Life Cycle Completion of Parasite Ascogregarina taiwanensis (Apicomplexa: Lecudinidae) in Non-Native Host Ochlerotatus japonicus (Diptera: Culicidae)

Abstract

Ascogregarina taiwanensis (Lien and Levine), a protist gut parasite of Aedes albopictus (Skuse), is not known to complete its life cycle within the potential competitor species, Ochlerotatus japonicus (Theobald). In a laboratory cross infection study we demonstrated that A. taiwanensis completed its life cycle within Oc. japonicus and remained infectious. We also sampled cohabitating mosquito larvae in Mercer County, NJ, and based on ribosomal DNA sequence data, we determined that Oc. japonicus cohabitating with Ae. albopictus can become infected with A. taiwanensis.

Full text

VECTOR/PATHOGEN/HOST INTERACTION,TRANSMISSION
Life Cycle Completion of Parasite Ascogregarina taiwanensis
(Apicomplexa: Lecudinidae) in Non-Native Host Ochlerotatus
japonicus (Diptera: Culicidae)
J. A. ERTHAL,
1
J. S. SOGHIGIAN, AND T. LIVDAHL
Lasry Center for Bioscience, Clark University, 950 Main Street, Worcester, MA 01610
J. Med. Entomol. 49(5): 000Ð000 (2012); DOI: http://dx.doi.org/10.1603/ME12018
ABSTRACT Ascogregarina taiwanensis (Lien and Levine), a protist gut parasite of Aedes albopictus
(Skuse), is not known to complete its life cycle within the potential competitor species, Ochlerotatus
japonicus (Theobald). In a laboratory cross infection study we demonstrated that A. taiwanensis
completed its life cycle within Oc. japonicus and remained infectious. We also sampled cohabitating
mosquito larvae in Mercer County, NJ, and based on ribosomal DNA sequence data, we determined
that Oc. japonicus cohabitating with Ae. albopictus can become infected with A. taiwanensis.
KEY WORDS Ochlerotatus japonicus,Ascogregarina taiwanensis,mosquito,parasite
Ascogregarina (Syn. Lankesteria; Ascocystis)isagenus
of protist gut endosymbionts of container-dwelling
mosquitoes (Ward et al. 1982, Voty´pka et al. 2009).
Ascogregarina can have signiÞcant Þtness conse-
quences, particularly under deÞcient nutrient condi-
tions (Comiskey et al. 1999) or high exposure levels
(Sulaiman 1992). Although some earlier research sug-
gests that Ascogregarina have little effect on the mor-
tality of their natural hosts (McCray et al. 1970,
Jacques and Beier 1982, Mourya and Soman 1985,
ReyesÐVillanueva et al. 2003), a more recent study of
infection by Ascogregarina showed alteration of the
competitive interactions between species (Aliabadi
and Juliano 2002), suggesting that the impact of As-
cogregarina parasites requires further investigation.
We address here the potential for an introduced As-
cogregarina species to infect non-native hosts in situ-
ations involving two invasive host species.
Species of Ascogregarina have been characterized as
ahost-speciÞc, meaning that each species completes
its life cycle in a single host species (Lien and Levine
1980, Beier and Craig 1985, Chen 1999). However,
more recent studies have demonstrated that some
Ascogregarina, while more reproductively efÞcient in
one particular species, are capable of life cycle com-
pletion in multiple host species (Munstermann and
Wesson 1990, Garcia et al. 1994). Frequently, these
cross infections in non-native species result in high-
host mortality (Walsh and Olson 1976, Munstermann
and Wesson 1990, Garcia et al. 1994, Comiskey and
Wesson 1997).
Ascogregarina taiwanensis (Lien and Levine) is a
well-characterized gregarine that primarily infects
Aedes albopictus (Skuse). It can also infect several
species across multiple Culicidae genera, but the ma-
jority of these infections occur without successful life
cycle completion of the parasite (Munstermann and
Wesson 1990, Garcia et al. 1994, Reeves and Mc-
Cullough 2002). In non-native hosts Ochlerotatus ep-
actius (Dyar and Knab !Aedes epactius; see (Reinert
2000), Ochlerotatus atropalpus (Coquillett) (Munster-
mann and Wesson 1990), and Ochlerotatus taenio-
rhynchus (Wiedemann) (Garcia et al. 1994) it has
been demonstrated to complete its life cycle and pro-
duce infectious oocysts (Fig. 1). In Þeld collection
studies, A. taiwanensis gamonts have been found in
Aedes aegypti (L.) (Fukuda et al. 1997) and Oc. epac-
tius larvae (Munstermann and Wesson 1990). Because
of similarities in morphology between species of As-
cogregarina, apolymerasechainreaction(PCR)-
based assay has been developed by Morales et al.
(2005) to distinguish speciÞcally A. taiwanensis from
similar gregarines Ascogregarina culicis (Ross) and As-
cogregarina barretti (Vavra). Using PCR primers tar-
geting the ITS region of ribosomal DNA they distin-
guish A. taiwanensis as a 450 bp amplicon on an
electrophoretic gel (Morales et al. 2005).
Previous research has delineated the life cycle of A.
taiwanensis within Ae. albopictus (Chen et al. 1997,
Roychoudhury and Kobayashi 2006). Ae. albopictus
larvae become infected upon ingesting A. taiwanensis
oocysts from the container habitat and are vulnerable
to gregarine infection at all larval instars (Roychoud-
hury and Kobayashi 2006). Approximately 1 hr after
ingestion, A. taiwanensis oocysts release sporozoites
into the larval gut (Roychoudhury and Kobayashi
2006). These sporozoites enter midgut epithelial cells
and use host cell mitochondria to supply the energy
required to mature into a trophozoite (Chen et al.
1997). Trophozoites generally mature concurrently
1
Corresponding author, e-mail: tlivdal@clarku.edu.
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with their host and upon the release of host molting
hormone 20-hydroxyecdysone, trophozoites exit
midgut epithelial cells, travel down the digestive tract,
and enter the Malpighian tubules (Chen 1999). Once
in the Malpighian tubules, the trophozoite becomes
segmented and divides into gametes (Chen et al.
1997). Gametes combine via syzygy, creating game-
tocysts that in turn bud oocysts (Chen et al. 1997).
When Ae. albopictus pupae eclose, or when adults
defecate or oviposit in container habitats, they release
these A. taiwanensis oocysts and expose the next gen-
eration of Ae. albopictus larvae to infection (Roy-
choudhury and Kobayashi 2006).
The invasive species Ae. albopictus was introduced
from Japan via the international used tire trade (Haw-
ley et al. 1987, Reiter and Sprenger 1987) and sustain-
ing populations were Þrst detected in the United
States in 1985 near Houston, TX (Sprenger and
Wuithiranyagool 1986). A. taiwanensis was not de-
tected in the United States until 1988 (Munstermann
and Wesson 1990). It has been conjectured that this
delay between the arrival of the host and parasite
occurs because hosts with rapid range expansion may
temporarily outrun and escape their parasite in newly
founded populations (Blackmore et al. 1995). Ae. al-
bopictus has been an efÞcient colonizer in the United
States and is a successful competitor with previously
established mosquito species (Ho et al. 1989, Livdahl
and Willey 1991, OÕMeara et al. 1995, Juliano and
Lounibos 2005). In the past 26 yr, Ae. albopictus has
expanded its range to 36 states and has invaded regions
of the Middle East, Europe, Africa, and Central and
South America (Enserink 2008).
Like Ae. albopictus, Ochlerotatus (Finlaya)japoni-
cus japonicus (Theobald) is a container-dwelling mos-
quito native to Japan. First detected in the United
States in 1998 in Connecticut, New York, and New
Jersey (Peyton et al. 1999, Andreadis et al. 2001), the
range of Oc. japonicus includes Japan, Korea, South
China, Taiwan, Russia, Canada, and at least 22 states on
both the east and west coasts of the United States
(Williges et al. 2008). Like Ae. albopictus, Oc. japonicus
appears to have been transported to the United States
via international tire shipping (Peyton et al. 1999) and
is also a successful competitor with resident mosquito
species (Burger and Davis 2008, Andreadis and Wolfe
2010).
The natural parasite of Oc. japonicus, Ascogregarina
japonicus (Roychoudhury, Isawa, Hoshino, Sasaki &
Kobayashi) was recently described in Japan (Roy-
choudhury et al. 2007b), but has not been reported in
the United States. However, because Oc. japonicus and
Ae. albopictus have overlapping ranges and occupy the
same container habitats, it seems likely that Oc. ja-
ponicus would be exposed to A. taiwanensis. Suscep-
tibility of Oc. japonicus to cross infection by the par-
asites of its potentially competing container-dwelling
mosquitoes has not yet been established. If Oc. ja-
ponicus could be infected by A. taiwanensis, the par-
asite could inßuence the competitive capacity and
range expansion of both mosquito species (Munster-
mann and Wesson 1990, Garcia et al. 1994, Blackmore
et al. 1995). In this study, we infected Oc. japonicus
with A. taiwanensis in the laboratory to investigate
whether A. taiwanensis can complete its life cycle in
Oc. japonicus. We also collected Oc. japonicus cohabi-
tating with Ae. albopictus to test for natural infections
of Oc. japonicus by A. taiwanensis.
Materials and Methods
Collection and Culturing of A. taiwanensis and Ae.
albopictus.Water samples containing Ae. albopictus
were collected from container habitats (e.g., buckets,
vases, tarpaulins, and tires) in St. Georges and Ham-
ilton, Bermuda by the Department of Vector Control
of the Bermuda Ministry of Health. We dissected Ae.
albopictus to determine the presence of Ascogregarina
and conÞrmed the identity of these gregarines as A.
taiwanensis via parasite morphology (Lien and Levine
1980) and PCR assays identical to those described
below. We ampliÞed A. taiwanensis stocks by infecting
multiple generations of Ae. albopictus with oocysts
collected from Ae. albopictus in Bermuda. The oocyst
homogenate was stored at 4"Cuntilneededforex-
perimental use or further ampliÞcation of the parasite.
Oocyst concentration was determined using a hema-
cytometer. Since 2005, Ae. albopictus has been the only
Aedes mosquito species on Bermuda (Kaplan et al.
2010), preventing any chance of cross infection with
any other gregarine.
Fig. 1. Cladogram of Culicidae mosquito hosts and sum-
marized reports of infectivity of A. taiwanensis in those hosts.
The relationships shown between species taken from the
Maximum Likelihood tree displayed by Shepard et al. (2006).
Species that were not included in Shepard et al.Õs study have
been placed at the genus level based on current classiÞcation
and indicated with dotted lines. (a) Munstermann and Wes-
son (1990), (b) Garcia et al. (1994), (c) Mourya and Soman
(2000), (d) Reeves and McCullough (2002), (e) this study.
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The Ae. albopictus colony used in this study to am-
plify A. taiwanensis and determine the viability of
oocysts passed through Oc. japonicus (see below) was
established from egg batches obtained in Bermuda
ovitraps. Because the Þeld populations of these mos-
quitoes were infected with A. taiwanensis, we rinsed
the egg slats with distilled water before hatching them
in 1 g/L of nutrient broth (Bisco) and removed then
after 20 h, placing them in distilled water. Larvae
dissected from this initial hatching were uninfected
(data not shown) and dissections of the next gener-
ation also suggested that the colony was uninfected
with Ascogregarina. The colony was maintained at
24"Cwith80%RHandaphotoperiodof16:8(L:D)h.
Collection of Oc. japonicus.Oc. japonicus adults
were captured in Hadwen Arboretum of Clark Uni-
versity, Worcester, MA (42 15#2$,%71 49#58[dprime),
bloodfed, and kept in cages with oviposition contain-
ers in 80% humidity. We collected egg sheets collected
from these adults and immersed them 1 g/L of nutrient
broth (Bisco) for 24 h to hatch larvae.
Experimental Design. We disbursed 10 Þrst instars
into 14, 10 cm petri dishes. each containing a 30 ml
solution of A. taiwanensis oocysts in distilled water
containing 1,000 oocysts per ml (3,000 oocysts per
larva). Larvae were not fed for the Þrst 20 h after
hatching to encourage oocyst ingestion after which
they were fed 0.05 g powdered yeast (Kal) every other
day. Larvae were reared at 24"C.
From each dish, we sampled and dissected Oc. ja-
ponicus larvae, as pupae, and as adults. On days 4, 7,
and 8, one larva from each dish was dissected, with the
Þnal two larvae being dissected on day 12 as they were
the only remaining mosquitoes in of the dishes. Dis-
sections of pupae occurred on days 8, 9, 10, and 11; no
more than one pupa per dish was dissected per day, if
there was a pupa available. Infection status of larvae
and pupae was conÞrmed through mid-gut dissection
and visual conÞrmation of A. taiwanensis gamonts in
the mid-gut lining. Before dissecting larvae and pupae,
we rinsed them in distilled water, after which we
placed specimens on a microscope slide in 0.05 ml
distilled water removed the head with a dissecting
needle, inserted one dissecting needle into the thorax
while pulling posteriorly with another dissecting nee-
dle inserted into the terminal abdominal segment. This
process separated the terminal segment from the
body, drawing out the Malpighian tubules and gut
along with it. Gut contents ßooded into the surround-
ing medium. We covered gut and Malpighian tubules
with a cover slip and examined at 400&under a com-
pound microscope.
In specimens reared to adulthood, pupae were re-
moved individually from the oocyst solution, rinsed in
distilled water and placed in 1.5 ml centrifuge tubes
with 0.5 ml distilled water which were plugged with
cotton. When the adults emerged they were trans-
ferred to a second 1.5 ml centrifuge tube in which they
were frozen. Adults were then homogenized within
the centrifuge tube using a motorized pestle and pos-
itive A. taiwanensis infection was conÞrmed and quan-
tiÞed using a hemacytometer. Homogenized pupal
exuviae of emerged adults were also analyzed because
Ascogregarina oocysts are frequently shed during eclo-
sion (Fellous and Koella 2009).
After running the single-round infectivity trial,
oocysts produced from Oc. japonicus adult and pupal
exuviae samples were collected for use in conÞrming
the viability of these oocysts through infection of Ae.
albopictus. Thirty newly hatched Ae. albopictus were
divided into two petri dishes, with one petri dish
containing 20 ml of 406 oocysts (produced by Oc.
japonicus)permlandtheothercontaining20mlof
distilled water. Four larvae from each petri dish were
dissected at the third instar to assess parasite success.
The remaining mosquitoes were reared to adulthood
and handled as described above to determine if the
oocysts from Oc. japonicus were able to complete their
life cycle in Ae. albopictus.
Confirmation of Cross Infection via PCR Amplifi-
cation. We conÞrmed the accuracy of hemacytometer
results by extracting DNA from samples of 12 homog-
enized adult samples and 12 homogenized pupal ex-
uviae, including both negative and positive infection
status. DNA was extracted using the E.Z.N.A Forensic
DNA Kit (Omega Biotek, Norcross, GA). We ampli-
Þed a sequence of rDNA encompassing the partial 18s,
complete ITS1, complete 5.8s, and partial ITS2 regions
using PCR with primers and a PCR cycle described by
Morales et al. (2005). We added a 10
!
Mprimer
speciÞcforthegenusAscogregarina AU (5#-ACC GCC
CGT CCG TTC AAT CG-3#)anda10
!
Mprimer
speciÞcforA. taiwanensis AT (5#-GAG AAG CCG
TCG TCA ATA CAG C-3#)toareactionmixcontain-
ing 12.5
!
lofGoTaqMasterMix(PromegaCorpora-
tion, Madison, WI) and 7.5
!
lofsterile,distilledwater.
We analyzed PCR products on a 1% agarose gel using
standard electrophoretic procedures (Sambrook et al.
1989), scoring samples as positive or negative based
the presence of or absence of an appropriately sized
amplicon ('450 bp) as determined by comparison
with PCR Markers DNA ladder (Promega Corpora-
tion, Madison, WI). For DNA samples that were neg-
ative for the presence of A. taiwanensis, we then am-
pliÞed using PCR with primers speciÞctoOc.
japonicus ITS2 and 28S regions of the rDNA, using 10
!
MofforwardprimerspeciÞcforthestartoftheITS2
region (5#-GCG TGC GCG TTT CAC TTC GG-3#)
and 10
!
MofreverseprimerspeciÞcforthestartofthe
28S region (5#-GCC TAC TGG AGT GTT ATA TGT
GGG C-3#)inareactionmixcontaining12.5
!
lof
GoTaq Master Mix (Promega Corporation) and 7.5
!
l
of sterile, distilled water. We analyzed PCR products
on a 1% agarose gel using standard electrophoretic
procedures (Sambrook et al. 1989), scoring samples as
positive or negative based the presence of or absence
of an appropriately sized amplicon ('267 bp) in com-
parison with PCR Markers DNA ladder (Promega
Corporation). After running the PCR products on the
agarose gel we detected the appropriate 267 bp Oc.
japonicus amplicon in all samples scored as negative
for A. taiwanensis and conÞrmed that the absence of
an A. taiwanensis amplicon was not because of irreg-
ularities with the DNA samples.
September 2012 ERTHAL ET AL.: GREGARINE CROSS INFECTION IN Ochlerotatus japonicus 3
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Field Collection and DNA Extraction. We collected
larvae from sampling sites in Trenton and Hopewell,
NJ (40.295655, %74.782884) with the assistance of the
Mercer County Department of Mosquito Control. We
selected this area because at this latitude, Oc. japonicus
and Ae. albopictus ranges overlap and both species
have been observed occupying the same containers at
the same time, providing a natural opportunity for
cross infection. We collected samples from habitats
where Oc. japonicus and Ae. albopictus were cohabi-
tating (childrenÕs pools, cemetery vases, etc.). Sam-
ples were washed in distilled water and dissected
under a dissecting microscope at 40&magniÞcation.
We extracted DNA from these Oc. japonicus samples
(and one Ae. albopictus sample) using the E.Z.N.A
Forensic DNA Kit (Omega Bio-Tek Inc., Norcross,
GA) and performed PCR as described above to test for
presence of parasite DNA.
Sequencing of 18s rDNA. While the morphology of
our samples from Oc. japonicus resembled that of A.
taiwanensis, we could not conclude deÞnitively from
morphology alone the identity of our samples because
there has been no published morphology on the tro-
phozoite life stage of A. japonicus. Instead, we sought
to compare the DNA sequences from our samples to
those of A. japonicus, A. taiwanensis, and other species
of Ascogregarina parasites in the eastern United States.
The only available nucleotide sequence for A. japoni-
cus on GenBank was an 1,800 bp fragment from the 18s
region, so we ampliÞed and sequenced this region of
rDNA from our samples collected from New Jersey, as
well as a sample of A. barretti from Ochlerotatus tri-
seriatus (Say) collected in Worcester, MA, and a sam-
ple of A. taiwanensis from Ae. albopictus collected in
Bermuda.
To amplify only rDNA from Ascogregarina, as our
DNA extractions contained mosquito DNA as well,
we used a universal 18s forward primer (5#-
CGAATTCAACCTGGTTG ATCCTGCCAGT-3#)
from Roychoudhury et al. (2007a) and an Ascogre-
garina-speciÞcreverseprimerAsco28sR(5#-CAG
TGG GTA GCC TTG TC-3#)thatbindstothebegin-
ning of the 28s region from Morales et al. (2005).
These primers ampliÞed roughly 1,800 nucleotides of
the 18s region along with the complete ITS1, 5.8s, ITS2,
and partial 28s rDNA regions. The PCR conditions
included an initial denaturing step at 94"Cfor2min,
then 35 cycles of 94"Cfor1min,50"Cfor30s,and72"C
for 3 min, with a Þnal extension step of 10 min at 72"C.
PCR products were sequenced directly using the uni-
versal 18s forward and universal 18s reverse (5#-CCG
GAT CCT GAT CCT TCT GCA GGT TCA CCT AC-
3)#primers, two 18s internal primers (5#-GGA GAG
GGA GCC TGA GAA-3#and 5#CTC TAA GAA GCG
ACG CCA-3#)describedbyRoychoudhuryetal.
(2007a), the Ascogregarina-speciÞcreverseprimer,
and an Ascogregarina-speciÞcforwardprimer
Asco18sF (5#-CGA CTG GAT GAT CCG G-3#)from
Morales et al. (2005) that binds near the end of the 18s
region.
We cleaned the ampliÞed PCR products via ethanol
precipitation and sequenced in both directions with
the above primers using BigDye 3.1 Terminator chem-
istry and manufacturerÕs protocols, and the sequenc-
ing reaction was cleaned using Agencourt CleanSeq
magnetic plate (Beckman Coulter Inc., Brea, CA). We
used a 3130 Genetic Analyzer (Applied Biosystems
Inc, Foster City, CA) to obtain DNA sequences.
We assembled the six reads for each sample in
Geneious (Drummond et al. 2012) and trimmed low
quality regions at the start and end of the reads re-
sulting in a 2070 bp sequence of rDNA from Ascogre-
garina collected in New Jersey, Massachusetts, and
Bermuda. We aligned resulting rDNA sequences in
Geneious using MAFFT (Katoh et al. 2002) and
trimmed to just the 18s region, resulting in 1733 bp (or
1735 bp in the case of our sample from A. barretti). We
aligned the trimmed sequences again in Geneious us-
ing MAFFT with four 18s Ascogregarina sequences
from GenBank (Table 1), trimming sequences from
GenBank to the length of our sequences. We exam-
ined the alignment visually to ensure that trimming
did not cause any alignment errors.
Data Analyses. We regressed infection status in Oc.
japonicus against time since hatch using a nominal
logistic model. We included larval and pupal dissec-
tion data, as well as adult and exuviae life cycle de-
tection. After Þnding signiÞcant differences in prev-
alence of A. taiwanensis among life stages of
experimentally infected Oc. japonicus by
"
2
square
contingency analysis, we tested for speciÞcdiffer-
ences among larvae, pupae and adults using pairwise
comparisons described by Zar (1999). We used Fisher
exact test to compare the prevalence of A. taiwanensis
Table 1. Parasite species, host, origin, ungapped sequence length in our alignment, and accession number for the rDNA sequences
used in this study
Parasite species Host collected in Origin Sequence length Accession no.
Ascogregarina armigerei Armigeres subalbatus (Coquillett) New Jersey 1733 DQ462459
Ascogregarina barretti Oc. triseriatus Massachusetts 1735 To be added
Ascogregarina culicis Ae. aegypti Columbia 1733 DQ462457
Ascogregarina japonicus Oc. japonicus Japan 1733 DQ462458
Ascogregarina sp. Oc. japonicus (J1) New Jersey 1733 To be added
Ascogregarina sp. Oc. japonicus (J6) New Jersey 1733 To be added
Ascogregarina sp. Ae. albopictus (J3) New Jersey 1733 To be added
Ascogregarina taiwanensis Ae. albopictus Bermuda 1733 To be added
Ascogregarina taiwanensis Ae. albopictus Japan 1733 DQ462454
Samples in bold were sequenced in this study.
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in dissections of Ae. albopictus larvae exposed to
oocysts from Oc. japonicus with those unexposed to
oocysts, frequency of life cycle completion by A. tai-
wanensis in experimentally infected Oc. japonicus with
experimentally infected Ae. albopictus, as well as prev-
alence of A. taiwanensis in Þeld collected Ae. albopic-
tus versus Oc. japonicus from New Jersey. We used
JMP v.9 (SAS Institute 2010) for contingency analysis,
logistic regression, and Fisher exact tests.
From the MAFFT alignment of our sequences and
the sequences for Ascogregarina armigerei (Lien and
Levine), A. culicis, A. japonicus, and A. taiwanensis
(Accession numbers: DQ462459, DQ462457,
DQ462454, and DQ462454, respectively), we created
adistancematrixofpairwisesimilaritytoidentifyeach
Þeld collected Ascogregarina sample and we con-
structed an unrooted maximum likelihood (ML) tree.
The ML analysis was performed with 100 bootstrap
replicates on the RAxML BlackBox server (http://
phylogench.vital-it.ch/taxml-bb/index.php; Stamata-
kis et al. 2008) using A. armigerei (DQ462459) as an
outgroup based on the previous phylogenetic analysis
of Roychoudhury et al. (2007). The resulting best
scoring ML tree was viewed in Dendroscope (Huson
et al. 2007).
Results and Discussion
Experimental Infection. Of the 44 Oc. japonicus
dissected as larvae, 42 (95%) were infected with A.
taiwanensis, all dissected on days 4, 7, or 8 (Table 2).
The two larvae that showed no signs of A. taiwanensis
infection in the midgut were dissected on day 12, and
as such, we expect they had ample time to consume
oocysts. Therefore, we assume that all of the Oc. ja-
ponicus larvae were exposed to A. taiwanensis, even if
they did not shown signs of infection at each life stage.
We dissected 28 Oc. japonicus as pupae, 39% were
infected with visible gamonts. In a sample of 53 ho-
mogenized adults, 30% had oocysts present in either
the exuviae or the homogenized adult tissue (Table 3).
These results demonstrate that A. taiwanensis is capa-
ble of completing its life cycle within Oc. japonicus.
Our logistic regression suggested that Oc. japonicus
is able to clear itself of infection, as prevalence of A.
taiwanensis decreased over time (Fig. 2;
"
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1
!31.39,
P(0.001). This clearance of infection may have oc-
curred during the pupa life stage because there was no
difference between prevalence in adults compared
with pupae, but parasite prevalence was signiÞcantly
greater in dissected larvae (Table 2). This seems com-
mon in experimental infection of non-native hosts, as
Garcia et al. (1994) found that despite a 100% prev-
alence of A. taiwanensis in experimentally infected Ae.
aegypti, Ae. albopictus, and Oc. taeniorhynchus larvae,
no oocysts were found in Ae. aegypti adults and only
30% of Oc. taeniorhynchus adults harbored oocysts,
whereas all 100% of Ae. albopictus adults had oocysts.
Further, Chen (1999) found that all of the trophozo-
ites in Ae. aegypti were lysed in the pupa life stage, but
despite 100% infection of pupae, roughly half of the
trophozoites in Ae. albopictus failed to migrate to the
Malpighian tubules and were lysed during the pupa
life stage.
We have conÞrmed the viability of A. taiwanensis
oocysts collected from parasitized Oc. japonicus be-
cause they produced a second-round infection in the
midgut lining of all four of the dissected Ae. albopictus
larvae exposed to those oocysts, while none of the
Table 2. Prevalence of A. taiwanensis in three life stages of Oc.
japonicus
Stage Number examined Prevalence
Larva 44 95%a
Pupa 28 39%b
Adult 53 30%b
Prevalence in different stages that are signiÞcantly different (P(
0.01) are marked by different letters.
Table 3. Presence of A. taiwanensis oocysts in adult and pupal
exuviae of Oc. japonicus by sex
Sex Total
individuals IP
a
IA
b
O
E
c
Female 17 24% 18% 24%
Male 36 25% 14% 33%
Combined 53 25% 15% 30%
Differences between sexes not signiÞcant (FisherÕs exact tests, P)
0.05).
a
I
P
Infected pupal exuviae.
b
I
A
Infected homogenized adult tissue.
c
O
E
Oocysts present in either pupal exuviae or homogenized adult
tissue.
Fig. 2. Logistic regression on prevalence of A. taiwan-
ensis in Oc. japonicus through time. Points are proportion of
infected Oc. japonicus by A. taiwanensis in dissections of
larvae and pupae or detection of life cycle completion in
adults. Pupa were dissected starting on day 8; adults began
emerging, and were checked for life cycle completion, be-
ginning on day 10. The predictive model equation is shown;
the coefÞcient for time (t), 0.73 *0.13, is signiÞcant (
"
2
1
!
31.39; P(0.0001).
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uninfected Ae. albopictus had any visible parasite in-
fection (Fisher exact test, P(0.03). Furthermore, of
the mosquitoes exposed to oocysts that reached adult-
hood, all eight had visible oocysts in the pupal exuviae,
while none of the ten unexposed mosquitoes that
reached adulthood did (Fisher exact test, P(0.0001).
Despite treatment with a much lower initial dosage of
oocysts (406/ml compared with 1,000/ml), all surviv-
ing Ae. albopictus showed evidence of life cycle com-
pletion by A. taiwanensis, compared with only 30% of
Oc. japonicus (Fisher exact test, P!0.002). Although
our study was not designed to thoroughly quantify the
level of life cycle completion of A. taiwanensis in Ae.
albopictus, these results indicate that Ae. albopictus
may be more susceptible to A. taiwanensis than Oc.
japonicus.
In previous cross infection studies, gregarine life
cycle completion could only be conÞrmed by visually
detecting oocysts or by producing second-round in-
fections in a new generation of mosquitoes. To help
resolve issues concerning the sensitivity of a visual
survey, we not only scored samples for the presence
of A. taiwanensis oocysts using a hemacytometer but
also conÞrmed results using A. taiwanensis primer se-
quences developed by Morales et al. (2005). Of eight
adult pupal exuviae samples tested with PCR ampli-
Þcation of parasite rDNA, six were scored as positive
based on presence of a 450 bp band (Fig. 3). Lane
three and lane nine were negative for infection in
the hemacytometer visual survey and were also neg-
ative for infection in the PCR assay. Lane 6, however,
was negative for infection in the hemacytometer vi-
sual survey but was positive for infection in the PCR
assay. This shows that while a hemacytometer can be
useful in detecting life cycle completion, it is not as
sensitive as a PCR ampliÞcation of parasite rDNA.
Field Collection and rDNA Sequencing. From the
Þeld samples of cohabitating Ae. albopictus and Oc.
japonicus larvae collected from Mercer County, NJ,
we were able to detect A. taiwanensis infection in Oc.
japonicus and Ae. albopictus both through visual iden-
tiÞcation of trophozoites in the larval midgut and
through PCR ampliÞcation and sequencing of DNA
obtained from dissected larvae. We observed what
appeared morphologically to be A. taiwanensis infec-
tion in three of seventeen Oc. japonicus sampled and
9of18Ae. albopictus (Fisher exact test P!0.075). We
were able to amplify parasite DNA from two of the
infected Oc. japonicus samples and one Ae. albopictus
sample. The 18s sequences from our three samples
collected from New Jersey were the most similar to
one another, with one to two base pairs different
between them (Table 4). These sequences had a two
to three base pair difference compared with the se-
quence from A. taiwanensis collected in Bermuda, but
afourtoÞve nucleotide difference in the same region
compared with the sequence of A. taiwanensis from
Japan. Because Ae. albopictus is thought to have been
introduced to Bermuda from the United States (Ka-
plan et al. 2010), we expected that out of the se-
quences examined in this study, this sequence of A.
taiwanensis from Bermuda would be the most similar
to the sequences from New Jersey if indeed our sam-
ples of Oc. japonicus were infected with A. taiwanensis.
Furthermore, the likelihood of our Þeld collected sam-
ples being any other species of gregarine found in the
eastern United States, or A. japonicus, seems quite low
given the much larger difference in nucleotides be-
tween those four other species and our three Þeld
collected samples. This is reßected in a maximum
likelihood tree comparing our New Jersey samples
from Oc. japonicus and Ae. albopictus to A. armigerei,
A. barretti, A. culicis, A. japonicus, and A. taiwanensis
(Fig. 4). This analysis strongly supports the mono-
phyly of the clade containing all three of our samples,
as well as both sequences from A. taiwanensis.
While our own phylogenetic analysis did not re-
solve the relationships among Ascogregarina species,
phylogenetic analyses of Ascogregarina spp. by Roy-
choudhury et al. (2007a) found that A. taiwanensis
from Ae. albopictus and A. japonicus from Oc. japonicus
were morphologically similar (in oocyst size) and
their 18S rDNA sequences revealed that they were
more closely related to each other than to A. culicis or
A. armigerei. The similarity between these two species
could be further investigated with host speciÞcity
Fig. 3. An ethidium bromide-stained 1% agarose gel un-
der ultraviolet light showing A. taiwanensis species-speciÞc
products. Lane L is the PCR Markers DNA ladder; lane 2,
male pupal exuvia; lane 3, male adult; lane 4, male pupal
exuvia; lane 5, male adult; lane 6, female pupal exuvia; lane
7, female adult; lane 8, female pupal exuvia. Œindicates
sample was previously classiÞed as noninfected with a he-
macytometer.
Table 4. Pairwise comparison of Ascogregarina rDNA se-
quences
Species 123456789
A. armigerei
DQ462459
15458635553555656
A. culicis
DQ462457
296.8 17 23 12 11 12 13 13
A. japonicus
DQ462458
396.698.9 28 19 18 19 20 20
A. barretti US MA 4 96.3 98.6 98.3 21 22 23 24 24
A. taiwanensis
DQ462454
596.799.298.898.7 4 4 5 5
A. taiwanensis BM 6 96.9 99.3 98.9 98.6 99.7 2 3 3
Ascogregarina NJ
Oc. japonicus (1)
796.799.298.898.699.799.8 1 1
Ascogregarina NJ
Ae. albopictus
896.799.298.898.599.699.799.9 2
Ascogregarina NJ
Oc. japonicus (2)
996.799.298.898.599.699.799.999.8
Percentage similarity (bottom) and no. of nucleotides different in
the alignment of the 18S region between New Jersey collected As-
cogregarina samples (4, 5, and 6), A. japonicus, and other Ascogregarina
found in the eastern United States.
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studies by cross infecting Ae. albopictus with A. ja-
ponicus from Oc. japonicus.
Concerning implications for mosquito ecology, pre-
vious studies have suggested that A. taiwanensis has
negligible inßuence on Ae. albopictus mortality, Ali-
abadi and Juliano (2002) showed that the gregarine
does have the capacity to alter Ae. albopictus Þtness
because Ae. albopictus is a superior competitor against
Oc. triseriatus when it is not burdened with an infec-
tion of A. taiwanensis. Our results suggest that Oc.
japonicus, while a viable host for A. taiwanensis, may
not be as competent a host as Ae. albopictus, though
further research is needed in this area. If Oc. japonicus
is a less competent host, the total output of oocysts
from a mixed community of Ae. albopictus and Oc.
japonicus may be lower than that of a community
containing just Ae. albopictus, potentially causing suc-
cessive generations of Ae. albopictus to be exposed to
fewer parasites in those habitats and thus escape par-
asitism. If larger numbers of Ae. albopictus were able
to escape parasitism, they could have an increased
competitive advantage in the Þeld. Armistead et al.
(2008) established that Ae. albopictus is already a su-
perior competitor relative to cohabitating Oc. japoni-
cus and we suggest that this competitive advantage
could be ampliÞed in wild populations where Ae. al-
bopictus can share the burden of infection with par-
asite-free Oc. japonicus, particularly if future studies
can elucidate the Þtness consequences of infection.
Given the ability of A. taiwanensis to complete its
life cycle in multiple Ochlerotatus host species, it is
possible that A. taiwanensis could experience a range
expansion outside of the current range of its native
host Ae. albopictus. This parasite range expansion
could inßuence Ochlerotatus host species abundance
and distribution, as it has been shown that several
Ochlerotatus species experience high mortality when
challenged with A. taiwanensis (Munstermann and
Wesson 1990, Garcia et al. 1994).
Future studies that quantify the degree of infectiv-
ity of A. taiwanensis in Oc. japonicus versus the natural
host Ae. albopictus should be performed, as well as
competition studies that investigate whether A. tai-
wanensis infection has a measurable negative effect on
Oc. japonicus Þtness to assess more accurately future
proliferation or range expansion for Ochlerotatus spp.
and Ae. albopictus.
Acknowledgments
We thank A. Farajollahi and his staff at the Mercer County
Mosquito Control department; R. Furbert and his staff at the
Vector Control unit, Bermuda Ministry of Health; D. Kendell,
M. Tseng, and Y. Tsuda for their comments or kind assistance
in either the Þeld or laboratory. D. Robertson and D. Hibbett
of Clark University, S. Juliano and two anonymous reviewers
provided useful comments on earlier versions of this manu-
script. Portions of this study were supported by the Depart-
ment of Biology at Clark University, as well as grants from the
Keck Foundation and the National Institutes of Health
(1R15AI092577-01A1) to T.L.
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58(Supplement)

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9—AQ9: Please provide a volume number and complete page range.
10—AQ10: Per ESA style, please provide a volume number and complete page range.
AUTHOR QUERIES
AUTHOR PLEASE ANSWER ALL QUERIES 1