Table Of ContentPROC. ENTOMOL. SOC. WASH.
109(2), 2007, pp. 385-399
OVIPOSITOR ULTRASTRUCTURE AND OVIPOSITION BEHAVIOR OF
THE CRYPTIC AND SYMPATRIC SPECIES, TRUPANEA NIGRICORNIS
(COQUILLETT), A POLYPHAGE, AND THE NARROWLY OLIGOPHAGOUS
T. BISETOSA (COQUILLETT) (DIPTERA: TEPHRITIDAE)
Khouzama M. Knio, Richard D. Goeden, and David H. Headrick
(KMK) Biology Department, American University of Beirut, P.O. Box 11-0236,
Riad El Solh, Beirut, Lebanon (e-mail: [email protected]); (RDG) Department of
Entomology, University of California, Riverside, CA, U.S.A.; (DHH) Horticulture
and Crop Science Department, California Polytechnic State University, San Luis
Obispo, CA, 93407, U.S.A.
—
Abstract. The flower head infesting tephritids, Trupanea nigricornis (Coquillett)
and T. bisetosa (Coquillett) are cryptic and sympatric species. Trupanea nigricornis is
a polyphagous species while T. bisetosa is a specialist on wild sunflowers. The two
species showed major differences in their oviposition behavior. Females of T.
nigricornis oviposited in various developmental stages of open immature heads of
Encelia spp. and always pierced the plant tissues during oviposition; whereas, T.
bisetosa females only oviposited in the early stages ofclosed buds ofwild sunflowers
and deposited their eggs loosely between the florets without injuring plant tissues.
Timing of oviposition without plant tissue injury by T. bisetosa was critical because
older buds were covered with hard bracts and exuded resin when injured. The period
of flower head suitability for oviposition was shorter for T. bisetosa than T.
nigricornis. The differences in oviposition behavior are reflected in the ultrastructure
of their ovipositors. The aculeus tip of T. nigricornis is pointed, whereas that of T.
bisetosa is rounded. The acanthae covering the ventral side of the eversible
membrane have pointed tips in T. nigricornis and are rounded in T. bisetosa.
Trupanea nigricornis has two pairs ofcentral ampulliform sensilla at the apex of the
aculeus while T bisetosa has three pairs. Therefore, ovipositor morphology reflects
oviposition behavior.
Key Words: Diptera, Tephritidae, Trupanea, oviposition behavior, ovipositor
structure, acanthae, sensilla, phenology, Encelia, Helianthus annuus
The flower head infesting tephritids, separate and are best identified accord-
Trupanea nigricornis (Coquillett) and T ing to their host plants (Knio et al.
bisetosa (Coquillett) occur in sympatry in 1996a). The adults show great morpho-
southern California (Foote et al. 1993). logical similarities. Males can be distin-
They are closely related, cryptic species guished by the color of their third
as they are similar morphologically and antennal segment and most females by
genetically, yet they do not interbreed the shape of the Y-shaped apical mark-
(Knio et al. 1996a, 2007). The immature ing on the wing (Cavender and Goeden
stages of both species are difficuk to 1983, Foote et al. 1993). Resource
386 PROCEEDINGS OF THE ENTOMOLOGICAL SOCIETY OF WASHINGTON
Utilization studies demonstrated that the ences in oviposition behavior between
larvae of both species exploited the the two species and the differences in the
flower heads of their host in a similar ultrastructure of their ovipositors. Sen-
manner and fed on a relatively similar sory structures on ovipositors play an
number of achenes (Knio et al. 2001). important role in determining host plant
Nevertheless, the two sympatric species suitability in tephritids (Schoonhoven
show major ecological differences. Tru- 1983). Further, timing of oviposition by
panea nigricornis behaves as a polypha- T. bisetosa females on their wild sun-
gous species infesting the heads of 33 flower hosts is critical for overcoming the
genera belonging to at least 8 tribes of problem of the hard bracts and plant
the Asteraceae, while T. bisetosa is resins. Thus, host plant usage and
narrowly oligophagous, attacking 6 spe- specialization is not only determined by
cies ofthe tribe Heliantheae, and mainly host plant chemistry, but by other
specializing on wild sunflowers in south- factors like the biophysical features of
ern Cahfornia (Goeden 1985, 1992). the plant, the synchronization of adult
Behaviorally, Trupanea nigricornis and emergence and female oviposition with
T. bisetosa show a number ofdifferences. host phenology, and availability of buds
Adult males differ subtly in courtship at stages suitable for egg laying (Berube
behavior and in the timing of mating in 1978b, Straw 1991, Zwolfer and Harris
the field. Males of T. nigricornis were 1971).
observed to court in the mornings In this study, we examine the phenol-
whereas those of T. bisetosa exhibited ogy of flower head development of the
courtship display in the afternoon (Knio most common hosts of T. nigricornis and
et al. 1996b). On the other hand, females T. bisetosa in relation to oviposition
differed greatly in their oviposition suitability and we investigate whether
behavior. Trupanea nigricornis females the differences in ovipositor morphology
always pierced the plant tissues during relate to differences in oviposition behav-
oviposition, such that the posterior end iors. This paper is the last of a series
ofthe egg was inserted into plant tissues. aimed at shedding light on the nature of
In most cases (ca. 80%), females de- polyphagy/monophagy in closely related
posited 1-3 eggs per flower head. On the sympatric and cryptic tephritids.
other hand, T. bisetosa females never
Materials and Methods
pierced or injured plant tissues during
oviposition and the eggs were vertically Phenology of flower head develop-
—
aligned loosely atop or among the ment. The phenology of flower head
corollas. In most cases (ca. 70%), T. development of Encelia farinosa (Gray)
bisetosa females placed 3-8 eggs per (Asteraceae) and Helianthus annuus L.
flower head (Knio et al. 1996b). (Asteraceae), the most common hosts of
Fecundity studies demonstrated that T. nigricornis and T. bisetosa, respective-
T. nigricornis females had a higher fe- ly, in relation to oviposition was studied
cundity than T. bisetosa. In no-choice in the field. The sites were: University of
CA
experiments, T. bisetosa did oviposit in California, Riverside Co., (site 1);
CA
the non-host flower heads of Encelia Casa Blanca, Riverside Co., (site 2),
CA
farinosa (Gray); however, T. nigricornis and Lake Perris, Riverside Co., (site
could not oviposit in the non-host flower 3). Only site 3 was observed for T.
heads ofHelianthus annuus L. because of bisetosa. At each location, 10-20 in-
the morphological features of this plant dividual flower heads on different spikes
(Knio et al., in press). These observations were labeled with masking tape attached
emphasize the importance of the differ- to the peduncle ofeach flower head. The
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109, 2 387
flower heads were in the earhest stage of at California Polytechnic State Universi-
development as 'unopened' buds. The ty, San Luis Obispo.
terminal flower heads were not labeled The terminology used in the descrip-
because they developed and matured tion of the ovipositor of T. nigricornis
faster than the apical ones found on and T. bisetosa follows that of White et
different peduncles of the same spike. al. (1999).
Development of these labeled flower
heads was followed in the field. Flower Results and Discussion
head diameters at their maximum width Phenology of flower head develop-
—
and lengths from the base of the re- ment. The development ofEnceliafari-
ceptacle to the tip of the florets were nosa flower heads was divided into the
measured using dial calipers at days 1,3, following stages: (1) 'closed bud' stage,
5, 10, 15, 18, 22, 28, 33, and 40. At each in which the bracts cover the immature
of these intervals, the labeled flower florets; (2) 'open, green bud' stage, in
heads were checked for oviposition which the immature florets are exposed,
wounds, and 20^0 other flower heads but still green; (3) 'open, light -green
at the same developmental stage were bud' stage, in which the immature florets
collected and dissected in the laboratory are light-green; (4) 'open, yellow bud'
to record the number of T. nigricornis or stage, in which the florets are turning
T. bisetosa eggs. yellow, but are not mature, and a few ray
Insect rearing for scanning electron florets are starting to develop; (5) 'blos-
microscopy. Trupaneanigricornis adults som' stage, in which the florets are at
were reared from flower heads of E. anthesis and the ray florets are fully
farinosa while those of T. bisetosa were developed; and (6) 'post-blossom' stage,
reared from wild sunflower heads, H. in which the achenes are mature and
annuus. The mature flower heads, con- hard, the florets begin to dry, and the ray
taining third instar larvae or puparia, flowers wilt (Fig. lA-F).
were placed in glass-topped, sleeved in- Trupanea nigricornis females ovipos-
sectary cages (34X32x35 cm) at the ited in the immature 'open' buds having
University of California, Riverside, at a green, light-green, or yellow color
60% RH and 12/12 (LD) photoperiod (stages 2, 3, and 4; Figs. IB-D). The
from 0500 1700 h. — flower heads that were suitable for
Ovipositor ultrastructure. The ovi- oviposition had mean diameters ranging
positors of T. nigricornis females (n = from 7.1-10.5 mm and mean lengths
4) and T. bisetosa females (n = 3) were ranging from 4.3-7.4 mm (Table 1).
fixed in 2% gluteraldehyde for 12 h, The period of time that a flower head
washed twice in distilled water, post- was suitable for oviposition was about
fixed in 2% osmium tetroxide overnight, 12 d: 8, 13, and 14 d in sites 3, 2, and 1,
washed twice in distilled water, dehy- respectively. This period of suitability
drated in an increasing series ofethanol, did not correspond to the entire bloom
then washed twice in absolute ethanol. period ofE. farinosa, as stems continued
The specimens were critically point- producing flower heads sequentially as
dried, mounted on stubs, and coated long as there was enough moisture in the
with a gold-platinum alloy before exam- soil. The total period of flower-head
ination with a scanning electron micro- development of E. farinosa, lasted ca.
scope (SEM), at 15 kV accelerating 40 d (5 7 weeks) in the field. The total
voltage. Micrographs were taken using period of development of T. nigricornis
Polaroid 55P/N ® films. The micrograph from oviposition to adult emergence
negatives are stored with D. H. Headrick from the mature heads ranged between
PROCEEDINGS OF THE ENTOMOLOGICAL SOCIETY OF WASHINGTON
Fig. 1. Stagesin flowerhead development ofEncelia.farinosa (bar = 1 cm). A, 'Closed bud' stage. B,
'Opengreenbud' stage'. C, 'Openlight-greenbud' stage; D, 'Openyellowbud' stage. E, 'Openyellowbud'
and 'blossom' stages. F, 'Post-blossom' stage.
25-30 d (ca. 4 weeks) in the field (Ta- in flower heads with light green florets;
ble 1). the rest (26% and 21%) were found in
Dissections ofE.farinosa flower heads heads with green and yellow florets,
A
confirmed the results ofthe field phenol- respectively (Fig. 2). correlation ex-
ogy experiment. The flower heads that isted between flower head size and
contained T. nigricornis eggs were open number ofeggs laid per head. The flower
buds with green, light-green or yellow heads with yellow florets were the largest
florets. These corresponded to the flower and contained the greatest mean number
head stages 2, 3, and 4. The majority ofeggs (2.9) per head. They were closely
(53%) of the eggs (n = 100) were found followed by flower heads with light-
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Table 1. Phenology of flower head development of Encelia farinosa at three locations in southern
California showing the stages in which eggs of Tnipcmea nigricornis are found.
PROCEEDINGS OF THE ENTOMOLOGICAL SOCIETY OF WASHINGTON
390
Fig. 2. Stages in flower head development ofHeliantlnis. anmms (bar = 1 cm). A, Small 'closed bud'
suitable for oviposition by T. bisetosa females. B, Advanced 'closed bud' stage'. C, 'Open yellow bud'
stage. D, 'Early blossom' stage. E, 'Late blossom' stage. F, 'Post-blossom' stage.
ed open flower heads, but that in the oviposition by T. nigricornis. This
field they preferred closed flower heads. suitability for egg laying covered only
The closed green buds that were suit- part of the entire flowering period of
able for oviposition by T. bisetosa wild sunflowers, as some plants flowered
females had diameters ranging from throughout the year in southern Califor-
mm
5.0-9.8 (Table 2). The period of nia under favorable environmental con-
time that a sunflower head was suitable ditions, e.g., mild frost-free winter and
for oviposition in the field was ca. 5 d ample rainfall. The total period of de-
(Table 2), and much shorter than the velopment for wild sunflower heads was
suitability period of E. farinosa for ca. 35^0 d in the field. Like T. nigri-
,
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109, 2 391
3.5
Yellow
LightGreen
2.5
2
Green
£ 1.5
0.5
4-6 6.1-9 1.1-11
Ftowerheaddiameter(mm)
Fig. 3. Mean number ofTrupaneanigricorniseggs found in three bud stages ofEnceliafarinosa heads
=
(n 100),
cornis, the period of development of T. closed buds with straight bracts (stage 1
bisetosa from egg to adult ranged from Fig. 3A). The mean diameter of these
mm
30-35 d (ca. 4-5 weeks). buds ranged from 5.0-7.8 and their
Dissections of field-collected sunflow- mean length ranged from 6.5-8.5 mm
er heads confirmed these findings on (Table 2). The dissected buds contained
phenology. The flower heads that con- mainly 3-5 eggs as previously reported
tained T. bisetosa eggs (n = 100) were all by Knio et al. (1996b).
Table 2. Phenology offlower head development ofHelianthusanimus showing the stages suitable for
egg laying by Trupanea bisetosa.
Days
—
392 PROCEEDINGS OF THE ENTOMOLOGICAL SOCIETY OF WASHINGTON
Similar to T. bisetosa, the window for of several Urophora species have been
oviposition suitability was narrow and found to be correlated with the diameter
restricted to a short phase, 'unopened of flower heads exploited, implying
buds', in Trupanea conjuncta (Adams), '"evolutionary responses of these phyto-
Tephritis dilacerata Loew and T.forniosa phages to a particular structure of the
Loew (Goeden 1987; Berube 1978a, b). host plants"(Zw61fer 1987). Moreover,
Trupanea conjuncta females only laid in addition to the length of the ovipos-
eggs in the small green buds of their itor, the aculeus tip bears a number of
host, Trixis californica Kellog (Astera- sensilla that are used to guide the female
ceae) and this stage lasted about 5 d during oviposition. In the following
(Goeden 1987). Tephritis dilacerata fe- section, we examine the ultrastructure
males deposited their eggs in the closed ofthe ovipositors of T nigricornis and T
buds of Sonclnis arvensis L. (Asteraceae) bisetosa in order to better understand the
and weaved their ovipositors slowly in differences in their oviposition behavior.
between the bracts in order to avoid Ultra-structure of the ovipositors.
piercing the host tissues and releasing The external anatomy of the ovipositors
sticky latex (Berube 1978a). Both T of T. nigricornis consists of the modified
dilacerata and T. formosa timed oviposi- seventh abdominal segment or oviscape
tion to coincide with the stage of un- (syntergosternite 7), an eversible mem-
opened buds when they were at their brane, and an aculeus (Norrbom and
maximum growth as this stage was the Kim 1988, White et al. 1999) composed
most suitable for gall induction by the of three, long, parallel processes, two
young larvae (Berube 1978b). On the ventral and one dorsal (Stoffolano and
other hand, similar to T. nigricornis, the Yin 1987), which are the eighth sternites
suitability period for oviposition by the or ventral flaps, and the eighth tergite,
tephritids, Tephritis bardanae (Schrank) respectively (White et al. 1999) (Fig. 4C,
and Cerajocera tussilaginis (F.) on Arc- F).
tium minus (Hill) Bernh. (Asteraceae) The oviscape of T. nigricornis is
was 10-11 d; however, there was no conical in shape and heavily sclerotized
overlap in the oviposition suitability (Fig. 4A). It measured (from tip to base
periods as T. bardanae oviposited early on the ventral side) 1.05 ± 0.014 (range:
in the smaller unopened buds while C 0.89-1.16) mm in length (n = 25).
mm
tussilaginis followed a late attack strate- The eversible membrane (0.21 as
gy and deposited eggs in the pre-flower- greatest width) of T. nigricornis also is
ing heads. Like T nigricornis and T heavily sclerotized (Fig. 4B). It is cov-
bisetosa, these flies never laid eggs in ered with acanthae, scale-like, cuticular
flower heads that had started to flower projections (Fig. 4D-E). When the ovi-
(Straw 1989). Females of Chaetostomella positor is retracted only the oviscape is
undosa (Coquillett) also oviposited in the apparent (Fig. 4A-B); the rest of the
closed and open buds of their host, ovipositor, including the eversible mem-
Cirsium cymosum (Greene) J. T. Howell brane and aculeus, is housed within the
(Asteraceae), but like T nigricornis, they seventh abdominal segment.
showed a preference to open buds (Steck The retractable eversible membrane
1984). connects the oviscape to the aculeus
In selecting heads suitable for ovipo- (Fig. 4A-C). The eversible membrane is
sition, female tephritids seem to compare visible only when the ovipositor is
their body size and length of their extended. It is covered with acanthae
oviscape to flower head dimensions that point anteriad. The acanthae grad-
(Straw 1989). The length of the oviscape ually increase in size from the tip of the
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109, 2 393
15KU X40 0087 100. 0U 15KU X180 0009 100. 0U
Fig.4. ScanningelectronmicrographsoftheovipositorofTrupcmeanigricornis. A, Ventralviewofthe
ovipositorshowing (1) the oviscape, (2) the eversible membrane, and (3) the tip ofthe aculeus. B, Ventral
view of the eversible membrane showing (4) the pointed acanthae. C, Ventral view of the extended
ovipositorshowing(1) the oviscape, (2) basal region and (5) distal region theeversible membrane, and (3)
the aculeus. D, The eversible membrane. E, Pointed acanthae on theeversible membrane. F, Ventral view
ofthe basal part ofthe eversible membrane, and the aculeus showing (6) the eighth tergite and (7) the
ventral flaps or eighth sternites.
distal region to the base of the basal The aculeus of T. nigricornis has
region of the eversible membrane a sharply pointed apex. It bears two
(Fig. 4D). These acanthae are sharply ventral sclerites (eighth sternites) and
pointed in T. nigricornis (Fig. 4E). a dorsal sclerite (eighth tergite) (Fig. 4F).
394 PROCEEDINGS OF THE ENTOMOLOGICAL SOCIETY OF WASHINGTON
Fig. 5. Scanning electron micrographs ofthe ovipositor of Trupanea nigricornis. A, Lateral aspect of
the dorsal process with the row of(1) hair-like sensilla. B, Hairhke sensillum on the dorsal process. C,
Apical region of the aculeus showing (2) the two ventrolateral grooves with (3) the elongated sensilla
(threepergroove), (4) theshallowampulliformsensilla, (5)theellipsoidalampulliformsensilla, and(6)the
central ampulliform sensilla. D, Central ampulliform sensillum sunken in an oval socket.
The ventral sclerites appear as two sensilla (Fig. 5A) that have blunt tips.
parallel, elongate structures with blunt The sensilla are surrounded by a shallow
ends on the ventral side ofthe ovipositor. depression (Fig. 5B). Such sensilla also
They are shorter than the dorsal sclerite occur on the ventral sclerites (Fig. 5B).
and only visible when the ovipositor is These hairlike sensilla are similar to
extended. The ventral sclerites, which are those described for the apple maggot
joined by a flexible, median and infolded (Stoffolano and Yin 1987). They are
membrane, do not completely meet numerous (50-60) on the dorsal and (11-
(Stoffolano and Yin 1987, Zacharuk et 12) ventral sclerites of the apple maggot
al. 1986); a ventral groove extends ovipositor; they were identified as mech-
between them and terminates in the anoreceptors innervated by a single neu-
cloaca, the common opening to the ron, and were not associated with
reproductive and digestive tract. The chemoreception (Stoffolano and Yin
dorsal sclerite (eighth tergite) measured 1987).
0.14 mm at greatest width (n = 4) and The tip of the dorsal sclerite (eighth
1.27 mm in length (n = 4) (Fig. 4F). It tergite) of the aculeus of T. nigricornis
bears a ventrolateral row of hairlike bears different types ofsensilla that show
