73.2, P < 0.001 and F3.108 14.8,
P < 0.001, respectively). However, due to the significant interaction between them (F6.108 8.9, P < 0.001), the two effects
were analysed separately; with the effect of the treatment
analysed for each day. Females fed either on water-soaked or
antibiotic-treated seeds oviposited a similar average of
approximately 56 eggs on the first day after inhabiting the
seed (F2,27 0.265, P > 0.05). However, the number of eggs
oviposited by females fed with water-treated seeds increased
over time, whereas the number of eggs oviposited by females
fed with antibiotic-treated seeds did not increase throughout
the experiment (F2,27 36.5, P < 0.001, F2,27 19.8,
P < 0.001 and F2,27 54.6, P < 0.001 for 9, 13 and
17 days, respectively) (Fig. 2). An analysis of the influence of
the treatments on the number of oviposited eggs revealed
significant effect (F2,117 415.47, P < 0.001), with more
eggs oviposited by females treated with water-soaked seeds
than by females fed antibiotic-treated seeds, and no difference
between the number of eggs oviposited during time between
the 4 and 17 days of antibiotic treatments (Tukey HSD multiple comparisons, P < 0.05) (Fig. 2).

offspring produced (ANOVA: F1,54 103.40, P < 0.001 and


F2,54 26.10, P < 0.001, respectively). Because progeny
numbers were also affected by the interaction between
these two factors (F2,54 31.75, P < 0.001), they were
analysed separately, using ANOVA followed by Tukey HSD
multiple comparisons.
Females age at oviposition had no effect on the number
of eggs laid by control females (F2,27 0.346, P > 0.05),
but a significant effect of female age was detected when
antibiotic-treated females were tested (F2,27 150.74,
P < 0.001) (Fig. 3).
The antibiotic treatment had a significant effect on the
number of eggs laid by females fed at 1-day and 7-days old
(F1,18 61.62, P < 0.001 and F1,18 98.09, P < 0.001,
respectively), but had no effect on females fed at 21-daysold (F1,18 0.001, P > 0.05). Almost no eggs were laid by
1-day-old females fed sliced date seeds treated with 3%
tetracycline; significantly more eggs were laid by 7-day-old
females fed antibiotics and still significantly more eggs
were oviposited by females fed with antibiotics when they
were 21-day-old (Tukey HSD multiple comparisons,
P < 0.05) (Fig. 3). Dissections of control females revealed
the presence of several oocytes in various maturation
stages, with no more than one mature egg ready to be
laid. By contrast, when antibiotic-treated females that had

60
a
No. of oviposited eggs

Thirty days after treatment with various antibiotics


concentrations, a significant difference in female survival was
detected (F3.36 180.1, P < 0.001). There was no difference in
numbers of surviving females after feeding on seeds treated with
either water, 1% and 3% antibiotic (mean  SE: 8.5  0.7,
7.8  0.94 and 7.6  0.67 females, respectively). However a
significant difference was detected in females feeding on seeds
treated with 5% antibiotic solution (2.0  0.74 females) (Tukey
post hoc, P < 0.05). Thus, the 3% tetracycline solution was used
for testing the effect of antibiotics on female oviposition.

40

Both antibiotic treatment and age of the female at the


onset of the experiment significantly affected the number of

3%

20
c
0
0

3%

1 day old

The effect of female age on antibiotic treatment outcome

3%

7 days old

21 days old

Fig. 3. Number of eggs oviposited by mated Coccotrypes dactyliperda females fed with date seeds treated with 3% tetracycline
(stripped columns) or water (clear columns) at different ages
(mean  SE).

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168 E. Zchori-Fein, C. Borad and A. R. Harari


not laid any eggs were dissected, no oocytes were detected
in the ovaries.
Parthenogenesis Virgin, 1-day-old females fed on date
seeds treated with 3% tetracycline solution for 10 days
did not lay a single egg throughout the 15-day laying
period, whereas virgin females at the same age which were
fed on water-treated seeds oviposited 4.25  0.52
(mean  SE) eggs (One sample t-test with a mean of 0,
t 8.13, d.f. 19, P < 0.001).

Discussion
It has been suggested that the maternally transmitted symbiotic bacterium Wolbachia affects the female-biased sex
ratio observed in the coffee berry borer and other beetles
within the subfamily Scolytinae (Vega et al., 2002). In one
such species, the arrhenotokous date stone beetle
Coccotrypes dactyliperda, the elimination of Wolbachia
was suspected to result in the production of a less femaleskewed sex ratio. However, in the present study, when both
mated and unmated C. dactyliperda females are treated
with antibiotics, they stop producing eggs (Fig. 2).
The adverse effect of antibiotic treatment on female oviposition is most evident in young females, whereas old
females are not affected at all and females of intermediate
age produce intermediate numbers of eggs (Fig. 3). It is
also found that the beetles are synovigenic, and the gradually maturing eggs are laid at an average rate of 3.4  0.9 a
day (Fig. 1). Combined with the observation that no signs
of developing oocytes are found in antibiotic-treated
females that did not lay eggs, the results suggest a positive
correlation between the presence of symbiotic bacteria and
oocyte development. If this symbiont-dependent egg
maturation hypothesis is correct, the prediction is that no
new oocytes will develop in the absence of symbionts,
whereas mature oocytes will not be affected. The results
support this prediction and show that the number of eggs
produced by older females is less affected by the antibiotic
treatment (Fig. 3).
Using PCR, the proteobacteria Wolbachia and Rickettsia
are found in whole-body extracts of untreated C. dactyliperda, but cannot be detected in antibiotic-treated females.
Although Wolbachia is generally recognized as an arthropods symbiont with a wide array of phenotypes, species of
the genus Rickettsia are usually referred to as vertebrates
pathogens vectored by arthropods (Weiss & Moulder,
1984). However, Rickettsia has been previously reported
to be associated with male-killing in the buprestid beetle
Brachys tessellates (Lawson et al. 2001), and the ladybird
beetles Adalia bipunctata and A. decempunctata (Werren
et al., 1994; von der Schulenburg et al., 2001). Rickettsia
has also been reported in insects such as the bruchid beetle
Kytorhinus sharpianus (Fukatsu & Shimada, 1999) and even
in plants (Davis et al., 1998), where its phenotype has not
been established yet. Whatever the role Rickettsia plays in

Journal compilation

these systems, the above hosts are unlikely to serve as


vectors to warm-blooded hosts.
Symbiotic bacteria associated with egg formation have
been reported in several insects. The first correlation
between egg formation and the presence of Wolbachia was
discovered in the parasitoid Asobara tabida (Hymenoptera:
Braconidae), where antibiotic treatments of Wolbachiacarrying females resulted in symbiont-free wasps that did
not produce mature oocytes (Dedeine et al., 2001).
Although no other physiological functions tested are
affected by the removal of Wolbachia, the ovaries of aposymbiotic females contain aborted egg chambers and there
is no indication of vitellogenesis. Because egg production in
A. tabida has also been shown to be strongly correlated
with Wolbachia density in the females, it was suggested that
this bacterium is essential for oogenesis. Starr & Cline
(2002) suggested a mechanism by which the symbiont can
affect oogenesis in its host. They observed that, in mutant
D. melanogaster females that were prevented from producing eggs by protein-coding lesions in Sex-lethal (Sxl) (i.e.
the master regulator of sex determination), fertility is
restored in the presence of Wolbachia. That study demonstrates the interaction between Wolbachia and a specific
host regulator protein in a way that can rescue Drosophila
oogenesis defects.
The present study shows that antibiotic-treated C. dactyliperda females do not produce mature oocytes and do not
lay eggs. It is suggested that bacterial symbionts are essential for oogenesis in this species. Both the results and the
interpretation are in agreement with data gathered from the
beetle Xyleborus ferrugineus, another member of the tribe
Scolytinae, in which an extensive study was conducted on
the association of the beetle and a complex of symbiotic
microorganisms (Peleg & Norris, 1972; Norris & Chu,
1980). One of these studies shows that surface-sterilized
eggs contain both Staphylococcus and a nonidentified rodshaped bacterium (Norris & Chu, 1980), but no molecular
analysis is performed. Antibiotic treatments of X. ferrugineus females results in lower population levels of the two
bacteria, accompanied by a significant reduction in the
percentage of ovipositing females and the number of progeny per female (Peleg & Norris, 1972). These authors
conclude that the symbiotic bacteria in X. ferrugineus are
required for oogenesis, and speculate that they take the role
of the sperm in activating the egg cell (Peleg & Norris,
1972). In light of the present findings and the electron
micrograph picture provided by Norris & Chu (1980), it is
believed that the symbiont responsible for egg formation
induction in X. ferrugineus is the rod-shaped bacteroid,
which may correspond to the Rickettsia or Wolbachia
found here, rather than the suggested Staphylococcus.
Because the two bacteria found in C. dactyliperda appear
to be transmitted from the mother to her offspring through
the egg, it is difficult to determine which is the one (or the
combination of the two) that affects the egg formation
process. Moreover, the current data do not permit the
exclusion of the possibility that other bacteria are present
in the study insect, and may also influence its phenotype.

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2006 The Royal Entomological Society, Physiological Entomology, 31, 164169

Beetle oogenesis depends on symbiotic bacteria


The study demonstrates, again, the importance of identifying the whole suit of symbionts associated with a given
species or line because, even in arthropods where
Wolbachia has been found, the possibility of double infections with an unrelated bacterium such as Rickettsia exists.
Special care must therefore be taken in assigning particular
effects on host reproduction to a particular bacterium.

Acknowledgements
The authors gratefully acknowledge Dr L. Kirkendall and
Dr F. Vega for their valuable comments on early drafts
of the manuscript. This research was partially supported
by The Israel Science Foundation (grant number 649/03 to
E. Z-F)

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Accepted 8 November 2005


First published online 27 March 2006

# 2006 The Authors


Journal compilation # 2006 The Royal Entomological Society, Physiological Entomology, 31, 164169

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