BioControl 47: 427–433, 2002.

© 2002 Kluwer Academic Publishers. Printed in the Netherlands.

Infectivity of beetle spiroplasmas for new host species

M. KLEIN1, Y. BRAVERMAN2, A. CHIZOV-GINZBURG2,

A. GOL’BERG1, D. BLUMBERG1, Y. KHANBEGYAN1 and
K.J. HACKETT3,∗
1 Department of Entomology, ARO, The Volcani Center, Bet Dagan 50250, Israel
2 Department of Entomology, Kimron Veterinary Institute, P.O.B. 12, Bet Dagan 50250, Israel
3 U.S. Department of Agriculture, Agricultural Research Service, Insect Biocontrol
Laboratory, Beltsville, Maryland 20705, USA
∗ Author for correspondence; e-mail: kjh@ars.usda.gov

Received 11 January 2000; accepted in revised form 24 July 2001

Abstract. Five beetle spiroplasmas, the Colorado potato beetle spiroplasma (CPBS, strain
LD-1), the Cantharis carolinus spiroplasma (CCBS, strain CC-1), the Ellychnia corrusca
firefly spiroplasma (FS, strain EC-1), the Diabrotica undecimpunctata corn rootworm spiro-
plasma (CRS, strain DU-1), and the Spiroplasma floricola fall flower spiroplasma (FFS), all
associated with beetles, were fed to beetles (Maladera matrida and Carpophilus humeralis)
and mosquitoes (Aedes aegypti and Culex pipiens). CPBS and CCBS were also injected into M.
matrida. Attempts to recover spiroplasmas from regurgitates and hemolymph were conducted
1–10 days after their introduction. After day 1, orally administered spiroplasmas could not
be recovered from M. matrida beetles; however, at 2–5 days, four out of five spiroplasmas
were recovered from adult C. humeralis. Injected spiroplasmas survived in the hemolymph of
M. matrida beetles for a relatively long period (at least 22 days). All five spiroplasmas were
recovered from mosquitoes 1 day post feeding, but only two (CCBS and CRS) survived for
five or more days. The results show short and variable persistence in orally challenged non-
host insects, with general failure to pass the gut barrier. Such evidence should be considered
when attempting to use these microbes in biocontrol programs.

Key words: Aedes Aegypti, biological control, Caprophilus humeralis, Culex pipiens,
Maladera matrida, spiroplasma

Introduction

Mollicutes (cell-wall-less bacteria), including spiroplasmas (helical, motile

mollicutes), are often isolated from insects, where they sometimes cause
pathology (Hackett and Clark, 1989; Tully, 1989). Natural infection rates can
be 100% in the gut or hemocoel, although rates of 30–80% are more typical,
with 50% being frequent, 10–20% common (Hackett et al., 1990), and <1%
not observed (K. Hackett, unpubl. data). To increase the biocontrol potential
of non-pathogenic spiroplasmas, the introduction of lethal genes has been
proposed (Hackett et al., 1992).
428 M. KLEIN ET AL.

The scarabaeid beetle, Maladera matrida is known to occur at very high

densities during summer in Israel and has become a pest in agriculture in the
central Coastal Plain and in the northwestern Negev Desert (Yathom et al.,
1994). Large numbers of nitidulid beetles, Carpophilus spp., also attack crops
in Israel. Mosquitoes are also pests, and biocontrol measures have proved to
be effective, but are still not commercial in Israel.
The current study was undertaken to determine whether beetle-associated
spiroplasmas might have biocontrol potential for beetle and mosquito pests
found in Israel which have not been reported to be hosts. Determination of the
fate of spiroplasmas fed to insects not known to be hosts would also provide
information that could be used to predict non-target effects.

Materials and methods

Test insects
M. matrida (Coleoptera: Scarabaeidae) is bred routinely in our laboratory
(Gol’berg et al., 1991). Adults were fed on a semi-artificial, semi-solid
medium based on dry beans and dry wheat germ. Grubs were fed on natural
food consisting of wheat roots grown in sterile soil. The insects were main-
tained at 25±2 ◦ C, 50–60% relative humidity, with a 10-h photoperiod.
Adults from this laboratory culture, as well as some occasionally collected
from rose flowers, were used in the experiments. During the experiments,
adults were separated into groups of 20–30 individuals, caged in plastic
containers (11 cm diameter, 7.5 cm height), and fed fresh rose flowers.
Carpophilus (Urophorus) humeralis (Coleoptera: Nitidulidae) beetles were
supplied from a laboratory culture maintained on semi-artificial Drosophila
medium (Blumberg et al., 1985). Prior to the test, adults were transferred to
containers (100 or more beetles per container) similar to those used for M.
matrida. New medium was added every 2–3 days. The females laid eggs in
the medium, and larvae completed their development there. Larvae and adults
were used in the experiments. Two species of mosquitoes, Aedes aegypti and
Culex pipiens (Diptera: Culicidae), were obtained from standard cultures at
Kimron Veterinary Institute, Bet Dagan and were bred according to standard
procedures (Braverman and Shlosberg, 1988). Adults were maintained on
10% sucrose. Females were fed on mice when needed.

Spiroplasma stocks
Spiroplasmas used in the experiments were: i) the Colorado potato beetle
spiroplasma (CPBS, strain LD-1), ii) the Cantharis carolinus beetle spiro-
plasma (CCBS, strain CC-1), iii) the Ellychnia corrusca firefly spiroplasma
 SPIROPLASMA HOSTS 429

(FS, strain EC-1), iv) the Diabrotica undecimpunctata corn rootworm spiro-
plasma (CRS, strain DU-1), and v) the fall flower spiroplasma (FFS,
Spiroplasma floricola). The first four spiroplasmas were obtained from the
collection of K. Hackett at Beltsville, MD, USA. They were grown for more
than 10 passages in MID medium and were shipped as lyophilized cultures.
The fifth spiroplasma was supplied by S. Rottem of the Hebrew University
of Jerusalem, Israel, who grew it for more than 30 passages in SP-4 medium
(Tully et al., 1977).
Prior to their use in infectivity experiments, the spiroplasmas were
cultured in SP-4 medium.

Bioassays

Using established procedures (Klein and Purcell, 1987), insects were chal-
lenged orally and by injection. For treatment of M. matrida adults, detached
petals of the rose flower were immersed in 24-h cultures of the spiroplasmas.
The petals were then transferred into small Petri dishes (5.5 cm diameter),
each containing 20–30 adult beetles, which were allowed to feed on the
petals for 18 h at a temperature of 25±2 ◦ C. Afterwards, the beetles were
fed every 1–2 days with five untreated petals. M. matrida was also injected
intrahemocoelically (20 individuals each time) with 1 to 2-day-old CPBS and
CCBS cultures. Isolations were also attempted from over 2,000 M. matrida
adults and larvae collected from fields in Israel, at various times throughout
the year.
For treatment of C. humeralis adults and larvae, 4 g of artificial medium
was homogenized with 2.5 ml of spiroplasma culture. This mixture was
offered fresh in Petri dishes (as above) to groups of approximately 100–200
adult beetles or larvae, which were allowed feed for 18 h.
For mosquito trials, spiroplasma cultures were mixed (1:1 v:v) with fresh
cow blood and fed to adults through chick-skin membranes. The FS was also
fed to mosquitoes by providing cotton rolls soaked with undiluted culture.
Treatments were for 18 h at 32 ◦ C.
For all bioassays (feeding or injection), uninoculated media was used as a
control (n = 50 for each treatment); there were no infections in the controls.
After treatment, beetles were returned to fresh diet and mosquitoes to sucrose
(10%).

Recovery of spiroplasmas from test insects

Recovery of the spiroplasmas was carried out on externally disinfected insects

(2-min immersion in 70% ethanol followed by two rinses in sterile distilled
water) after intervals of 1–10 days following treatment. From M. matrida
430 M. KLEIN ET AL.

beetle adults, hemolymph was collected in sterile glass needles after piercing
the insect membrane between head and thorax. Regurgitates were collected
from the beetle’s mouth parts by slightly pressing on its body. Hemolymph
and regurgitates were sampled separately from 10 insects and inoculated into
spiroplasma growth medium. Samples were inoculated into media containing
a standard mixture of antibiotics (the antibiotics and concentrations are
detailed in Chastel and Humphrey-Smith, 1991) and incubated at 32 ◦ C for
30 days. In addition to hemolymph and regurgitate samples, two whole adults
(after removal of their wings) or two adult intestines were macerated in 5 ml
medium. This was followed by clarification by 5 min centrifugation at 2,500
g and filtration through a 0.45-µl membrane. The filtrates were cultured as
above.
Since Carpophilus and mosquito adults are fairly small, their hemolymph
and regurgitates were not sampled. Instead, samples of five beetles were
crushed in SP-4 medium with no subsequent centrifugation or filtering.
The presence of spiroplasmas (positive results) was determined by acidi-
fication of media (as indicated by a red to orange color change in the phenol
red indicator) and by dark-field microscopic examination of cultures for
motile spiroplasma cells.

Results

Of over 2,000 M. matrida adults and larvae sampled during a 2-year period
covering all seasons in Israel, none were found to harbor cultivable spiro-
plasmas. For orally challenged M. matrida beetles, only three spiroplasmas
(CPBS, CCBS, and CRS) were detected in regurgitates, and then only at day
one, while for C. humeralis four out of five spiroplasmas were recovered 1–5
days after being fed (Table 1). Spiroplasmas were not present in hemolymph,
and isolation was not enhanced by the use of whole insects or dissected
intestines. There were no differences in results between treated laboratory and
field-collected beetles. We assume that spiroplasmas in the gut of inoculated
insects were present only as Type A contaminants (sensu T.B. Clark, 1984),
i.e., as transient infections.
In the case of intrahemocoelically inoculated M. matrida adults, spiro-
plasmas were not recovered from regurgitates, and were only rarely (five
times) recovered from hemolymph at an early stage (less than 5 days) after
administration. Two inoculated beetles did have hemocoel infections that
persisted for 22 days (the longest period sampled).
In the case of orally challenged C. humeralis beetles, recovery was more
successful: CPBS was detected in all batches of adult beetles at 5 days, but
 SPIROPLASMA HOSTS 431
Table 1. Recovery of spiroplasmas provided in diet of Maladera matrida and Carpophilus
humeralis

Spiroplasma tested Maladera matrida Carpophilus humeralis

No. of tests Days after feeding No. of tests Days after feeding
(no. of insects) 1 2 3 4 5 10 (no. of insects) 1 2 3 4 5 10

Colorado potato beetle 6 (250) +a − − − − − 2 (110) + + + + + −

spiroplasma (CPBS)
Cantharis carolinus beetle 1 (90) + − − − − − 1 (90) + + + − − −
spiroplasma (CCBS)
Ellychnia corrusca beetle 2 (200) − − − − − − 2 (200) − − − − − −
spiroplasma (FS) − − − − − − 2b (120) − − − − − −
Diabrotica undecimpunctata 1 (90) + − − − − − 1 (120) + + − − − −
beetle spiroplasma (CRS) 1b (120) + − − − − −
Spiroplasma floricola (FFS) 3 (150) − − − − − − 2 (200) + + + − − −

−: Spiroplasma not detected

+: Spiroplasma detected (generally in all batches)
a CPBS was isolated from only one batch of M. matrida beetles.
b Tests on larvae of C. humeralis beetles.

Table 2. Recovery of spiroplasmas provided in diet of Aedes aegypti and Culex pipiens
mosquito adults

Spiroplasma tested Aedes aegypti Culex pipiens

No. of tests Days after feeding No. of tests Days after feeding
(no. of insects) 1 2 3 4 5 10 (no. of insects) 1 2 3 4 5 10

Colorado potato beetle 3 (110) + + + − − − 2 (100) + + − − − −

spiroplasma (CPBS)
Cantharis carolinus beetle 3 (300) + + + − − − 3 (300) + + + + + −
spiroplasma (CCBS)
Ellychnia corrusca beetle 3 (300) + − − − − − 3 (300) + − − − − −
spiroplasma (FS)
Diabrotica undecimpunctata 3 (300) + + + + + − 3 (300) + + + + + +
beetle spiroplasma (CRS)
Spiroplasma floricola (FFS) 3 (150) + + − − − − 2 (100) + + + − − −

−: Spiroplasma not detected

+: Spiroplasma detected generally in all batches

not 10 days after feeding; CCBS and FFS were detected until 3 days, and the
CRS spiroplasma until 2 days after feeding (Table 1). FS was again negative.
Spiroplasmas were more persistent in mosquitoes than in beetles. From A.
aegypti and C. pipiens, CRS was recovered 5 and 10 days (but not 15 days)
after feeding, respectively, while CPBS, CCBS and FFS could be recovered
after 2–5 days and the FS spiroplasma only on day one (Table 2).
432 M. KLEIN ET AL.

Discussion

Typically, spiroplasmas infect and multiply in insect guts; some spiroplasmas

penetrate the gut barrier to the hemocoel, multiply in hemolymph and tissues,
infiltrate the salivary glands, and are transmitted through the saliva (Hackett
and Clark, 1989). In M. matrida beetles, spiroplasmas did not persist in the
gut, yet did persist for up to 22 days in the hemocoels of inoculated beetles.
Spiroplasmas did not cross from the gut into the hemocoel, or from the hemo-
coel of inoculated beetles into the gut or saliva (i.e., none were recovered from
the regurgitates of inoculated insects). Failure of spiroplasmas to survive in
orally treated insects may be due to: i) unsuitability of the gut environment,
or ii) inability of the spiroplasmas to cross the gut barrier.
Absence of living spiroplasmas at day 10 in orally-challenged beetles
and mosquitoes is discouraging from a biocontrol standpoint. However, if
this lack of persistence is due to spiroplasma host specificity, it could be an
advantage in situations in which the spiroplasmas are developed for use in
biological control of their natural hosts, e.g., the corn rootworm and Colorado
potato beetle.

Acknowledgements

Supported by Binational Agricultural Research and Development Grant No.

US-1902-9OR. This is a contribution from the Agricultural Research Organi-
zation, Institute of Plant Protection Bet Dagan, Israel. No. 537/999-E, 1999
series.

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