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Plant–Insect Interactions
A New Gall Midge Species of Asphondylia (Diptera:
Cecidomyiidae) Inducing Flower Galls on Clinopodium
nepeta (Lamiaceae) From Europe, Its Phenology, and
AssociatedFungi
UmbertoBernardo,1 FrancescoNugnes,1 LiberataGualtieri,1 RosarioNicoletti,2,3
PaolaVarricchio,2 RaffaeleSasso,4 and GennaroViggiani2,5
1CNR, Institute for Sustainable Plant Protection, SS of Portici, Portici (NA), Italy, 2Department of Agriculture, University of Naples
‘Federico II’, Portici, Italy, 3Council for Agricultural Research and Agricultural Economy Analysis, Rome, Italy, 4ENEA C.R. Casaccia,
Laboratorio SSPT-BIOAG-SOQUAS, Roma, Italy, and 5Corresponding author, e-mail: genviggi@unina.it
Subject Editor: JoeLouis
Received 14 November 2017; Editorial decision 12 February 2018
Abstract
A new gall midge, Asphondylia nepetae sp. n.Viggiani(Diptera: Cecidomyiidae), causing flower gall on Clinopodium
nepeta (L.) Kuntze(Lamiaceae), is described from Europe. The morphological characteristics of adult, larvae, and
pupa are described and illustrated. Molecular approach (by sequencing 28S-D2, ITS2, and COI) confirmed that
A.nepetae is a distinct species. The development of the gall is always associated with the presence of the fungus
Botryosphaeria dothidea(Moug.: Fr.) Ces. and De Not. (Botryosphaeriales: Botryosphaeriaceae). The new species
can complete several generations per year, on the flowers of the same host plant and its adults emerge from late
spring to autumn. Pupae overwinter inside peculiar flower galls in a state of quiescence. The impact of the pest
is highly variable with a percentage of flowers infested that ranged between 3 and 57.5% in the sampled years.
Insect mortality was, at least in part, due to parasitoids that attack the young stages of the midge. Among them, the
dominant species was Sigmophora brevicornis (Panzer)(Chalcidoidea: Eulophidae).
Key words: Botryosphaeria, Cladosporium, lesser calamint, overwintering, quiescence
Lesser calamint (Clinopodium nepeta (L.) Kuntze [=Calamintha
nepeta (L.) Savi)], Lamiaceae) is a bushy, rhizomatous medicinal herb
growing in South, Western and Central Europe, and East England
(Bozovic and Ragno 2017). During a study started in 2013 on the
egg parasitoids of the leafhoppers associated with this plant (Nugnes
etal. 2017), some owers transformed in galls were observed. From
them, adults of a gall midge, subsequently identied as Asphondylia
sp., emerged. This interesting nding suggested extending our study
also on this midge because at that time no literature data reported on
the association lesser calamint-Asphondyliaspp.
The genus Asphondylia Loew (Diptera: Cecidomyiidae)
belongs to the tribe Asphondyliini, a wide group of gall makers,
and includes 320 nominal species (Gagné and Jaschhof 2014). Due
to morphological similarities, the adult discrimination of different
species remains very difcult (Silvestri 1908, Kolesik et al. 2010).
In some cases, morphological characters of larvae and pupae are
useful in distinguishing even among species associated with a cer-
tain genus or species of the host plant (Gagné and Waring 1990).
At present, the DNA analysis is considered the most effective tool
for the species discrimination when combined with biological, eco-
logical, distributional, and morphological information (Yukawa
etal. 2003, Kolesik etal. 2010).
Unfortunately, most of the species of Asphondylia remain poorly
known; among them, there are the species associated with Lamiaceae.
On the other hand, the taxonomy of many genera of this plant family
is in a state of continuous ux (Bräuchler etal. 2008). Among the 20
Asphondylia species recorded on Lamiaceae (Gagné and Jaschhof 2014),
17 are from the Palaearctic region; 8 of them were described by Fedotova
(1985, 2003, 2008). Unfortunately, some species, such as A.calaminthae
(Kieffer 1909), have been named solely based on the host plant and on
the induced gall, which does not show any peculiarity thus preventing
any comparison. Again, the taxonomic characters of many other species
are undened and useless. In conclusion at present, at least in this group
of species, the host plant remains the only valuable reference.
The only species of Cecidomyiidae inducing ower galls on
C. nepeta mentioned as Cecidomyiidae sp1 nov. and probably an
Asphondylia sp., was recorded by Cerasa (2015) in Sicily. The pres-
ent paper aim is to characterize the species reared on C. nepeta
Environmental Entomology, 47(3), 2018, 609–622
doi: 10.1093/ee/nvy028
Advance Access Publication Date: 9 March 2018
Research
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by an integrative approach, laying the foundations for a complete
integrative systematic revision of species belonging to Asphondylia
genus associated with Lamiaceae. The specimens emerged from
C.nepeta in the course of the present study, in several Italian regions
and Croatia were morphologically and molecularly analyzed, and
several biological and ecological characteristics were observed and
described. The integrative approach involves the characterization of
the fungal symbiont and its ecological role, as well as the identica-
tion of the complex of parasitoids developing on the new species.
The new species is named here, and taxonomic data are given for
its identication.
Material and Methods
Insect Sampling and Morphological
Characterization
Samples of stems with owers of C.nepeta were collected in several
locations of Italian regions (Apulia, Basilicata, Calabria, Campania,
Latium) from September 2014 to January 2017. Amore intensive
sampling was made in Rivello (Basilicata) and Portici (Campania) to
study the unknown biology of the gall midge. To collect the emerged
specimens, the sampled material was maintained in bags or boxes
kept at room temperature and photoperiod (from November to
April: 20–22°C and 10:14 (L:D) h; from May to October: 23–25°C
and 16:8 (L:D) h). Flowers of different ages were dissected to collect
the young stages of the gall midge and to observe their development
and behavior. Adults, larvae, and pupae were preserved in 70% etha-
nol. The adults for molecular analysis were singularly placed in vials,
containing 95% ethanol and preserved at −20°C until use. Several
adults, larvae, and pupae were slide mounted using Balsam-phenol
as a permanent medium. Mounted specimens were examined and
measured under a Zeiss Axiophot microscope.
Flowers of C. nepeta were randomly sampled, for each stem,
to evaluate the percentage of both the owers infested by the gall
midge and the parasitic activity. Afurther sampling of ower galls
was carried out during autumns 2015–2016 and winter 2016 to
study the overwintering of the gall midge. For this purpose single
ower galls were isolated in vials to obtain the emergence of the
organisms living inside. Some of them were kept at room temper-
ature and others at 25°C. Other overwintering ower galls were
measured and dissected.
All data are presented as mean values with standard deviation (±SD).
Molecular Characterization of GallMidge
Total genomic DNA was extracted from whole single specimens
listed in Table1 by using a Chelex and proteinase K based method as
in Gebiola etal. (2009). After DNA extraction, samples were rinsed
with distilled water, and slide-mounted as above described.
Three genes were sequenced: the mitochondrial cytochrome c
oxidase subunit I (COI), the Internal Transcribed Spacer 2 (ITS2)
and the expansion segment D2 of the 28S ribosomal subunit (28S-
D2). The COI region was amplied by using primer pair LCO-1490/
HCO-2198 (Folmer etal. 1994) with the thermocycler condition as
in Gebiola etal. (2009).
The ribosomal gene ITS2 was amplied with primers ITS2F
(Campbell etal. 1993) and ITS2Rev-Trich (Stouthamer etal. 1999)
in PCR reactions as in Gebiola etal. (2010). For the amplication
of the 28S-D2 portion, primers D2F and D2R (Campbell et al.
1993) setting the PCR reactions and cycling conditions were used as
described in Gebiola etal. (2009).
PCR products were checked on a 1.2% agarose gel stained with
SYBR Safe (Invitrogen) and directly sequenced. Chromatograms
were assembled using BioEdit 7.0 (Hall 1999) and edited manu-
ally. COI sequences were virtually translated into amino acids to
detect frameshift mutations and nonsense codons using EMBOSS
Transeq (http://www.ebi.ac.uk/Tools/st/emboss_transeq/ [accessed 3
April2017]). COI sequences were aligned manually, ITS2 sequences
and 28S-D2 sequences were aligned using the G-INS-I algorithm in
Table1. Specimens used for this study and analyses performed
Code Species Locality Longitude Latitude Altitude
m a.s.l.
Host Sex Mth Genbank accession code
COI 28S-D2 ITS2
An_1
Asphondylia
nepetae
sp. n.
Rivello, Italy 40°03′ N 15°45′ E 435
Clinopodium
nepeta
♂a MF479666 MF479684 MF479648
An_2 ♂a MF479667 MF479685 MF479649
An_3 ♂a MF479668 MF479686 MF479650
An_4 ♂a MF479669 MF479687 MF479651
An_5 ♂a MF479670 MF479688 MF479652
An_6 ♀a MF479671 MF479689 MF479653
An_7 Pozzuoli, Italy 40°50′ N 14°04′ E 2 ♂b MF479664 MF479682 MF479646
An_8 ♀a MF479672 MF479690 MF479654
An_9 ♂a MF479673 MF479691 MF479655
An_10 Napoli,
Camaldoli,
Italy
40°51′ N 14°12′ E 323 ♂a MF479674 MF479692 MF479656
An_11 Portici, Italy 40°48′ N 14°20′ E 29 ♂a MF479675 MF479693 MF479657
An_12 ♂a MF479676 MF479694 MF479658
An_13 Orria, Italy 40°17′ N 15°11′ E 236 ♀a MF479677 MF479695 MF479659
An_14 ♀a MF479678 MF479696 MF479660
An_15 ♀a MF479679 MF479697 MF479661
An_16 ♂a MF479680 MF479698 MF479662
An_17 ♂a MF479681 MF479699 MF479663
Cr_9 Slatine,
Croatia
43°29′ N 16°20′ E 15 ♀c MF479665 MF479683 MF479647
Mitochondrial haplotype: (Mth).
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MAFFT 7 (Katoh and Standley 2013). Generated sequences were
deposited in GenBank with accession numbers reported in Table1.
Only one of multiple identical DNA sequences was retained for phy-
logenetic analysis.
In addition to our specimens of Asphondylia, to perform a com-
plete phylogenetic reconstruction of this group, all the veried and
codifying sequences of other related species available in Genbank
were included in the analyses, while Pseudasphondylia matatabi
(Yuasa and Kumazawa) was used as outgroup to root the COI
trees. Due to the lack of homologous ITS2 sequences of adequately
related taxa in GenBank database, Asphondylia caudicis Gagné and
Asphondylia atriplicis (Townsend) were used as outgroups as sug-
gested by preliminary phylogenetic analyses using midpoint-rooting
option.
Phylogenies on ITS2 and COI alignments were reconstructed
using maximum likelihood (ML) and Bayesian inference (BI), uti-
lizing RAxML 7.0.4 (Stamatakis 2006) and MrBayes 3.2 (Ronquist
etal. 2012), respectively. The GTR+G and GTR+G+I evolutionary
models selected by jModeltest (Posada 2008) were used for the ITS2
and COI datasets, respectively.
ML branch support was based on 10,000 rapid bootstrap pseu-
doreplicates, and clades were considered supported when bootstrap
was >70%. For BI, two parallel runs of four simultaneous Monte
Carlo Markov chains were run for 1 million generations for ITS2
and 8 million generations for COI, trees sampled every 1,000 gen-
erations with a burnin value set at 25%. Tracer 1.6 (Rambaut etal.
2014) was used to assess the convergence.
Uncorrected intra- and interspecic p-distances based on COI
were calculated using MEGA4 (Tamura et al. 2007). Taxa were
grouped based on most recent references (Yukawa etal. 2003, Joy
and Crespi 2007, Dorchin etal. 2015, Uechi etal. 2017).
Parasitoids
Flower galls of C.nepeta were dissected to study the young stages of
the parasitoids and their relationship with the host. The parasitoids
emerged from the ower galls were mounted on pins and slides for
their identication according to Graham (1987).
Fungi Associated With Gall Development
Isolations from the mycelium developing inside the galls, from seeds
and different ower parts, and from the body surface of larvae and
pupae of the gall midge and its parasitoids were carried out on pota-
to-dextrose agar (PDA, Oxoid) amended with 200mg/liter strepto-
mycin sulphate. The fragments of gall or ower tissues to be placed
on the agar medium were cut and transferred by using pins previ-
ously sterilized in 96% ethanol. Insect larvae and pupae were placed
onto the agar medium without preliminary dissection. Hyphal tips
from the emerging fungal colonies were transferred to fresh PDA
plates for morphological identication and storage of pure cultures.
Sporulation of sterile isolates of the botryosphaeriaceous morpho-
type was induced in cultures prepared in plates containing 2%
water agar (WA) topped with sterilized pine needles, which were
kept at room temperature under near-UV illumination (Crous etal.
2006). Besides preliminary observations of morphological features
through the microscope (Olympus BX51), species identication
was performed through rDNA-ITS sequencing. To this regard, total
genomic DNA was extracted from fresh mycelium taken from either
pure cultures or directly from the galls as described in Cubero etal.
(1999). Concentration and purity of DNA samples were assessed by
measuring the absorbance with Varioskan Flash (Thermo Scientic).
PCR amplication was carried out with the specic primers ITS1-F
(Gardes and Bruns 1993) and ITS4 (White et al. 1990). Cycling
parameters were 40 cycles of a denaturation at 94°C for 1 min,
annealing at 53°C for 45s and elongation at 72°C for 1 min. To
ensure good quality sequences over the entire length of the ampli-
cons, the forward and reverse sequences were aligned with MUSCLE
(Edgar 2004), and only overlapping regions were used as a query for
BLASTn searches in the NCBI nr/nt database.
The eventual establishment of antagonistic relationships between
isolates of the most common species was assessed in vitro in dual
cultures prepared on PDA and WA, respectively to evaluate inhibi-
tion of growth and mycoparasitic interactions. Cultures were grown
in the dark at 25°C until mycelium from the opposed colonies
merged. Rectangular blocks from the area where hyphae interacted
in WA cultures were cut, stained with lactophenol cotton blue, and
observed through the microscope at 600× magnication.
Results
The specimens of Asphondylia emerging from ower galls on
C.nepeta (Fig.1a), collected in several Italian regions and Croatia,
were recognized as belonging to an undescribed species and it is here
described.
Asphondylia nepetae sp. n.Viggiani Morphological
Characterization
Male
Eye facets close together, hexagonoid, eye bridge 8 to 10 facets long
medially. Palpus 3-segmented, rst segment about half-length of
the second, third segment 1.5–2.3 longer than the second (Fig.1b).
Occipital area grey with a whorl of long setae. Antenna 2+12 seg-
mented, with scape subtrapezoidal, distally enlarged, twice as long as
the transverse pedicel (Fig.1c); agellomeres cylindrical, with dense
circumla; rst agellomere four times as long as wide, the subse-
quent gradually shorter, distal two agellomeres as in Fig.1d. Wing
2.4 as long as wide, with R5 vein ending near wing apex and other
characters as in Fig.1e. Legs with the following length ratio between
femur, tibia and tarsomeres: foreleg (50:53:6:39:17:11:8), mid-leg
(48:45:6:30:13:8:7), hind leg (57:55:7:33:18:10:7); ventrodistal spine
of the rst tarsomere bent at distal half (Fig.1f). Claws simple, slightly
longer than empodium (Fig.1g). Genitalia showing the typical shape
for Asphondylia (Fig. 1h); male terminalia slightly wider than long
(5:4.5); gonocoxite compact, one-third longer than wide, with subtri-
angular and setose lobes, twice as wide as long and with long setae on
the distal margin; gonostylus ovoidal, slightly longer than wide, with
two apical and sclerotized teeth (Fig. 1i); cerci developed into two
subcircular and setose lobes; hypoproct smaller, distally divided into
two triangular and setose lobes; aedeagus with a large base followed
by a distally pointed rod about nine times as long aswide.
Body color: grey-brown, with black eyes, fronto-vertex yel-
low-grey. Area of the wing sclerites and not sclerotized parts of the
abdomen orange.
Body length: 2.3–2.7mm (n=10).
Female
Antenna 2+12 segmented, about one-fourth shorter than in male,
agellomeres with the following length/maximum width ratio: F1
(43/7), F2 (33/8), F3 (32/8), F4 (30/8), F5 (30/8), F6 (28/8), F7
(28/7), F8 (21/8), F9 (18/8), F10 (13/10), F11 (10/10), F12 (8/8); last
agellomere subglobular (Fig. 1j). Abdominal tergites 1–7 rectan-
gular, covered with sparse setae of different length; tergite 7 about
1.2 times as long as tergite 6; tergite 8 small, saddle-like, without
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setae; seventh abdominal sternite 2.0–2.2 longer than sixth (Fig.1k).
Terminalia typical of Asphondylia (Fig.1l), length of the needle part
of the ovipositor in average 1.21±0.082mm (n=20), 2.6 (2.3–3.2)
times as long as the length of the seventh sternite. Other characters
as in the male.
Molecular Characterization of GallMidge
Sequenced mtCOI fragments showed low haplotype variability,
indeed only three different haplotypes were recorded. Mitochondrial
haplotype (Mth a) resulted the most common (Table1) indeed it was
shared by the 89% (16 on 18)of the analyzed specimens. Differences
between the three haplotypes were minimal, only 2 to 3bp but there
was a difference in the deduced amino acid residues. Only two spec-
imens with haplotypes 2 and 3 were recorded: the rst (Mth b) was
shown by one out three specimens from Pozzuoli (Lago d’Averno),
the second (Mth c) from a Croatian locality where a single specimen
was reared (Table1).
ML and BI analyses of COI resulted in trees of identical topology
and suggested that the specimens collected on C.nepeta belong to
the same species that is very different from all other species charac-
terized up to now by molecular approach (Fig.2).
All the analyzed specimens shared the same ITS2 sequence.
Both ML and BI phylogenetic reconstructions showed a strongly
supported clade highly distinct from the other Asphondylia species
(Fig. 3). Furthermore, ITS2 tree demonstrated that sometimes dif-
ferent Asphondylia species share the same ITS2 sequences as in the
cases of A.clavata - A.villosa - A.pilosa and A.fabalis - A.silicula.
A unique sequence of the 28S-D2 genes was in common between
all the specimens reared from C.nepeta. The unique complete sequence
available in GenBank belonged to Asphondylia sarothamni (Loew)
and showed a 2% of difference with our sequence (8bp and 3gaps).
Uncorrected p-distances based on COI within and between
groups are reported in Supplementary Table S1. The mean distance
across the genus was 15.7%. The highest interspecic distance was
between A.silva and A.verbasci (26.5%) while the lowest between
A.pilosa and A.clavata (0.23%).
The lowest and the highest p-distances between the specimens
reared from C.nepeta and the other taxonomical groups were respec-
tively 9.3% with A.conglomerata and 17.3% with Asphondylia sp.2.
The mean intraspecic distance was 1.5%, A.auripila showed the
highest value (7.5%) and, among the species not sharing a unique
haplotype, A.rosetta showed the lowest values (0.14%). The intraspe-
cic distance between specimens reared from C.nepeta was 0.4%.
Examined Material
Holotype
Male (on slide). ITALY: Rivello (PZ), 15 May 2015, from flower
gall on C. nepeta collected on 4 May 2015; coll. G.Viggiani.
Fig.1. Asphondylia nepetae: (a) female. Male: (b) head, front view; (c) basal antennal segments; (d) last antennal segments; (e) wing; (f) first tarsomere; (g) claw;
(h) terminalia; (i) gonostylus. Female: (j) last antennal segments; (k) abdomen, lateral view of the seventh sternite; (l) terminalia.
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Paratypes: 1♂, same data of the holotype, but flower gall col-
lected on 2 March 2015 and laboratory emergence on 20 May
2015; 1♂, same data, but flower gall collected on 21 February
2015 and laboratory emergence on 14 May 2015; 1♂, same data,
but flower gall collected on 4 May 2015 and laboratory emer-
gence on 12 May 2015; 1♂, same data, but flower gall collected
on 30 March 2015 and laboratory emergence on 20 May 2015;
1 ♂, same data, but flower gall collected on 25 August 2014
and laboratory emergence on 1 September 2014; 1♂, Corbara
(SA), from flower gall collected on 21 April 2016 and laboratory
emergence on 9 June 2016; 1♂, same data of the holotype, but
flower gall collected on 29 March 2016 and laboratory emer-
gence on 11 May 2016; 1 ♂, same data, but flower gall col-
lected on 20 September 2015 and laboratory emergence on 29
November 2015 2♀, S. Giorgio a Cremano (NA), 1 October
2014, from flower gall collected on 20 September 2014; 1♀,
Rivello, 21 May 2015, from flower gall collected on 30 March
2015; 1♀, Napoli, Soccavo, 20 April 2016, from flower gall col-
lected on 18 November 2015; nine slides with last instar larvae;
eight slides with pupal cases.
Fig. 2. Maximum likelihood tree based on cytochrome c oxidase subunit I data. Bootstrap values ≥70 and posterior probabilities ≥0.95 for a topologically
identical majority rule Bayesian consensus tree are shown above branches. ns=not significant.
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DNA (Table1)
Holotype and paratypes are deposited in the entomological col-
lection of the Dipartimento di Agraria dell’Università degli Studi
‘Federico II, Portici, Napoli, Italy’.
Other Information and Descriptions
Etymology
The specic name nepetae is referring to the host plant C.nepeta.
Distribution
ITALY: Basilicata (Rivello, PZ, Matera); Calabria (Paola, CS),
Campania (Caserta; Corbara, Orria-Santoianni [SA]; Napoli-
Bagnoli; Napoli-Camaldoli; Napoli-Soccavo; Portici, Pozzuoli-
Astroni, Pozzuoli-Lago d’Averno, Pozzuoli-Lucrino, Procida-Vivara,
S.Giorgio a Cremano [NA]); Lazio (Bracciano, RM), Puglia (Bari),
Umbria (Terni); CROATIA: Čiovo island (Slatine).
YoungStages
Egg
The newly emerged females show an ovary with about 25 ovarioles,
each with 7–8 mature oocytes (Fig.4a). They are oval and whitish.
Dimensions (n=20): length (average): 0.19±0.045mm and width:
0.10 ± 0.014 mm. The deposited egg (Fig. 4b) has similar shape
and color, without apparent sculpture on the chorion. Dimensions
(n=10): 0.15±0.018mm × 0.10±0.008mm.
First InstarLarva
Body: elongate, tapering anteriorly, rather well segmented (Fig.4c and
d); Color: cream; Head: small, with short antennae (Fig.4e). Thorax and
abdomen without apparent dermal structures, except a pair of robust and
ventrally curved spines on the last body segment and a U-shaped sclerite,
laterally with two small spine-like projections on penultimate sternite
(Fig.4f and g). No apparent tracheal system and spiracles. Dimensions
(n=20): length: 0.30±0.082mm and width: 0.07±0.018mm.
Second InstarLarva
Similar to the last instar larva, but smaller, with a smooth cuticle
and without sternal spatula (Fig.4h). Dimensions (n= 9): length:
1.00±0.223mm and width: 0.35±0.091mm.
Last InstarLarva
Body: orange, deeply segmented, tapering posteriorly (Fig. 4i);
Head: small with very short conical antennae. Sternal spatula quad-
ridentate (Fig.4j), the inner pair of teeth usually slightly shorter than
the outer. Five lateral papillae with setae are present on each side of
the spatula, one single and two pairs. Body segments present dense
verrucose sculpture on dorsum and are without spines. Last segment
distally slightly bilobed and without apparent setae and papillae
(Fig.4k). Aperipneustic respiratory system with nine pairs of spira-
cles (on prothorax and the eight abdominal segments). Dimensions
(n=20): length: 2.70±0.580mm and width: 1.10±0.009mm.
Fig.3. Maximum likelihood tree based on ITS2 data. Bootstrap values ≥70 and posterior probabilities ≥0.95 for a topologically identical majority rule Bayesian
consensus tree are shown above branches. ns=not significant.
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Pupa
At the beginning of its formation the pupal body appears ochreous
yellow, then brick red; when mature, head, and antennae, wing, and
leg cases are blackish (Fig.5a and b). Antennal horns short, subtrap-
ezoidal, closely approximated at base, obliquely trunked (Fig.5c).
Upper frontal horn bid; lower frontal horn (Fig.5d) like a small
crest around grooved, with a middle tooth and some smaller lateral
projections. Lower face usually with three papillae on each side,
one of which with a rather long seta (Fig.5c). Prothoracic spiracles
length 50–75µm, trachea at basal third. Abdominal segments I–VII
dorsally with a verrucose sculpture on a basal and distal band; seg-
ments II–VIII with two main rows of stout spines in the middle part;
distal row regular with spine length of 15–25 µm, pre-distal row
not always regular and with slightly smaller spines; last abdominal
Fig.4. (a) Ovarioles. (b) Laid egg (arrow). (c) The full embryo in the egg (arrow indicates the terminal spine-like projections). (d) First instar larva: (e) head;
particular of last abdominal segments with the terminal spines, (f) lateral view and (g) dorsal view. (h) Second instar larva. (i) Last instar larva: (j) head and
sternal spatula; (k) last abdominal segment.
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segment, in addition, with a pair of stouter and curved spines lat-
erally the last row of spines (Fig.5e). Dimensions (n= 20): length:
2.10±0.307mm and width: 0.90±0.163mm.
Pupae can be sexed according to the length of the antennal cases
and the character of the last abdominal segment in a back view. In
the female pupa (Fig.5f) the distance between the end of the anten-
nal cases and the distal angle of metasternum is not longer than the
length of the latter; in the male pupa (Fig.5g) this distance is longer.
In addition, the back view of the last segment in the female pupa
shows the track of two small, not well-marked, ventral lobes sepa-
rate by a scar (Fig.5h). In the male, the ventral lobes are larger, well-
marked, and with the scar placed above them (Fig.5i).
Gall
The ower gall (Fig. 6a, left) is very similar in size (length:
3.21± 0.705mm, width: 1.66±0.348 mm) and external shape to
the uninfested mature ower (Fig.6a, right). The uninfested mature
ower of C. nepeta can be recognized from the ower gall as its
lateral proles are not uniformly convex, but show a distal concave
prole followed basally by a more pronounced convexity.
The ower gall is formed by the normal calyx that wraps the
true gall. The latter is made by the coalescent, except the distal part,
modied petals, forming an ovoidal body in which the midges and
the associated fungi develop. The ower galls found from June to
late autumn show externally a normal, greenish or brownish calyx
and internally an ochreous or ferruginous corolla. The wall of the
gall (modied corolla) is rather soft, without any remains of other
internal organs.
Seasonal Activity
In our study area the lesser calamint owers from late May up to
November, and in some warmer places even later (Fig.6b). The o-
ral racemes develop from the top of each stem. Aspondylia nepetae
females start to oviposit in the closed owers of 2.5–4mm in length
Fig. 5. (a) Pupa, dorsal view. (b) Mature pupa, lateral view. Pupa: (c) antennal and facial horns; (d) particular of the lower facial horn, (e) last abdominal
segments, dorsal view. (f) Female pupa, ventral view of head and thorax. (g) Male pupa, ventral view of head and thorax. (h) Female pupa. Last abdominal
segment in the back view. (i) Male pupa. Last abdominal segment in the back view.
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when the petiole of the main oral raceme reaches the length of
0.5–1cm. Females oviposit in each ower a single egg near the basis
of the ovarium (Fig.4b). The young larva (Fig.6c) starts feeding on
this organ and then on others internal ower elements. In the mean-
time, a white fungal mycelium develops in the ower cavity (Fig.6d).
The midge larva develops feeding on the internal content of the ower
and at the end of its development remains in the cavity formed by the
modied petals (Fig.6e), internally covered with a white or blackish
fungal mycelium. The pupation takes place in this cavity, and the new
adult emerges from the top of the infested ower through an exit
hole prepared by the pupal horns (Fig. 6f). The rst generation of
A.nepetae develops from late May-beginning of June up to late June-
beginning of July. Then several generations overlap, probably 4–5,
until late autumn. In the laboratory, adults of A.nepetae emerged
from sampled ower galls from late June up to December. Eggs, rst
larvae and other young stages of the midge have been reared every
month from the beginning of June to December, showing the continu-
ous reproductive activity of A.nepetae, but from late December and
in some places only overwintering pupae have beenfound.
The gall midge is widespread; the position of the ower galls on
stems, whorls, and racemes and the degree of ower infestation are
very variable (3–57.5%) (Tables 2 and 3).
Overwintering
The oral racemes of C.nepeta become progressively drier starting
from the late autumn as well as the ower galls of A. nepetae in
which the pupae of the midge overwinter. The overwintering ower
galls show externally a dried calyx covering an ovoidal hard body,
the true gall, which is like a seed (Fig.6g) with the gall wall inter-
nally covered with a thick and compact layer of fungal mycelium.
The overwintering ower galls can remain attached to the plant stem
or fall to the soil. It seems an efcient structure to protect the midge
pupa for at least 4–5 months (Fig.6h). The dried calyx has a normal
shape and covers the true gall, which appears like a seed with a
tough wall. In fact, in this case, the gall plays the role of puparium.
Frequently, as consequence of the pupal activity, a cup is detached
from the top of the gall, and the adult can emerge through this large
exit hole. The pupae overwinter in a state of quiescence (not in a dia-
pause). In fact, this state can be overcome with temperatures in aver-
ages around 20°C (Table4). At the constant temperature of 25°C,
the pupal quiescent state is overcome in 1 month(Table5).
Parasitoids
The reproductive activity of the parasitoids of A. nepetae starts
as soon as the midge oviposits in the calamint owers in late
Fig. 6. (a) Flower gall of Asphondylia nepetae (left) and uninfested flower of Clinopodium nepeta (right). (b) Flowering C. nepeta. (c) First larval instar of
A.nepetae in a new flower gall. (d) White fungal mycelium of Botryosphaeria dothidea in a young flower gall of A.nepetae. (e) Flower gall of A.nepetae during
the reproductive period of the midge. (f) Pupal exuvia of A.nepetae remained attached to a flower gall after the emergence of the midge (frontal and lateral
views). (g) Overwintering true flower galls of A.nepetae. (h) Dissected flower gall of A.nepetae during the quiescent period of the gall midge.
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May-beginning of June and continues up to the late autumn. The
most abundant parasitoid was Sigmophora brevicornis (Panzer)
(Hymenoptera: Eulophidae), a species associated with several
Asphondylia species (Graham 1987, Noyes 2017). The complex of
the other species is under study. According to some sampling data
(Table2), the percentage of the ower galls with evidence of para-
sitic activity varied from 0 to 92.3%.
Fungal Association
The presence of a white cottony mycelium lining the inner wall was
observed when galls were cut for inspection of their content (Fig.6d).
Walls in older galls harboring pupae usually were hard and darkened
(Fig.6h), with the mycelium visible as a residual degraded layer. The
direct observation of the mycelium inside galls at any stage showed the
Table2. Sampling at random of flowers of C.nepeta
Collecting site Date of sampling Number of
examined owers
% infested owers
by Asphondylia
% of ower galls of Asphondylia
with parasitic activity
Rivello 1 Sept. 2014 107 17.7 15.8
Rivello 6 June 2015 20 50 20
Rivello 16 June 2015 40 57.5 45
S Giorgio a Cremano 25 June 2014 225 8.9 45
S. Giorgio a Cremano 18 June 2015 33 3 100
Table3. Dissection of closed flower galls
Location Collecting and
dissection date
Number of ower
galls dissected
Number of ower
galls with larvae of
Asphondylia
Number of ower
galls with pupae of
Asphondylia
Number of ower galls
with larvae, pupae or
tracks of parasitic activity
% of ower galls of
Asphondylia with
parasitic activity
Pozzuoli, Lago
d’Averno
8 Nov. 2015 40 25 6 9 22.5
Portici 28 Jan. 2015 80 0 4 33 41.2
Rivello 11 Feb. 2015 20 0 20 0 0.0
Rivello 21 Feb. 2015 20 0 9 9 45.0
Rivello 2 Mar. 2015 50 0 36 5 10.0
Rivello 20 Oct. 2015 30 9 16 5 16.6
Rivello 9 Nov. 2015 22 1 17 4 18.2
Rivello 29 Nov. 2015 28 4 15 9 32.1
Rivello 22 Feb. 2016 44 0 36 8 18.2
Rivello 29 Mar. 2016 25 0 15 10 4.0
S. Giorgio a
Cremano
18 Nov. 2015 20 1 1 18 90.0
S. Giorgio a
Cremano
4 Feb. 2016 13 0 1 12 92.3
Table4. Emergence of A.nepetae from overwintering flower galls in the laboratory at room temperature (around 20–25°C)
Location Collecting date Number of sampled
ower galls
Period or date of
adult emergence
Number of
Asphondylia specimens
♀ ♂ T
Corbara (SA) 21 April 2016 7 9 June 2016 9 June 2016 0 1 1
Napoli, Camaldoli 3 Dec. 2014 15 1 April 2015 5 May 2015 2 3 5
Napoli, Soccavo 28 Nov. 2015 13 27 May 2016 27 May 2016 1 0 1
Matera 25 Feb. 2016 14 7 April 2016 27 April 2016 4 2 6
Portici 11 Jan. 2016 60 9 Feb. 2016 16 Mar. 2016 8 1 9
Pozzuoli, Lago d’Averno 30Nov. 2014 10 28 April 2015 6 May 2015 0 2 2
Pozzuoli, Lucrino 16 April 2016 19 20 May 2016 27 May 2016 7 3 10
Palma Campania 16 Jan. 2016 93 4 May 2016 27 May 2016 15 4 19
Rivello (PZ) 21 Feb. 2015 13 23 April 2015 14 May 2015 3 1 4
Rivello 22 Mar. 2015 40 22 April 2015 15 May 2015 5 11 16
Rivello 30 Mar. 2015 45 28 April 2015 3 June 2015 5 15 18
Rivello 8 Apr. 2015 10 7 May 2015 22 May 2015 0 3 3
Rivello 4 May 2015 20 12 May 2015 19 May 2015 3 5 8
Rivello 15 Nov. 2015 12 12 April 2016 27 April 2016 2 4 6
Rivello 29 Nov. 2015 4 12 April 2016 19 April 2016 2 1 3
Rivello 23 Jan. 2016 87 31 Mar. 2016 27 April 2016 9 21 30
Rivello 22 Feb. 2016 153 2 April 2016 27 May 2016 49 51 100
Rivello 29 Mar. 2016 78 15 April 2016 10 May 2016 15 13 28
Rivello 9 May 2016 30 20 May 2016 27 May 2016 10 2 12
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absence of conidial structures. Therefore, identication of the gall-as-
sociated fungi was based on the results of isolations on PDA, yield-
ing a quite similar species assortment from every location sampled.
Besides a few sparse strains belonging to miscellaneous genera (e.g.,
Epicoccum, Fusarium, Lecanicillium, Penicillium), Cladosporium
spp. resulted from the majority of the isolation attempts (approxi-
mately 40%); Alternaria-like fungi were also recovered from all the
locations but to a lesser extent. Another series of isolates (about
1/3 of the total) did not sporulate on PDA and shared a common
morphotype characterized by a dense mycelium with irregular mar-
gins, gray-olivaceous and darkening with age, recalling species in
the Botryosphaeriaceae. These isolates successfully formed pycnidia
in 1–2 weeks on pine needles in WA cultures (Fig.7a), producing
narrowly fusiform hyaline conidia (Fig.7b) whose size was within
the range reported for the anamorphic stage (Fusicoccum aesculi) of
the species Botryosphaeria dothidea (Moug.: Fr.) Ces. and De Not.
(Botryosphaeriales: Botryosphaeriaceae) (Slippers et al. 2004). This
taxonomic ascription was conrmed using rDNA-ITS sequencing.
BLAST search in the NCBI nr/nt database yielded 100% homology
with deposited sequences from some B.dothidea strains from differ-
ent sources. Sequences obtained from a couple of strains and myce-
lium directly taken in a gall collected at Astroni have been deposited
in GenBank (accessions MF092877, MF092878 and MF092879).
Several attempts were made to rear larvae of both A.nepetae and
S.brevicornis taken from fresh galls on mycelium of B.dothidea in
Petri dishes. Although in some cases they survived for even 1week,
possibly feeding on the mycelium, larvae of both species always
failed to pupate and were in the end colonized by the underlying
fungus. It is evident that culturing on agar media provides a power-
ful support to the saprophytic fungal development, unlike what can
be presumed to occur in galls undergoing senescence, where the fun-
gus is somehow constrained by both the depletion of the nutritional
support by the plant tissues and the trophic pressure by the larva.
Unfortunately, the possible fundamental effect exerted by the for-
mation of a hardened gall wall in promoting metamorphosis, or an
eventual regulatory role by other associated microbial entities are
even more difcult to be ruledout.
Observations concerning morphology and conidial production
by the Cladosporium isolates showed all of them to belong to the
Cladosporium cladosporioides species complex. Unlike B.dothidea,
on C.nepeta this fungus was also isolated from achenes and from
ovaries collected from normal owers, and from a thin mycelium
sometimes visible crowning the pistil base in ower buds which
apparently had not been colonized by the midge.
Discussion and Conclusion
It is well known that the morphological similarities of several spe-
cies of the genus Asphondylia and their induced galls prevent their
discrimination (Yukawa et al. 2003, Uechi and Yukawa 2004,
Kolesik etal. 2010, Uechi etal. 2017). In these cases, as in that of
Asphondylia associated with the ower of Lamiaceae, the integra-
tion of the adult, larval and pupal morphology, with molecular data,
life history, and ecological traits, is essential to allow reliable identi-
cation. According to Kolesik etal. (2010) and in contrast to what
happens to Dasineura, in Asphondylia molecular analysis provides a
better species recognition than morphology. Unfortunately, to date,
only a few Asphondylia species were characterized based on the
aforementioned integration of data. Moreover, as suggested by some
authors, the best approach to gain a greater supported phylogenetic
reconstruction should include cytochrome oxidase I(COI) sequences
(Uechi and Yukawa 2004, Veenstra-Quah etal. 2007, Kolesik and
Veenstra-Quah 2008), in addition to nuclear gene sequence data
such as the internal transcribed spacer gene (ITS2) (Kolesik et al.
Table5. Overwintering flower galls of Asphondylia nepetae collected on Clinopodium nepeta and maintained in Petri dishes at 25°C from
13 Jan. 2016
Location Collecting date Number of ower galls Emergence period of
Asphondylia specimens
Number of
Asphondylia specimens
♀ ♂
Rivello 22 Oct. 2015 12 10–18 Feb. 2016 5 2
Rivello 29 Nov. 2015 55 10–16 Feb. 2016 1 1
Rivello 29 Nov. 2015 81 10–23 Feb. 2016 14 6
Rivello 29 Nov. 2015 10 12–23 Feb. 2016 5 3
Rivello 11 Jan. 2016 85 10–20 Mar. 2016 9 4
Rivello 22 Feb. 2016 33 22–28 Mar. 2016 6 8
Fig.7. (a) Pycnidia of B.dothidea. (b) Conidia of B.dothidea (Fusicoccum stage).
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2010). However, ITS2 portions present often long deletions or intro-
gressions whereby the simultaneous use of a third more conserved
sequence as 28S could allow better results (Gebiola etal. 2012). Our
molecular data pointed out that A.nepetae is a new species in the
genus Asphondylia. Indeed both COI and ITS2 supported the species
delimitation. By now, due to the lack of other homologous sequences
in gene databases, the 28S-D2 portion gave us just the chance to
characterize the A.nepetae and to distinguish it from A.sarothamni,
but no phylogenetic reconstruction of the genus Asphondylia was
possible.
Phylogenetic reconstruction suggests that there are several sys-
tematic problems in the genus, as evidenced for A.pilosa, which is
split into two separate and distant clades (Fig.2); besides, several
sequences deposited in GenBank as A.gennadii belong to another
species (A.capsicicola) (Uechi etal. 2017). These results conrm the
importance of an integrative approach that uses different markers
and the need for a genus revision.
Hitherto, almost all the descriptions of the species belonging to
Asphondylia genus have been limited to describing adult, pupa and
mature larva. To our knowledge, the rst larva was described and
gured in details only for A.sarothamni H. Loew (Parnell 1964).
The description of the rst larva of A.nepetae in the present paper
conrms the features shown by the previous species, in particu-
lar, the dermal structures on the last two abdominal segments of
unknown function (movement and/or scratching?) (Fig. 4f and g)
and the absence of a tracheal respiratory system. It should be inter-
esting to extend the study of these aspects on other species.
The possibility to distinguish the sex of the pupa or that of its
exuvia in Asphondylia species, rstly pointed out in the present
paper, can be used for taxonomic and sampling purposes.
Biology of many species of Asphondylia associated with Lamiaceae
is unknown; in fact, for most of the species, just some host plants and
gall shapes are known. There is only one exception because a bio-
logical and ecological study has been published recently by Malagaris
(2011) on Asphondylia coridothymi Skuhravá, inducing ower galls
on Coridothymus capitatus (L.) Reichenbach (=T. capitatus (L.)
Hoffmanns and Link. (Lamiaceae) in the island of Samos, Greece.
This univoltine species shows a long larval development, mostly of
the rst instar larva (beginning of June to end of September). The
pupae appear in the middle of October and overwinter in the ower
galls until April of the following year. In the type species of the genus
Asphondylia, A.sarothamni H.Loew, the midge overwinters as an egg
in the host plant buds (Parnell 1964). Differently, A.nepetae overwin-
ters as a pupa on C.nepetae and is multivoltine (4–5 generations/yr)
and active almost throughout the year; besides, not having diapause
(Table4) can develop rapidly as soon as climatic conditions allow it.
More research is needed on several interesting biological traits (over-
wintering etc.) of other Asphondylia species because new knowledge
can be important in managing these species that sometimes damage
various ofcinal species. Moreover, further research is necessary to
reconstruct the food web of the different species and therefore their
host range. The elimination of wild plants on which polyphagous spe-
cies belonging to Asphondylia overwinter could reduce the inoculum
for successful infestations. In this regard, with the exclusion of the
latest studies (Uechi etal. 2003, 2017; Tokuda etal. 2005; Yukawa
etal. 2016), most researchers have always treated the various species
as monophagous, and this has probably led to an overestimation of
the species described. Conversely, the parasitoid S.brevicornis that
resulted the most common and frequent parasitoid of A.nepetae is
a polyphagous species reared from many other species of the genus
(Graham 1987, Noyes 2017), and this can have profound implica-
tions for biological control strategies of some ofthem.
Findings on C. nepeta provide a further conrmation that
B. dothidea represents the fungal symbiont in Asphondylia galls,
matching with equal observations recently gathered from diverse
plant species (Adair etal. 2009, Heath and Stireman 2010, Kobune
etal. 2012, Lebel etal. 2012, Chao and Liao 2013), and our con-
current determination on Thymus vulgaris (Zimowska etal. 2017).
However, unlike strains obtained from T. vulgaris, none of the iso-
lates examined in the present study produced muriform conidia
corresponding to the Dichomera synanamorph (Barber etal. 2005),
even when observations were repeated throughout 1 month, or when
pycnidial production was stimulated by UV illumination in cultures
onPDA.
As in the case of galls produced by Asphondylia serpylli (Kieffer)
on T. vulgaris (Zimowska et al. 2017), the constantly associated
nding of B.dothidea did not clarify its actual relationships with
the insect biont, especially about a possible inoculation during ovi-
position. Conversely, we were able to assess that the presence of
C. cladosporioides in the galls does not depend on the associated
insects since this fungus was also frequently recovered from healthy
owers. This taxon is composed of over 40 described species, with
several additional cryptic entities yet need to be named (Bensch etal.
2010). The association of Cladosporium with cecidomyiids could be
more than just occasional, considering that these fungi were previ-
ously recovered from the body surface of Asphondylia and allied
midges, and from their galls (Docters van Leeuwen 1929, Adair etal.
2009, Kobune et al. 2012, Lebel et al. 2012). Possible antagonistic
relationships between B.dothidea and C.cladosporioides have also
been excluded by in vitro observations in dual cultures on PDA/WA,
where hyphae of the opposed strains grew intermingled, without any
evidence of inhibitory or mycoparasitic reactions, which might have
represented an indirect conrmation of this hypothesis.
SupplementaryData
Supplementary data are available at Environmental Entomology
online.
Acknowledgments
We thank Dr. Adriano Stinca, Università degli Studi di Napoli ‘Federico II’,
Dipartimento di Agraria, for the plant identication.
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