Parasitoids of the eucalyptus gall wasp Leptocybe spp.: a global review

Abstract

The genus Leptocybe Fisher & La Salle (Hymenoptera: Eulophidae) and its type species L. invasa were first described in 2004. Leptocybe spp. are global pests of eucalyptus plantations, and parasitoids play an important role in their control. In this review, we describe the species, distribution, biology, ecology and parasitism levels of Leptocybe spp. parasitoids and the problems associated with biological control programmes against Leptocybe spp. Additionally, prospects for the use of conservation or augmentative biological control programmes against Leptocybe spp. are discussed. Worldwide, approximately 23 species of parasitoids of Leptocybe spp. in 7 families and 10 genera have been found to date. Comparing the parasitism levels of the parasitoids showed that Quadrastichus mendeli, Selitrichodes neseri and several (approximately 4) native Megastigmus spp. could be exploited to manage Leptocybe spp. Available information on the Leptocybe spp. parasitoids is expected to improve our understanding related to ongoing studies of biological control programmes against Leptocybe spp.

Full text

REVIEW ARTICLE
Parasitoids of the eucalyptus gall wasp Leptocybe spp.: a global review
Zong-You Huang
1
&Jun Li
1
&Wen Lu
1
&Xia-Lin Zheng
1
&Zhen-De Yang
2
Received: 23 April 2018 /Accepted: 27 August 2018 /Published online: 3 September 2018
#Springer-Verlag GmbH Germany, part of Springer Nature 2018
Abstract
The genus Leptocybe Fisher & La Salle (Hymenoptera: Eulophidae) and its type species L. invasa were first described in 2004.
Leptocybe spp. are global pests of eucalyptus plantations, and parasitoids play an important role in their control. In this review, we
describe the species, distribution, biology, ecology and parasitism levels of Leptocybe spp. parasitoids and the problems asso-
ciated with biological control programmes against Leptocybe spp. Additionally, prospects for the use of conservation or aug-
mentative biological control programmes against Leptocybe spp. are discussed. Worldwide, approximately 23 species of para-
sitoids of Leptocybe spp. in 7 families and 10 genera have been found to date. Comparing the parasitism levels of the parasitoids
showed that Quadrastichus mendeli,Selitrichodes neseri and several (approximately 4) native Megastigmus spp. could be
exploited to manage Leptocybe spp. Available information on the Leptocybe spp.parasitoids is expected to improve our
understanding related to ongoing studies of biological control programmes against Leptocybe spp.
Keywords Eucalyptus .Leptocybe spp. .Parasitoids .Quadrastichus mendeli .Selitrichodes spp. .Megastigmus spp.
Introduction
The genus Leptocybe Fisher & La Salle (Hymenoptera:
Eulophidae) was first recorded in Italy in 2000, and its type
species was described in 2004 as a new species (Mendel et al.
2004). DNA barcode data revealed that Leptocybe spp. com-
prise two genetically separate lineages (lineages A and B) and
originate from Australia (Nugnes et al. 2015; Dittrich-
Schröder et al. 2018). Researchers consider lineage A, which
is present in Israel, South Africa, Uganda, Mozambique, Italy,
Brazil and Kenya, to be Leptocybe invasa, while all other
groups are referred to as Leptocybe spp. (Dittrich-Schröder
et al. 2018). Leptocybe spp. are global invasive pests of euca-
lyptus plantations that damage the leaves and stems of new
growth in eucalyptus, eventually weakening trees or stunting
their growth (Viggiani et al. 2000; Aytar 2003; Lawson 2012).
To date, Leptocybe spp. have been found in 45 countries from
five continents where eucalyptus grows through a ‘beach-
head effect’and globalized trade. This pest is likely to have
invaded many other countries worldwide where it has not
been reported (Zheng et al. 2014). Leptocybe spp. have
rapidly expanded worldwide and poses important chal-
lenges to forest growers around the world. Different man-
agement strategies have been explored to control Leptocybe
spp., including biological control (Kim et al. 2008), chem-
ical control (Javaregowda et al. 2010), breeding and the
selection of resistant material (Dittrich-Schröder et al.
2012), cultural control (Odhiambo et al. 2013)andsilvicul-
tural management (Zheng et al. 2016). However, there are
no effective methods to control this insect, and as a result,
biological control is considered to have long-term ecologi-
cal and economic benefits in terms of controlling exotic
pests (De Clercq et al. 2011). The literature contains many
examples of native parasitoids exploiting exotic hosts
(Cooper and Rieske 2007). Two previous surveys have
documented classical biological control against gall-
inducing wasps. One such species, the chestnut gall wasp,
Dryocosmus kuriphilus, was successfully controlled by
Torym u s s i n e n sis, which was introduced from China to
Japan for that purpose (Moriya et al. 2003). Another exam-
ple was the eucalyptus gall wasp, Ophelimus maskelli,
which was successfully controlled by Closterocerus
chamaeleon,Stethynium ophelimi and Stethynium
breviovipositor (Mendeletal.2017).
Responsible editor: Philippe Garrigues
*Xia-Lin Zheng
zheng-xia-lin@163.com
1
Guangxi Key Laboratory of Agric-Environment and Agric-Products
Safety, College of Agriculture, Guangxi University,
Nanning 530004, China
2
College of Forestry, Guangxi University, Nanning 530004, China
Environmental Science and Pollution Research (2018) 25:29983–29995
https://doi.org/10.1007/s11356-018-3073-0

Leptocybe spp. have rapidly expanded, and its worldwide
distribution can be attributed to the absence of specific para-
sitoids in new areas (Protasov et al. 2008). Although parasit-
oids can sometimes cross country boundaries to the invaded
regions without being released (Nugnes et al. 2016), re-
searchers and research institutions have had to introduce na-
tive parasitoids (e.g. Quadrastichus mendeli,Selitrichodes
kryceri and S. neseri)tocontrolLeptocybe spp. populations
(Kim et al. 2008). Thus, many more studies of biological
management options using native parasitoids are needed to
control Leptocybe spp. In this review, we focused on (1) up-
dates of the available information on the parasitoids of
Leptocybe spp. worldwide, including their distribution, ecol-
ogy and biology; (2) the parasitism levels of Leptocybe spp.
parasitoids; and (3) the problems associated with biological
control programmes against Leptocybe spp.
Parasitoids of Leptocybe spp.
The species
Because of the severe damage caused by Leptocybe spp. to
eucalyptus plantations worldwide, a search for its native par-
asitoids began in 2000 (Viggiani et al. 2000). At present, ap-
proximately 23 species of parasitoids belonging to 7 insect
families in Hymenoptera (Torymidae, Eulophidae,
Pteromalidae, Scelionidae, Mymaridae, Cynipidae and
Platygastridae) have been found in the regions invaded by
Leptocybe spp. The current investigation indicated that para-
sitoids have expanded to at least 17 countries since 2000
(Table 1), and parasitoids are likely to exist in many other
countries worldwide invaded by Leptocybe spp., but they have
not been reported. Among the reported parasitoids,
Megastigmus sp. is the most dominant, followed by
Aprostocetus sp. Other parasitoids collected include
Parallelaptera sp., Telenemous sp. and Zeala sp., but their
populations are less abundant, and only a few individuals have
been collected from Leptocybe spp. galls (Xu et al. 2008;
Kumari 2009). Cynipidae and Platygastridae were reported
in a previous study, but it is not known which species were
encountered (Udagedara and Warusapperuma 2014).
Ecological characteristics
The ecological characteristics of parasitoids of Leptocybe spp.
are associated with the relationships between the parasitoids
and their hosts (Table 2). In recent studies, the hosts of the
parasitoids were assessed by studying the larval morphologies
of the hosts and by dissecting the galls from which the para-
sitoids emerged (Doğanlar and Hassan 2010). The parasitoid
species are identified by evaluating the larval mandibles and
adult morphologies (Dr. Doğanlar, pers. comm.). It was found
that most parasitoids of Leptocybe spp., such as M. thitipornae,
are ectoparasitoids, feeding on the larval and pupal stages of
Leptocybe spp. (Sangtongpraow and Charernsom 2013).
Adult longevity
Longevity, one of the main factors affecting the performance
of biological control agents, refers to the length of the parasit-
oid lifespan from adult emergence to death. Several studies
suggest that longevity is lower in ovipositing females because
of the high physiological cost of oviposition (Protasov et al.
2007; Rizzo et al. 2015). In fact, the longevity of parasitoids is
significantly affected by the range of temperature and nutrient
conditions (Table 3). For example, the longevity of S. neseri
adults ranged from 8 to 15 days at 24.0 and 30.0 °C, and adult
longevity was less than 3 days at 29.1 to 30.0 °C (Masson
et al. 2017). When maintained at a temperature of 18.0–
33.0 °C, the lifespan of Q. mendeli gradually decreased as
the temperature rose (Feng 2016). Second, the survival pat-
terns of adult parasitoids differ significantly among feeding
treatments under the same temperature conditions. For exam-
ple, the maximum longevity of Q. mendeli was observed
when fed on honey + water solution or on young healthy
foliage + a honey solution, without significant differences
between these conditions. However, the minimum longevity
was recorded in no-food treatments and was significantly low-
er than that in other treatments (Kim et al. 2008).
Developmental time
ThedevelopmenttimeofQ. mendeli wasaffectedbytemper-
ature and the gall stages of Leptocybe spp. (days after oviposi-
tion) (Tables 4and 5). For example, the development time of
S. neseri rangedfrom12.0to31.0daysinthelaboratoryat24.0
to 30.0 °C (Masson et al. 2017). Feng (2016) reported that the
longest development time of Q. mendeli was 73.1 ± 4.5 days at
18 °C and that the shortest development time was 6.3 ± 1.3 days
at 33.0 °C under greenhouse conditions (RH = 75%; L:D =
14:10). Furthermore, the development time of parasitoids dif-
fered among the different gall stages of hosts. For example, the
mean development time of Q. mendeli reached a maximum in
prepupae to pupae (30.0 ± 2.0 days) and a minimum in young
larvae (28.0 ± 1.8 days) in ventilated glasshouses and glass
cages at 25 °C and 80% RH, conditions under which the mean
development time of M. viggianii reached a maximum in ma-
ture larvae (44.0 ± 1.6 days) and a minimum in prepupae to
pupae (42.0 ± 2.5 days) (Shivaraju 2012).
Fecundity
Quadrastichus mendeli,S. kryceri and S. neseri are able to
oviposit immediately after emergence and continue to develop
eggs and oviposit until they die (Dittrich-Schröder et al. 2014;
29984 Environ Sci Pollut Res (2018) 25:29983–29995

Table 1 Species of parasitoids, distribution for Leptocybe spp.
Continent Country Species of parasitoids First time
of detection
References
Asia Cambodia Q. mendeli 2014 (Lawson et al. 2014)
China Zeala sp. 2008 (Xu et al. 2008)
Aprostocetus sp. 2008 (Li et al. 2010)
A. causalis 2014 (Yang et al. 2014; Zheng et al. 2016)
Q. mendeli 2016 (Feng 2016; Zheng et al. 2016)
M. sichuanensis 2016 (Zheng et al. 2016;Doğanlar et al. 2017)
India M. viggianii 2000 (Viggiani et al. 2000; Ankita and Poorani 2008; Yousuf et al. 2017)
Megastigmus sp. 2008 (Kumari 2009; Kulkarni 2010; Jacob et al. 2015)
Aprostocetus sp. 2008 (Kumari 2009; Vastrad et al. 2010)
Parallelaptera sp. 2008 (Kumari 2009; Vastrad et al. 2010)
A. gala 2008 (Kumari 2009; Vastrad et al. 2010)
Q. mendeli 2008 (Shylesha 2008; Jacob et al. 2015; Yousuf et al. 2017)
S. kryceri 2008 (Shylesha 2008)
M. dharwadicus 2010 (Narendran et al. 2010; Verghese et al. 2013; Ramanagouda and Vastrad 2015)
Iran A. gala 2014 (Shafiee et al. 2014)
Israel Q. mendeli 2007 (Lawson 2012;Mendeletal.2017)
S. kryceri 2007 (Lawson 2012;Mendeletal.2017)
M. lawsoni 2007 (Doğanlar and Hassan 2010;Lawson2012; Mendel et al. 2017)
M. zvimendeli 2007 (Doğanlar and Hassan 2010;Lawson2012; Mendel et al. 2017)
Megastigmus sp. 2008 (Protasov et al. 2008)
M. leptocybus 2013 (Doğanlar 2015; Mendel et al. 2017)
Lao PDR Q. mendeli 2014 (Lawson et al. 2014)
S. neseri 2017 (Lawson et al. 2014)
Sri Lanka Megastigmus sp. 2012 (Udagedara and Warusapperuma 2014)
Tele n o mus sp. I 2012 (Udagedara and Warusapperuma 2014)
Tele n o mus sp. II 2012 (Udagedara and Warusapperuma 2014)
Parallelaptera sp. 2012 (Udagedara and Warusapperuma 2014)
Pteromalidea 2012 (Udagedara and Warusapperuma 2014)
Cynipidae 2012 (Udagedara and Warusapperuma 2014)
Platygastridae 2012 (Udagedara and Warusapperuma 2014)
Thailand M. zebrinus 2006 (Doğanlar 2015)
M. thitiporna 2010 (Doğanlar and Hassan 2010; Sangtongpraow and Charernsom 2013;Doğanlar 2015)
A. causalis 2012 (Sangtongpraow and Charernsom 2012)
Q. mendeli 2014 (Lawson et al. 2014)
M. thailandiensis 2015 (Doğanlar 2015)
Turkey Megastigmus sp. 2008 (Protasov et al. 2008)
M. zvimendeli 2010 (Doğanlar and Hassan 2010;Doğanlar 2015)
M. lawsoni 2010 (Doğanlar and Hassan 2010)
M. pretorinensis 2013 (Doğanlar 2015)
M. leptocybus 2013 (Doğanlar 2015)
Vietnam Q. mendeli 2014 (Lawson et al. 2014)
America Brazil M. brasiliensis 2013 (Doğanlar et al. 2013;Doğanlar 2015)
S. neseri 2015 (Masson et al. 2017)
Argentina M. zebrinus 2013 (Hernández et al. 2015)
Europe Italy Megastigmus sp. 2008 (Protasov et al. 2008)
2010 (Doğanlar and Hassan 2010)
Environ Sci Pollut Res (2018) 25:29983–29995 29985

Zheng et al. 2016). However, in M. thitipornae, it was shown
that all eggs in the ovaries of newly emerged females (age
12 h) were immature, and oviposition was observed on the
second day after emergence; oviposition then gradually de-
clined until the 10th day (Sangtongpraow and Charernsom
2013). Q. mendeli,S. kryceri and M. thitipornae are solitary
parasitoids (Kim et al. 2008; Sangtongpraow and Charernsom
2013). Q. mendeli and S. kryceri successfully parasitize ap-
proximately 2.5 and 2.2 gall units per day, respectively, at
25 °C and 70–75% RH (Kim et al. 2008). The average fecun-
dity of S. neseri females was 3.0 ± 0.1 eggs per day (range
1.0–5.0 eggs per female), and the overall maximum number
of offspring was 39 eggs/female under laboratory conditions
(Dittrich-Schröder et al. 2014).
Sex ratio
Under laboratory conditions, the offspring of M. thitipornae ex-
hibit a male-biased sex ratio; the average ratio of female to male
offspring was 4.3:8.9, or approximately 1.0:2.0 (Sangtongpraow
and Charernsom 2013). Under greenhouse conditions, the sex
ratio of both species of Megastigmus sp. was close to 1:1
(Protasov et al. 2008). Q. mendeli is a uniparental parasitoid,
and no males have been found to date (Kim et al. 2008).
The sex ratio of parasitoids is affected by temperature. In
Brazil, under laboratory conditions, a female-biased sex ratio
in S. neseri was recorded at 29.1–30.0 °C (80:20 female/male
offspring), but the sex ratio was 23:77 (female/male offspring)
at 25.1–26.0 °C (Masson et al. 2017). However, at the same
temperature (26.0 °C) under laboratory conditions, the sex
ratio of S. neseri in Brazil was lower than that found in
South Africa (33:67 female/male offspring) (Dittrich-
Schröder et al. 2014;Massonetal.2017). The sex ratio of
parasitoids may be affected by many other factors (Fuester
et al. 2003). The differences in the sex ratio between studies
may be due to differences in the conditions (Kenis 1996).
Future studies of parasitoids should focus on factors affecting
the sex ratio.
Parasitism levels of Leptocybe spp. parasitoids
Parasitism under laboratory or greenhouse conditions
In many studies, Q. mendeli has been found to display the
highest per cent parasitization among the parasitoids of
Leptocybe spp. (Fig. 1). Kim et al. (2008)andShivaraju
(2012) reported that the per cent parasitization of Q. mendeli
under laboratory conditions varied from 7.9 to 84.2%
(25.0 °C; 70–75% RH) and from 58.6 to 79.3% (25.0 °C;
80% RH), respectively. The different results can be attributed
to different conditions. Feng (2016) reported that the parasit-
ism by Q. mendeli toward early-instar larvae of Leptocybe
spp. (86.3%) was significantly higher than that toward mature
larvae (58.7%) and pupae (6.6%). The parasitism success of
S. kryceri varied from 3.2 to 67.4% under laboratory
Ta bl e 1 (continued)
Continent Country Species of parasitoids First time
of detection
References
M. lawsoni
M. leptocybus 2013 (Doğanlar 2015)
Q. mendeli 2014 (Nugnes et al. 2016; Gualtieri et al. 2017)
Africa Kenya Q. mendeli 2014 (K.E. Mutitu pers. Comm; Dittrich-Schröder et al. 2014)
Megastigmus sp. 2014 (Lawson et al. 2014)
Morocco Megastigmus sp.2009 (Maatouf and Lumaret 2012)
South Africa M. zebrinus 2006 (Doğanlar and Hassan 2010;Doğanlar 2015;Bushetal.2017)
S. neseri 2012 (Hurley 2012; Dittrich-Schröder et al. 2014;Bushetal.2017)
M. pretorinensis 2015 (Doğanlar 2015;Bushetal.2017)
Q. mendeli 2017 (Bush et al. 2017; Hurley et al. 2017a,b)
Oceania Australia Q. mendeli 2008 (Kim et al. 2008;Lawsonetal.2014)
S. kryceri 2008 (Kim et al. 2008)
Megastigmus sp. 2008 (Kelly et al. 2012;Lawsonetal.2014)
S. neseri 2010 (Hurley 2012; Kelly et al. 2012)
M. judikingae 2010 (Doğanlar and Hassan 2010;Doğanlar 2015)
M. lawsoni 2010 (Doğanlar and Hassan 2010;Doğanlar 2015)
M. leptocybus 2010 (Doğanlar and Hassan 2010)
M. zvimendeli 2010 (Doğanlar and Hassan 2010;Doğanlar 2015)
29986 Environ Sci Pollut Res (2018) 25:29983–29995

conditions (Kim et al. 2008). The per cent parasitization by
A. gala was lower than by the abovementioned parasitoids,
ranging from 0 to 20.0%, with a mean of 16.5% under green-
house conditions (Kulkarni et al. 2010). Under greenhouse
conditions, most Megastigmus species showed relatively low
rates of parasitization. Kulkarni et al. (2010)reportedthatthe
parasitism of Megastigmus sp. varied from 20.0 to 30.0%, and
Shivaraju (2012)reportedthatthepercentparasitizationby
M. viggianii ranged from 9.6 to 21.7%. Studies on
M. dharwadicus indicated a gradual increase in the per cent
parasitization under greenhouse conditions, ranging from 10.8
to 28.3% (Ramanagouda 2012). The per cent parasitization
was calculated using the following formula:
Per cent p arasitization
¼No:of parasitoid adults emerged
Total no:of adults ðgall wasps þparasitoidsÞ100
Table 2 Ecological characteristics of parasitoids in Leptocybe spp.
Species of
parasitoids
Parasitic stage Host
feeding
Solitary (S)
or gregarious
(G)
Idiobiont (I) or
koinobiont (K)
Ectoparasitoids
(EC) or
endoparasitoids
(EN)
References
Eulophidae
Aprostocetus sp. Larva and pupa - S - - (Vastrad et al. 2010;Lietal.2010)
A. causalis Larva and pupa Yes S K EN (Yang et al. 2014; Zheng et al. 2016)
A. gala - No - - - (Kumari 2009; Ramanagouda 2012)
Q. mendeli Larva Yes S I EC (Kim et al. 2008;Nugnesetal.2016)
S. neseri Late larva, pupa, and
callow adults
No G I EC (Dittrich-Schröder et al. 2014;Bushetal.
2017)
S. kryceri Young and mature
larva
No S I EC (Kim et al. 2008;Mendeletal.2017)
Torymidae
Megastigmus sp. Larva and pupa
§
-S I EC
§
(Protasov et al. 2008)
M. brasiliensis Larva and pupa
§
-- - EC
§
(Doğanlar et al. 2013)
M. dharwadicus Larva and pupa
§
-- - EC
§
(Ramanagouda 2012)
M. judikingae Larva and pupa
§
-- - EC
§
(Doğanlar and Hassan 2010)
M. lawsoni Larva and pupa
§
-- - EC
§
(Lawson 2012;Mendeletal.2017)
M. leptocybus Larva and pupa
§
Yes - - EC
§
(Doğanlar 2015)
M. pretorinensis Larva and pupa
§
-- - EC
§
(Doğanlar 2015)
M. sichuanensis -Yes---(Doğanlar et al. 2017)
M. thailandiensis Larva and pupa
§
-- - EC
§
(Doğanlar 2015)
M. thitipornae Mature larva and pupa No S I EC (Sangtongpraow and Charernsom 2013)
M. viggianii Larva No S K - (Ankita and Poorani 2008; Shylesha 2008)
M. zebrinus Larva and pupa
§
No - - EC
§
(Bush et al. 2017)
M. zvimendeli Larva and pupa
§
-- - EC
§
(Lawson 2012;Mendeletal.2017)
Scelionidae
Tele n o mus sp. - - - - - (Kumari 2009)
Tele n o mus sp. I - - - - - (Udagedara and Warusapperuma 2014)
Tele n o mus sp. II - - - - - (Udagedara and Warusapperuma 2014)
Pteromalidae
Zeala sp. - - - - - (Xu et al. 2008; Liang et al. 2010)
Mymaridae
Parallelaptera sp. - - - - - (Kumari 2009; Vastrad et al. 2010)
Cynipidae
- - - - - - (Udagedara and Warusapperuma 2014)
Platygastridae
- - - - - - (Udagedara and Warusapperuma 2014)
B-^indicates undocumented. B
§
^indicates personal communication (Dr. Mikdat Doğanlar)
Environ Sci Pollut Res (2018) 25:29983–29995 29987

Table 3 Longevity of parasitoids of Leptocybe spp. with different temperature and nutrient conditions
Species of parasitoids Sex No food Honey and
water solution
Young leaves and
honey solution
Galled leaves and
honey solution
Young leaves Flowers Water Temperature (°C) References
A. causalis ♀- 15.9 ± 1.9 - - - - - 27.0 ± 1.0 (Zheng et al. 2016)
♂- 11.5 ± 1.6 - - - - -
♀- 18.7 ± 1.9 - - - - - - (Yang et al. 2014)
♂- 13.3 ± 1.8 - - - - -
Q. mendeli ♀2.7 ± 0.2 6.0 ± 0.6 6.0 ± 0.6 2.7 ± 0.2 2.7 ± 0.2 4.5 ± 0.5 2.7 ± 0.2 25.0 (Kim et al. 2008)
2.2 3.7 5.3 3.7 3.5 2.7 25.0 (Shivaraju 2012)
- 5.6 ± 1.2 - - - - - 27.0 ± 1.0 (Zheng et al. 2016)
- 9.3 ± 0.6 - - - - - 18.0 (Feng 2016)
- 8.8 ± 0.3 - - - - - 21.0
2.4 ± 0.5 7.2 ± 0.7 7.4 ± 0.5 3.2 ± 0.4 2.9 ± 0.4 - 3.1 ± 0.5 25.0
- 7.0 ± 0.2 - - - - - 28.0
- 4.3 ± 0.9 - - - - - 33.0
S. neseri ♀2.7 23.3–26.1 - - - - - 25.8 ± 0.0 (Dittrich-Schröder et al. 2014)
♂2.7 23.3–26.1 - - - - -
♀-8.0–15.0 - - - - - 24.0–30.0 (Masson et al. 2017)
♂-8.0–15.0 - - - - -
S. kryceri ♀2.6 ± 0.2 6.5 ± 0.7 6.5 ± 0.7 2.6 ± 0.2 2.6 ± 0.2 4.0 ± 0.7 2.6 ± 0.2 25.0 (Kim et al. 2008)
♂2.8 ± 0.2 6.4 ± 0.7 6.4 ± 0.7 6.4 ± 0.7 2.8 ± 0.2 4.1 ± 0.3 2.8 ± 0.2
M. thitipornae ♀1.0 ± 0.0 9.8 ± 0.6 9.2 ± 0.5 - - 1.0 ± 0.0 1.2 ± 0.2 26.0–32.0 (Sangtongpraow and
Charernsom 2013)♂1.0 ± 0.0 7.8 ± 0.5 8.0 ± 1.6 - - 1.0 ± 0.0 1.0 ± 0.0
M. sichuanensis ♀- 5.5 ± 1.1 - - - - - 27.0 ± 1.0 (Zheng et al. 2016)
♂-3.6±1.0- - - --
M. viggianii - 2.0 5.2 3.8 2.7 3.0 2.3 25.0 (Shivaraju 2012)
Megastigmus sp. ♀3.0–4.0 4.5–5.0 4.5–5.0 3.0–4.0 3.0–4.0 - 3.0–4.0 25.0 (Protasov et al. 2008)
♂2.0–3.0 - - - 2.0–3.0 2.0–4.0 2.0–3.0
B-^indicates undocumented. The units of longevity: days and values are presented as the means ± SD
29988 Environ Sci Pollut Res (2018) 25:29983–29995

Parasitism under natural conditions
In China, three hymenopteran parasitoids of Leptocybe spp.
have been found: Q. mendeli,A. causalis and M. sichuanensis.
In the field, the parasitism of Q. mendeli was 19.5, 10.9, 9.1,
5.8 and 3.0% in Guangxi, Guangdong, Hainan, Sichuan and
Fujian Provinces, respectively. The evaluation of A. causalis
in the field showed that the per cent parasitization was 26.4,
5.5, 3.8, 3.1 and 2.3% in Sichuan, Guangxi, Jiangxi, Hainan
and Guangdong Provinces, respectively. The per cent parasit-
ization by M. sichuanensis was 24.9% in Sichuan Province
(Zheng et al. 2016).
Table 5 Development time of parasitoids of Leptocybe spp. with
different gall stages of hosts
Species of
parasitoids
Development
stage (days after
oviposition)
Developmental
time (egg to
adult) (days)
References
Q. mendeli 15.0 0.0 (Kim et al. 2008)
50.0 30.0 ± 1.4
75.0 29.0 ± 2.3
100.0 28.0
115.0 0.0
15.0 0.0 (Shivaraju 2012)
50.0 28.0 ± 1.8
75.0 29.2 ± 2.4
100.0 30.0 ± 2.0
115.0 30.0 ± 2.0
M.viggianii 15.0 0.00 (Shivaraju 2012)
50.0 43.0 ± 0.4
75.0 42.0 ± 2.5
100.0 44.0 ± 1.60
115.0 43.0 ± 0.2
Megastigmus
sp.
14.0 0.0 (Protasov
et al. 2008)50.0 41.0
75.0 44.0 ± 1.7
100.0 42.0 ± 2.2
115.0 43.0
The units of development time: days and values are presented as the
means ± SD
Table 4 Development time of parasitoids of Leptocybe spp. with different temperatures
Species of parasitoids Sex Developmental time (egg to adult) (days) Temperature (°C) References
A. causalis -12.92±0.92 - (Yangetal.2014)
Q. mendeli ♀30.0 25.0 (Kim et al. 2008)
28.0 ± 1.8–30.0 ± 2.0 25.0 (Shivaraju 2012)
29.0 - (Jacob et al. 2015)
73.1 ± 4.5 18.0 (Feng 2016)
45.1 ± 3.5 21.0
29.9 ± 2.3 25.0
23.8 ± 1.8 28.0
6.3 ± 1.3 33.0
S. neseri ♀19.1 ± 0.3 25.8 (Dittrich-Schröder et al. 2014)
♂19.9 ± 0.5 25.8
-18.0–30.0 26.0 (Kelly et al. 2012)
12.0–31.0 24.0–30.0 (Masson et al. 2017)
S. kryceri - 40.0 25.0 (Kim et al. 2008)
M. thitipornae - 17 ± 0.44 26.0–32.0 (Sangtongpraow and Charernsom 2013)
M. viggianii - 42.0 ± 2.5–44.0 ± 1.6 25.0 (Shivaraju 2012)
Megastigmus sp.-41.0–43.0 25.0 (Protasov et al. 2008)
B-^indicates undocumented. The units of development time: days and values are presented as the means ± SD
Fig. 1 A solitary ectoparasitoid of Leptocybe invasa,Quadrastichus
mendeli
Environ Sci Pollut Res (2018) 25:29983–29995 29989

In India, Vastrad et al. (2010) recorded several hymenop-
teran parasitoids, including A. gala, Aprostocetus sp.,
Megastigmus sp. and Parallelaptera sp., emerging from eu-
calyptus plant material infested by L. invasa under natural
conditions. Among these parasitoids, Megastigmus sp. was
dominant (90.7%), followed by Aprostocetus sp. (6.5%) and
A. gala (2.7%), and the values of combined parasitization
ranged from 49.0 to 74.0%. Kulkarni et al. (2010)reported
that parasitism of A. gala and Megastigmus sp. varied from
21.4 to 34.1%, respectively. The same authors found that
Megastigmus sp. was dominant, followed by A. gala, and
the total per cent parasitization ranged from 1.8 (Rampur) to
61.1% (Machagondanahalli) in different areas. Ramanagouda
et al. (2011) reported that the total per cent parasitization due
to A. gala and M. dharwadicus increased over a period of
10 months, ranging from 0 to 100.0% under natural condi-
tions, and the per cent parasitization by A. gala and
M. dharwadicus ranged from 3.1 to 15.8% and from 20.7 to
93.2%, respectively. Shivaraju (2012) reported that the per
cent parasitization of M. viggianii ranged from 18.1
(Tumkur) to 31.8% (Mysore). Verghese et al. (2013)reported
that the highest parasitism by M. viggianii was 24.0%, in
Karnataka.
In Italy, Q. mendeli was accidentally introduced and has
become so widespread in central and southern Italy that in
some places, the gall wasp has almost completely disap-
peared. The per cent parasitization by Q. mendeli was 30.2
± 8.1, 38.0 ± 9.0 and 50.5 ± 6.2% in Rome, Gallipoli and San
Giorgio a Cremano, respectively (Nugnes et al. 2016). In Sri
Lanka, a total of six species of parasitoids were found, includ-
ing Megastigmus sp., Telenomus sp. I, Telenomus sp. II, a
member of the Pteromalidae family, a species of Cynipidae
and a species of Platygastridae. The most abundant wasp was
Megastigmus sp., which is observed throughout the year in Sri
Lanka. The parasitization rates of all species of parasitoids in
different samples ranged from 25.0 to 100.0%, with a mean of
69.1 ± 8.0% (Udagedara and Warusapperuma 2014).
Biological control programmes against Leptocybe
spp.
Biological control programmes against Leptocybe spp. have
already been applied in many countries. The exotic parasitoid
Q. mendeli is currently under quarantine tests in Brazil, India,
Israel, Italy, South Africa, Thailand and Turkey (Mendel et al.
2017;Bushetal.2017), and efforts are also being made to
import this parasitoid species in the Mekong region (Lawson
et al. 2014). The management of Leptocybe spp. can work
well if biological control agents are introduced during the
early part of the invasion (Lawson et al. 2014). Classical bio-
logical control of L. invasa in Israel and Italy led to reductions
in L. invasa populations and eventually to the establishment of
a population balance between L. invasa and its introduced
native parasitoids (Mendel et al. 2017). There are some para-
sitoids that shifted from local host species to Leptocybe spp.,
and their potential as biocontrol agents could be potentially
exploited to control the eucalyptus gall wasp. In general, cli-
mates in different states and countries are suitable for the
survival of different species of parasitoids. At present,
Q. mendeli,S. neseri and several (approximately 4) native
Megastigmus spp. could potentially be used for the manage-
ment of Leptocybe spp.
Exploitation of native parasitoids
It has been reported that Megastigmus species were not orig-
inally associated with eucalyptus, and they have adapted to
develop on Leptocybe spp. (Protasov et al. 2008). Since
Megastigmus spp. are the most dominant and widespread na-
tive parasitoids of invasive Leptocybe spp., they could be used
for the management of this pest. Native Megastigmus species
are known to parasitize Leptocybe spp. in Africa, Asia,
Europe, America and Oceania, including Australia, Brazil,
China, Morocco, India, Israel, Italy, Kenya, Sri Lanka, South
Africa, Thailand and Turkey (Viggiani et al. 2000; Protasov
et al. 2008;Doğanlar and Hassan 2010; Maatouf and Lumaret
2012; Udagedara and Warusapperuma 2014; Lawson et al.
2014). In India, M. viggianii and M. dharwadicus are impor-
tant parasitoids of L. invasa and were collected in 2000 and
2010, respectively. M. viggianii was reared and released at
gall-infested locations in Punjab, and the results indicate that
the gall intensity among all eucalyptus clones in nurseries and
plantations was mostly below 10.0% (Shylesha 2008; Yousuf
et al. 2017). M. dharwadicus has successfully been reared
under laboratory conditions. The effect of its release in the
field is evident, as indicated by a substantial reduction in the
number of galls, which may inflict severe damage on host
plants, and by a significant increase in the per cent parasitiza-
tion after several releases (Ramanagouda and Vastrad 2015).
For example, the number of galls was substantially reduced,
coupled with a significant increase in per cent parasitization
(95.0%), after the third release (Ramanagouda and Vastrad
2015).
Exotic parasitoids: introduction and field release
Quadrastichus mendeli was collected in Australia in 2008
when searching for parasitoids for biological control of
Leptocybe spp. (Kim et al. 2008). Thelytokous species have
a greater potential for biological control (Silva et al. 2000), but
the parasitism levels of Q. mendeli are mainly due to its short
life cycle. Indeed, under greenhouse conditions, the develop-
mental period (egg-adult) of Q. mendeli lasts approximately
30 days (Feng 2016), a period that is approximately five times
shorter than that of its gall wasp host (132 days) at room
temperature (Mendel et al. 2004). In Africa, Asia, Europe,
29990 Environ Sci Pollut Res (2018) 25:29983–29995

America and Oceania, including in Australia, China,
Cambodia, India, Israel, Italy, Lao PDR, Thailand and
Vietnam, Q. mendeli has now become successfully
established (Lawson et al. 2014; Yousuf et al. 2014; Bush
et al. 2017;Mendeletal.2017). In India, Q. mendeli displays
the highest occurrence frequency among the parasitoids of
L. invasa and effectively controls L. invasa populations in
the field (Vastrad et al. 2010; Shivaraju 2012). In addition, it
is likely that Q. mendeli exists but has not yet been reported in
many other countries worldwide where Leptocybe spp. occur.
In Lao PDR, India and Israel, Q. mendeli was released, and the
parasitoid seems to be able to control Leptocybe spp. because
gallshavebecomeveryrareinsomeplaceswherethepestwas
abundant until 2 years ago (Lawson et al. 2014;Mendeletal.
2017). Although Q. mendeli was never officially released in
China or Italy, it has been reported in both China and Italy and
is assumed to have been accidentally introduced along with
the pest (Hurley et al. 2017a). In South Africa and Kenya,
initial efforts to establish populations of this parasitoid in quar-
antine facilities have failed (Dittrich-Schröder et al. 2014;
Nugnes et al. 2015).
Selitrichodes neseri was collected in Australia in 2010
when searching for parasitoids for the biological control of
Leptocybe spp. (Hurley 2012). S. neseri may be a suitable
biological control agent due to its high rates of parasitism on
Leptocybe spp., the lack of a pre-oviposition period, a short
developmental time, a long adult lifespan, the ability to utilize
a range of gall ages and a high level of host specificity
(Dittrich-Schröder et al. 2014). In Africa, Asia, America and
Oceania, S. neseri has now become successfully established,
including in Australia, Brazil, Lao PDR and South Africa
(Kelly et al. 2012; Anonymous 2013; Dittrich-Schröder
et al. 2014;Massonetal.2017). In Lao PDR, the parasitoid
S. neseri was imported from South Africa to Wattay
International Airport in March 2017, and a colony was suc-
cessfully established and reared through two generations un-
der quarantine. In South Africa, S. neseri has successfully
been reared under laboratory conditions, even in mature galls
on severed shoots (Kelly et al. 2012), and has become well
established throughout the country (Hurley et al. 2017a). In
addition, S. neseri was successfully released and has success-
fully become established in a eucalyptus plantation in Brazil
(Masson et al. 2017).
The impact of the introduction and release of exotic para-
sitoids such as Q. mendeli in the field is clearly evident, as
indicated by the drastic reduction in the number of galls and
the substantial increase in per cent parasitization in Israel
(Mendel et al. 2017). Classical biological control programmes
against Leptocybe spp. have been implemented in many coun-
tries around the world, including India, Cambodia, Lao PDR,
Thailand, Vietnam, Israel, South Africa and Brazil (Shylesha
2008;Lawsonetal.2014;Mendeletal.2017). In India,
Q. mendeli and S. kryceri collected in Queensland,
Australia, were introduced in 2008 (Shylesha 2008). During
2010–2011, investigations were carried out to assess the im-
pact of Q. mendeli on Leptocybe spp. Seventy days after the
first field release of the exotic parasitoid Q. mendeli, the per
cent parasitization was 73.1, 64.0, 37.8 and 64.0% in
Bangalore, Rajanakunte, Malur and Hoskote, respectively
(Shylesha 2008). Verghese et al. (2013) reported that the per
cent parasitization by Q. mendeli increased to 90.0% after its
release in Bangalore, Kolar, Tumkur, Mysore and Malur. In
the Mekong region, Q. mendeli was introduced and released
as a part of a biological control programme. Roadside and
smallholder surveys across Cambodia, Lao PDR, Thailand
and Vietnam revealed that Q. mendeli was widespread across
all countries (Lawson et al. 2014). In Israel, Q. mendeli,
S. kryceri,M. lawsoni and M. zvimendeli collected in New
South Wales, Australia, were introduced to control
Leptocybe spp. (Kim et al. 2008;Mendeletal.2017). After
the release of large numbers of these parasitoids, the
Q. mendeli occurrence rate ranged between 14 and 28 wasps
per 100 galls. S. kryceri occurred at low frequencies, with
values between 3 and 5 wasps per 100 galls. Occurrences of
M. lawsoni were scarce, and the occurrence of M. zvimendeli
was 7–13 wasps per 100 galls (Mendel et al. 2017). Although
S. neseri is native to Australia, it was first described in 2012 in
South Africa (Kelly et al. 2012). The first releases of S. neseri
were in July 2012 in Zululand; subsequent releases have been
made in all the major areas infested by L. invasa, and releases
currently continue (Dittrich-Schröder et al. 2014). The para-
sitism rate observed for S. neseri ranged from 9.7 to 71.8%. In
Brazil, S. neseri was imported from South Africa and first
released in March 2015. Although the numbers of S. neseri
adults released were low when sampled 171 days after initial
release, reports showed that in Brazil, this parasitoid was able
to reproduce in the field (Masson et al. 2017).
However, it was ultimately hard to predict which parasit-
oids were the best, so the introduction of more than one nat-
ural enemy species benefits biological control programmes
(De Bach and Rosen 1991; Gibbs et al. 2011). In Israel, the
biological control of L. invasa seems to be efficient, which
could be attributed to the activity of four introduced species of
parasitoids of L. invasa (Mendel et al. 2017). In India, the
introduction of multiple parasitoids was attempted, and in ad-
dition to a significant increase in per cent parasitization, the
number of galls was substantially reduced (Ramanagouda
2012; Yousuf et al. 2017).
Problems associated with biological control
programmes against Leptocybe spp.
Currently, there are few biological control programmes
against Leptocybe spp. available in some regions of the world.
Therefore, more research needs to be carried out to exploit the
Environ Sci Pollut Res (2018) 25:29983–29995 29991

potential of native parasitoids. In general, alien invasive in-
sects that expand their ranges from their original homeland are
attacked by native parasitoids, and host shifts are a common
phenomenon (Nicholls et al. 2010). Doğanlar and Hassan
(2010) recorded several Megastigmus parasitoids of gall in-
ducers on eucalyptus in invaded countries. However, the ex-
istence of an array of Megastigmus spp. utilizing Leptocybe
spp. as a host needs to be explored further (Ankita and Poorani
2008; Ramanagouda et al. 2011).
There are few data from the field related to the spread of
Leptocybe spp. parasitoids into neighbouring regions/coun-
tries, possibly because the relationships between most parasit-
oids and non-target gall makers are not clear (Mendel et al.
2017). In light of increasing evidence of non-target hosts and
the associated threats to native biodiversity, classical biologi-
cal control needs to be carefully evaluated (Louda et al. 2003).
However, testing host specificity has been a major challenge
because host specificity testing requires a great deal of time,
which also decreases the number of native parasitoids pro-
posed for release and slows the process (Ramanagouda 2012).
Many other factors may also affect the application of bio-
logical control programmes. Most developing countries have
not yet adopted classical biological control programmes
against Leptocybe spp. and are reluctant to issue permits for
introducing exotic parasitoids (Lawson et al. 2014). In devel-
oping countries, a major difficulty with biological control
programmes is that while introduced biological control agents
provide ‘free’pest control over the long term, the testing re-
quired for the introduction, release and establishment of
agents in the field can be quite costly (Lawson et al. 2014).
Another major challenge in the introduction of biocontrol par-
asitoids is the import request. The limits on the application of
biological control have included regulations affecting the ex-
port of potential biological control agents from their area of
origin. These rules are generally applied to a specific country
but are not effective in neighbouring countries (Hurley et al.
2017a). For instance, although the monophagy of Q. mendeli
assures the absence of non-target effects, the introduction and
inoculation of Q. mendeli in accordance with international
directives and national laws would require a long and expen-
sive risk assessment process (Nugnes et al. 2016).
Conclusions
Although parasite species and their per cent parasitization of
parasitoids against Leptocybe spp. worldwide have been clar-
ified, many problems associated with biological control
programmes need to be resolved. The actual distribution of
Leptocybe spp. and its parasitoids in every country must be
mapped, identified and evaluated. The parasitoids already
present in the region (either native or accidentally introduced)
need to be assessed through field discovery, collection and
preliminary evaluation (Lawson et al. 2014). Monitoring par-
asitoid establishment and effectiveness in the field, such as
determining the impacts of these releases on Leptocybe spp.
populations and evaluating regionally appropriate biotypes,
can further facilitate the selection of potential parasitoids from
among those already employed elsewhere (Mendel et al.
2017). Studies of the biology and ecology of the emerging
native parasitoids under laboratory conditions and successful
establishment of a laboratory colony are now needed to eval-
uate the applicability of each such parasitoid as a classical
biological control agent for L. invasa (Sangtongpraow and
Charernsom 2013). Although there have been considerable
advances in recent years, very little is known about the biolo-
gy and ecology of the emerging native parasitoids associated
with Leptocybe spp. due to their relatively short research his-
tory (Zheng et al. 2014).
Parasitoids that attack Leptocybe spp. are diverse, but the
most important of these parasitoids are the larval parasitoid
Q. mendeli and the larval and pupal parasitoids S. neseri and
several native Megastigmus spp. (Lawson et al. 2014;Bush
et al. 2017). These parasitoids induce high parasitism rates in
host populations, despite the fact that parasitism levels vary
depending on the local breeding site and on seasonal condi-
tions (Ramanagouda 2012; Shylesha 2008; Dittrich-Schröder
et al. 2014). The parasitism rates of Q. mendeli and
M. dharwadicus can reach 90.0 and 95.0%, respectively, at
some sites in India, indicating that parasitoids may be a pri-
mary mortality factor in Leptocybe spp. populations (Verghese
et al. 2013; Ramanagouda and Vastrad 2015). Thus, it will be
important to examine parasitism levels to obtain an accurate
picture of the use of conservation or augmentative biological
control programmes against Leptocybe spp.
Most parasitoids are widely distributed but have not yet
been recovered from any of the plant galls screened
(Ramanagouda 2012), suggesting the need for a much wider
exploration of native galls to identify their primary hosts.
Preliminary host specificity testing under quarantine condi-
tions supports release programmes overseas. Among the re-
leased parasitoids, those that are host specific, particularly at
the genus level, might also be the most effective. The prob-
lems associated with the damage caused by Leptocybe spp.
worldwide necessitate a mechanism for cooperation among
eucalyptus growers and scientists from around the world.
For example, scientists in Australia have been helping to de-
velop biological control of Leptocybe spp., building on this
knowledge to control and test parasitoids and the effectiveness
of released agents at establishing in the Mekong region
(Lawson et al. 2014). Communication among countries needs
to be significantly enhanced to develop cooperative biological
control programmes.
Genetic studies may, therefore, be useful for quickly iden-
tifying biocontrol species of Leptocybe spp. parasitoids.
Genetic studies have been carried out on Quadrastichus
29992 Environ Sci Pollut Res (2018) 25:29983–29995

spp., Selitrichodes spp. and Megastigmus spp., all parasitoids
that are associated with Leptocybe spp. in Australia and that
have been introduced overseas as biocontrol agents
(Ramanagouda 2012; Shivaraju 2012;Lawsonetal.2017).
For parasitoids, it is important to know if there was one single
introduction or if there were multiple introductions (Vargo
et al. 2006). Genetic studies are also being used to elucidate
the interactions among gall formers, parasitoids and inquilines
that co-inhabit the galls of Leptocybe spp. (Hurley et al.
2017b).
In many cases, the negative impacts that have emerged
were not expected. Unfortunately, negative impacts of biolog-
ical control have emerged, which is likely to lead to future
losses. In fact, worldwide, for the more than 2000 biological
control agents introduced against invasive pests, there have
been few cases of negative impacts on ecosystem functions
(Hajek et al. 2016). Biological control methods for addressing
Leptocybe spp. have provided many positive outcomes.
Governments should not only correctly guide growers to
adopt scientific cultivation methods and avoid the use of sus-
ceptible clones but also subsidize and encourage the coopera-
tion between scientists and growers to search for local native
parasitoids and build strength in surveillance, reduce
Leptocybe spp. invasion and apply the principles of
biosecurity/quarantine on the ground. The release of biocon-
trol parasitoids can be used by farmers, tree growers and
forest-based industries in their nurseries to significantly re-
duce the high cost of containing gall wasp outbreaks and
avoid the loss of planting material.
Acknowledgements We are grateful to Prof. & Dr. Mikdat Doğanlar
(Biological Control Research Station, Adana, Turkey) for providing very
useful information and thank Dr. Brett P. Hurley (University of Pretoria,
Pretoria, South Africa) for providing references and publications. We also
thank the anonymous reviewers for their valuable comments on an earlier
version.
Funding This work was supported by the National Natural Science
Foundation of China [grant number: 31870634 and 31560212] and the
Natural Science Foundation of Guangxi [grant number:
2018GXNSFAA138099].
Compliance with ethical standards
Conflict of interest The authors declare that they have no competing
interests.
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