Cynthia Jensen
November 10, 2011
Dr. Hoback
Investigating the modes of transmission of Wolbachia pipientis on the the Japanese
beetle-Popillia japonica Newman
Cynthia Jensen
Abstract
Wolbachia is an endosymbiont that resides in the reproductive cells of many arthropods
and nematodes. It plays an important role in the reproductive success of these organisms,
and the mechanisms by which it accomplishes this can be determined by investigating
infection rates among males and females in a population. To evaluate the role Wolbachia
plays in the reproductive success of Japanese beetles, one hundred-eighty-two beetles
were collected from five different plant species over a period of two months. Using the
Wspec primer to detect the presence of the ribosomal DNA of Wolbachia produced an
overall infection rate of 11.7%. Overall the infection rate for males was higher than
female beetles, but in some instances it was not significant. The data suggests the
possibility that cytoplasmic inhibition might be partially responsible for the reproductive
success and polyphagous behavior of Japanese beetles, but it may only be one factor
among many.
Introduction
The Japanese beetle, Popillia japonica, is considered to be a pest with serious economic
impact. First discovered in nursery stock in New Jersey in 1916, it has spread throughout
the eastern United States with the USDA currently establishing regulations to limit its
spread beyond the Mississippi River (United States Department of Agriculture 2004). In
the Orient where the beetle is believed to have escaped in root balls, it is kept in check by
natural enemies like the Tiphia wasp (Gardner 1938; Reding 2007). However, it has no
natural enemies in the U.S. allowing it to proliferate. Since its discovery, many methods
have been devised in an attempt to control not only adult populations, but also the larval
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stages. Since the beetle feeds in almost every stage of its life cycle, there is reason to seek
effective management. Additionally, there is evidence that the beetle might also be a
contributing factor in damage to grape crops by June beetles and yeast infections
(Hammonds et al. 2009).
The forelegs of the beetle can be used to distinguish males from females. Both have tiny
hairs along leg areas helping them to adhere to their food source, but the male has sharp
spurs on his forelegs used to hold on to the female during mating. Although the female
has spurs, too, they are not as big or pointed (United States Department of Agriculture
2004). The adult female deposits “translucent white to cream” eggs that are “elliptical
and about 1/16 inch in diameter” (Frank et al. 2009) in several sessions rather than all at
once. Forty to sixty eggs will be deposited in soil that is 4-6 cm. deep (Shetlar n.d.). Soil
composition and moisture content appear to play a role in egg deposition by the female
(Allsopp et al. 1992). After hatching, beetles go through three larval stages in the soil.
These grubs progress from the first instar to the third eating their way through the roots of
grasses and turf. Like many other grubs, they are c-shaped and creamy colored, but can
be distinguished from other larvae by several rows of hairs forming a ‘V’ on their
posterior end. The head end is a yellowish brown (Frank et al. 2009). Reaching the third
instar, they are approximately 32 mm in length and have moved up through the soil from
their winter quarters below the frost line. Before emerging as adults, they will pupate and
stop feeding for a short period (Shetlar n.d.). Adults feed on a variety of plants, and
generally during the warmer times of the day (Kreuger and Potter 2001).
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Wolbachia pipientis is an endosymbionic bacteria that generally lives within the sex cells
of insects. First discovered in 1924 by Wolbach (Hertig and Wolbach 1924), and later
characterized in 1936 by Hertig (Hertig 1936), it has become one of several
endosymbionic bacteria of interest because of possible mechanisms of control that it
might offer for the reduction and control of mosquitoes carrying viruses, filarial
nematodes (Bandi et al. 2001, Lo et al. 2007), or ticks with parasites causing Lyme
disease. Wolbachia, an obligate parasite, is maternally transmitted and causes
reproductive changes in its host usually benefiting both species. It has been shown that it
promotes male-killing, feminization, cytoplasmic incompatibility (CI), and
parthenogenesis in its hosts (Stouthamer et al. 1999). While this might benefit the
bacteria and its host, increasing fecundity, there is evidence that in doing so, an infection
might lead to the arthropod’s genetic diversity (Werren et al. 2008). Recent studies show
that there may be a “tripartite association” including a bacteriophage that plays a role in
subduing the control Wolbachia has over the host when the phage is in its lytic stage
(Bordenstein et al. 2006).
Japanese beetles have proven themselves to be highly successful in their invasion of the
U.S., but it is not clear if they possess an endosymbiont like Wolbachia providing them
with assistance, or if they do, what mode of transmission is affecting the insects. Close
relatives like the Japanese adzuki bean beetle, Callosobruchus chinensis, have been
shown to carry the bacteria (Kondo 2002). However, it is possible that the Japanese
beetle is one of the phytophagous beetles that is successful not because of reproductive
assistance from an endosymbiont, but because it is capable of feeding on numerous types
of plants. There is evidence that beetles in this category are capable of producing plant
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cell wall degrading enzymes (PCWDEs) fairly rapidly, thus not limiting their food
sources (Pauchet et al. 2010).
Effective management of this unwanted pest can begin with a thorough understanding of
not only its sexual and feeding behavior, but also its molecular and symbionic
requirements.
Materials and Methods
Collection:
Male and female Japanese beetles were collected from raspberry leaves, ferns, roses,
Japanese knotweed, and purple loosestrife. To eliminate error in sexing and reduce time,
only mating beetles were chosen. Beetles were immediately dropped into test tubes with
95% ethyl alcohol. Collection started during the third week of July and continued until
the last week in August to account for their entire adult life cycle. The collections were
divided up into three “emergences,” each lasting approximately two weeks.
DNA Extraction:
Since there were so many samples collected at different times, from different places, as
well as separating males and females, a numbering and labeling system had to be devised.
For most plants there were ten female and ten male beetles representing the population
found there at that particular time. Each beetle was blotted with Kimwipes to remove
ethanol, followed by excision of the reproductive organs in the lower portion of the
abdomen.
Using a Qiagen Dneasy Tissue Culture Kit (#69504), the reproductive
organs were macerated in 180 microliters (l) of ATL buffer using microtube pestles or
pipet tips. Proteinase K (20l) was added immediately following this step to reduce
destruction of DNA. After adding 200 l of AL lysis buffer, the entire eppendorf tube
was vortexed and then incubated at 70° C for 10 minutes. The addition of 200 l of
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ethanol precipitated the DNA. To remove the cellular debris, the liquid was pipeted into a
labeled spin column and centrifuged for 1 minute at 8,000 rpm. The flow through liquid
waste was discarded and the collection tube placed back under the filter. Next 500 l of
AW1 wash buffer was added to the spin column followed by centrifugation at 8,000 rpm
for 1 minute. The flow through liquid waste was discarded. After the addition of 500 l
of AW2 wash buffer, the mixture was centrifuged at 13,000 rpm for 3 minutes and the
waste discarded. This time the collection tube was discarded and a fresh collection tube
added. Adding 100l AE buffer rinsed and eluted the DNA into the new collection tube
after centrifuging for 1 minute at 8,000 rpm. The spin column containing the filter was
discarded and the labeled collection tube saved and stored at 4° C until PCR was
performed. DNA was extracted from positive and negative controls as well. These
controls consisted of infected and uninfected Nasonia sp. obtained from Marine
Biological Laboratory (MBL), Woods Hole, MA.
PCR:
PCR was performed using PCR Ready tubes (GE#27-9557-01). To each labeled tube
containing the master mix pellet, 15 l of sterile distilled water was added followed by 2
l of primers of W-spec forward, W-spec reverse, CO1 forward, CO1 reverse and 2 l of
the DNA template. The CO1 primers flank the gene for cytochrome oxidase in the insect.
Wspec primers identify ribosomal DNA in Wolbachia. PCR samples were run as follows:
1 cycle of 1 minute at 94° C: 38 cycles of 1 minute at 94° C, 1.5 minutes at 45° C, and 1
minute at 72° C: 1 cycle of 5 minutes at 72° C.
Electrophoresis:
PCR products were run on 2% gels with 2 l of GelRed stain. For each sample, 2 l of
Orange G loading dye was added to 10 l of the PCR product. The samples were pipeted
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into wells along with positive and negative controls and a DNA ladder. Gels ran at
approximately 100 volts for about 30-40 minutes. Gels were then viewed on a
transilluminator.
Results:
After viewing the first gels, it was discovered that there was a problem with the Nasonia
controls. The positive controls (red arrow) were showing negative results and the
negative controls (black arrow) were positive (Fig. 1). Additionally the positive
Wolbachia DNA (blue arrow) was not apparent or very light on some of the gels making
it difficult to confirm Wolbachia in any of the samples (Figs. 2 & 3). Products that
appeared positive were then rerun with the Wspec primers only. Since it was discovered
that similar problems had occurred with other researchers (Kang and Dempsey 2011),
new controls were ordered from Marine Biological Laboratories and PCR was again
performed on 27 selected products. Problems with the controls continued with the new
supply and none of the selected samples showed any bands. However, there were
suspicious heavy bands near the top of the wells and smears that ran from the wells to the
trimer dimers in some lanes. These matched the positive control (red arrow) (Fig. 4).
After speaking with Michele Bahr at MBL, it was determined that there might be
problems with the thermal cycler rather than the primers, so the samples were rerun at
MBL. In the first run of 20 samples, there was one clear positive (blue arrow) and seven
possible positives (Fig. 5). Five of these were male beetles and two were female. The
next gel also showed several positives. And again, these were from male beetles. Two
more gels run at MBL also showed positive results (Figs. 6 & 7). DNA from positives
and suspected positives will now be sequenced to confirm results.
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Statistically the number of males verses females in each of the three emergences (Fig. 8)
is not significant and data might be attributed to random sampling variability, however,
scientifically the fact that there are more males than females in the first two emergence
samplings might suggest that further study is warranted. It is also curious that in
emergence three the number of positives is greater than in emergence one or two, and
females and males are almost equal in number. It was noted that many of the beetles
(both male and female) in this emergence appeared smaller than the first two emergences.
Although they were not measured and sizes compared, this too, suggests further
investigation.
The number of males verses females feeding on raspberries and knotweed shows
statistical significance at the 0.1 confidence level with a P value of 0.05935 and 0.053
respectively. Interestingly, beetles were found on raspberry leaves during all three
emergences, and the other plants were host to the beetles for only one or two. Loosestrife
and roses are not statistically significant, but the sampling values are low (Fig. 9). These
values also suggest further study.
Discussion
Wolbachia belongs to the Class alpha-proteobacteria. Proteobacteria encompass a large
group of bacteria that includes pathogenic members from the Rickettsia and Ehrlichia
genera. The ancestors of this group were some of the earliest life forms branching off to
fill numerous niches from photoautotrophs to chemotrophs, living in marine and
terrestrial environments. Many evolved to form symbionic relationships and numerous
studies evaluating mtDNA (Gray et al. 1999, Markov and Zakharov 2006), protein
sequences and molecular clocks indicate that through some of these relationships
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mitochondria appeared as an energy producer for eukaryotic cells approximately 23001800Ma (Hedges et al. 2004).
Wolbachia is an obligate endosymbiont taking up residence in the reproductive cells of
approximately 20-30% of all arthropods worldwide (Bordenstein et al. 2005). Although it
is not the only bacteria infecting insects, it is the most prevalent. The effect that
Wolbachia and other maternally inherited bacteria, like Cardinium, have had on their
insect hosts can be important when considering the biology of both organisms (Duron et
al. 2008). Some endosymbionts like Buchnera transmitted vertically in aphids are not
harmful, but instead provide service, whereas Wolbachia can sometimes have a negative
effect on its host (Herr et al. 1999). Wolbachia is also found in some nematodes and is of
interest due to their ability to cause diseases in humans and animals (Fenn et al. 2007).
Additionally, these filarial worms from the family, Onchocercidae, suggest an
evolutionary relationship with insects since they use insects as vectors to further their life
cycle.
Wolbachia’s relationship with its host can cause several different reproductive scenarios.
Perhaps one of the most intriguing is cytoplasmic incompatibility (CI), which is the
mitotic disruption of fertilized eggs such that they fail to mature. It is caused when an
infected male mates with an uninfected female and is believed to be the original
reproductive scenario used by the common ancestor (Stouthamer et al. 1999). Other
reproductive changes include feminization of males, parthenogenesis, and the killing of
male offspring (Charlat et al. 2007, Stouthamer et al. 1999). The evolutionary pathway of
both the host and the symbiont can be altered based on the type of infection and the type
of infection can be different in each insect species (Rokas 2000).
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Wolbachia is generally vertically transmitted, but there is current research that suggests
horizontal or lateral transfer is also occurring (Hotopp et al. 2007). Some horizontal
transfer has been attributed to phage vectors (Bordenstein et al. 2007), and recently, it has
been suggested that environmental factors might favor transmission among different
species that are geographically isolated, frequent similar locations (Stahlhut et al. 2010 ),
or rely on the same food sources (Lo et al. 2001).
It has been suggested that although Wolbachia is part of the same clade of obligate
endosymbionts as Rickettsia and Ehrlichia, it has evolved in a different manner (Gupta
and Mok 2007). While other members of this group also infect arthropods, they only use
them as intermediate hosts on the way to infection of vertebrates. Anderson and Karr
(2001) maintain that “the manipulation of host reproduction is an evolutionary novelty
acquired by the Wolbachia lineage, which meanwhile has lost the ability, manifest in
Ehrlichia and Rickettsia species, to infect vertebrate, and in particular, mammalian
hosts.” When Wolbachia separated from its sister bacteria, it has been suggested that it
first took up residence with filarial nematodes and other primitive worm-like
invertebrates (Lo et al. 2002). Subsequently, it went on to infect arthropods and different
forms evolved called supergroups. Using the more conserved gene sequence for ftsZ
(Baldo et al. 2002), instead of 16S rDNA, it is estimated that there are six supergroups
(Lo et al. 2002), although there are other estimates of eight or more (Casiraghi et al.
2007). However, some researchers suggest that the number of supergroups may be
inaccurate due to the reliability of the primers used and the possibility of recombination
occurring among different variants ( Jiggins et al. 2001). Supergroups A and B are
generally associated with arthropods and are often distinguished from each other using
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the wsp gene (Lo et al. 2002), which codes for a surface protein. This gene has been
shown to display considerable variation among arthropods, but little change among
nematodes (Baldo et al. 2002). Supergroups C and D are associated with nematode
phylogeny. Groups E and F are associated with termites and springtails, members of
Collembola. Group F is considered significant since it infects both arthropods and
nematodes and could, with increased research, provide clues about the direction of
bacterial transfer and approximately how long ago it occurred (Casiraghi et al. 2007).
Interestingly, hosts of supergroups C and D have evolved to the extent that they are
dependent on each other and if the Wolbachia is eliminated using antibiotics such as
tetracycline (Grenier et al. 2002), the host dies. This is not the case with supergroups A
and B (Lo et al. 2001) and in a study using microarray-based comparative genome
hybridization (mCGH) comparing Wolbachia in different species of Drosophila,
researchers found diverse strains indicating recent lateral transfer (Ishmael et al. 2009).
This suggests that perhaps Wolbachia infected nematodes first, and over time they have
become mutualists due to lateral gene transfer. In fact, bacterial genes have been found
within the filarial host genomes, although they are degenerate and have no function
(Hotopp et al. 2007). Curiously, groups A and B have larger genomes than supergroups C
and D (Lo et al. 2001). Although more research is necessary to investigate the genomes
of the other supergroups in greater depth, this, too, might mean that the infections are
more recent. Over time through lateral gene transfer to the host genome some DNA
might be lost or through multiple infections the bacterium might have transferred
additional DNA. Surprisingly, it was found that Wolbachia genes of the reproductive
cells were laterally transferred into the genome of C. chinensis, a bean beetle (Nikoh et
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al. 2008). Additionally, in a study involving heteroptera (true bugs) known to harbor
endosymbionts in their gut regions, several subspecies of Wolbachia were found
suggesting lateral transfer into cells there (Kikuchi and Fukatsu 2003). Previously, lateral
gene transfer was considered rare in multicellular eukaryotes, however, recent studies are
showing that it is a more common occurrence than previously thought (Fenn et al. 2006).
Unlike the filarial nematodes, it is thought that Wolbachia infection of arthropods
occurred multiple times. This can occur when infected host populations crash. Selfish
genetic elements can be harmful to organisms when their sole purpose is to promote their
continued existence at the expense of the host (Hurst and Werren 2000, Werren et al.
2008). It has been theorized that if reproduction is “perfect” with a male killing
Wolbachia, and the host produces all female progeny, the population could crash if the
host population lacks the phenotypic fitness to survive (Bonte et al. 2008). Another
example of this ‘selfish genetic element’ is found with cytoplasmic incompatibility. In
this situation, only infected insects should survive. However, this form of infection could
be responsible for the speciation of many insects (Prout 1994). If uninfected females mate
with uninfected males, a separate species could evolve. In one study using deterministic
and stochastic models to evaluate the evolution of CI in arthropods the authors suggest
that selection favoring infected arthropods may be weak and can be selected against by a
subspecies that displays high rates of fecundity (Haygood and Turelli 2009). In a study
examining Drosophila recens and Drosophila subquinaria that are reproductively
isolated by Wolbachia it was determined that cytoplasmic inhibition was partially
responsible, but not the sole cause (Shoemaker et al. 1999). Additionally, it has been
suggested that different forms of Wolbachia can infect an insect species causing
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reproductive isolation as well (Telschow et al. 2002). And in a study examining
cytoplasmic incompatibility in Drosophila infected males of varying ages, it was found
that the effect of Wolbachia was greater when younger infected males were mated with
uninfected females (Reynolds and Hoffmann 2002).
Some researchers suggest that other endosymbionts and nuclear effects may play a more
important role in speciation and the evolution of invertebrates (Weeks et al. 2002). There
has also been evidence that another endosymbiont that behaves much like Wolbachia
might be causing some reproductive effects. This Cytophaga-like organism (CLO) was
found to have a 7% infection rate among the species tested and was sometimes found in
conjunction with Wolbachia (Weeks et al. 2003).
Conclusion:
Although there has been little research performed on Japanese beetles and their
relationship with endosymbionts like Wolbachia, this research shows that they are
harboring the bacteria and it may be having an effect on their reproductive success. It is
not entirely clear if cytoplasmic incompatibility is responsible or if other endosymbionts
might also be infecting the beetles. While the number of males verses females in this
study is generally not significant, there is the suggestion that with further investigation
with increased sampling it could be. There are other factors to consider as well. Since the
beetles are known to feed on such a variety of plants, perhaps they are capable of
producing multiple enzymes that allow them to digest different plant byproducts. Many
researchers suggest that Wolbachia may be just one factor in insect success, so clearly,
this suggests more study is required.
Acknowledgements:
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I wish to thank Michele Bahr and Chris at Marine Biological Laboratories for their
assistance and support through out this entire project. I’d also like to thank Cheryl Wright
for her advice and assistance in statistics. Most importantly, I am grateful for the support
given me by the Wolbachia Project, funded by grants from HHMI. Without this
assistance, teachers in secondary institutions could not perform authentic research of this
caliber with their students.
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http://ohioline.osu.edu/hyg-fact/2000/2001.html Accessed June 14, 2009.
43. Stahlhut JK, Desjardins CA, Clark ME, Baldo L, Russell JA, et al. (2010) The
mushroom habitat as an ecological arena for global exchange of Wolbachia.
Molecular Ecology. 1940-52.
44. Shoemaker DD, Katju V, Jaenike J (1999) Wolbachia and the evolution of
reproductive isolation between Drosophila recens and Drosophila subquinaria.
Evolution. 53(4): 1157-1164.
45. Stouthamer R, Breeuwer JA, Hurst GDG (1999). Wolbachia pipientis: microbial
manipulator of arthropod reproduction. Annual Review of Microbiology. 53: 71102.
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46. Telschow A, Hammerstein P, Werren JH (2002) The effect of Wolbachia on
genetic divergence between populations: models with two-way migration. The
American Naturalist. 160: S54-S66.
47. United States Department of Agriculture. Japanese Beetle Program for Airports
http://www.aphis.usda.gov/import_export/plants/manuals/domestic/downloads/ja
panese_beetle.pdf Accessed June 14, 2009.
48. Weeks AR, Turelli M, Reynolds KT, Hoffmann AA (2002) Wolbachia dynamics
and host effects: what has (and has not) been demonstrated? Trends in Ecology
and Evolution. 17(6): 257-262.
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parasite to mutualist: rapid evolution of Wolbachia in natural populations of
Drosophila. PloS Biology. 5(5): e114.
50. Weeks AR, Velten R, Stouthamer R (2003) Incidence of new sex-ratio-distorting
endosymbiotic bacterium among arthropods. Proceedings of the Royal Society of
London: Biological Science. 270:1857-1865.
51. Werren JH, Windsor DM (2000) Wolbachia infection frequencies in insects:
evidence of a global equilibrium? The Royal Society, Proceedings: Biological
Sciences. 267: 1277-1285.
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invertebrate biology. Nature Reviews Microbiology. 6(10): 741-751.
Figures and Tables:
Fig.1
Fig.2
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Fig.3
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Fig.4
17
Cynthia Jensen
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Fig. 5
November 10, 2011
Dr. Hoback
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decompressor
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Fig. 6
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Fig. 7
Fig. 8
Table 2: Chi Square Values- One-Tailed Analysis of Sex and Time (Emergence of
Adults)
Emergence
Male
Female
Chi-Square
1
4
1
Total
26
29
0.17665
2
4
1
18
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Total
3
Total
1 and 2 vs. 3
Total
November 10, 2011
38
5
14
10
134
41
6
13
11
27
Dr. Hoback
0.17985
1.0
0.0006
Fig. 9
Table 3: Chi Square Values-One-Tailed Analysis of Sex and Location (Plant Species)
Plant
Male
Female
Chi-Square
Raspberry(R)
4
0
Total
56
60
0.05935
Loosestrife(L)
4
7
Total
15
12
0.2378
L vs R
11
4
Total
27
120
<.0001
Knotweed(K)
4
0
19