A first record of egg parasitism in Orius laevigatus: the parasitoid

Erythmelus funiculi disrupts biological control in pepper

Amir H. Grosman1, Amit Sade1, Michael Salinger-Bubnov1, Shiri Even Haim, Miriam
Kishinevsky2, Shimon Steinberg1,
1
BioBee Sde Eliyahu LTD., Sde Eliyahu, Israel
2
Department of Biology and the Environment, University of Haifa – Oranim, Tivon, Israel

Abstract: Since more than two decades, biological control became a commercial standard in
greenhouse sweet pepper operations in Israel and the anthocorid predatory bug Orius laevigatus plays
a key role in this system. In the past few years, a puzzling phenomenon has been observed: after a
successful establishment, O. laevigatus populations gradually decrease. The age distribution becomes
biased towards adults and eggs, with relatively few nymphs. In many cases, the predator population
collapses, despite repeated releases. Preliminary work has shown that this was not due to chemical
sprays, lack of alternative food or reduced survival, fertility or fecundity of the predators. A few years
later, a minute parasitic wasp has been observed at the vicinity of O. laevigatus eggs on the crop,
parasitizing the eggs. The wasp was identified as Erythmelus funiculi (Mymaridae), the first host
record for this species.
Bioassays have confirmed that E. funiculi can successfully parasitize and complete its development in
eggs of O. laevigatus. An extensive field monitoring program has demonstrated that the parasitoid
occurs in the main cultivation areas – the arid Negev and Arava regions in Israel's south . Parasitism
was found in 17 of the 19 sampled farms, with an average parasitism rate of 52 ± 4% per sample
(mean ± se, n = 119). Parasitized Orius spp. eggs were found also in close proximity to pepper farms,
on Verbesina encelioides plants, a wide-spread alternative host plant of Orius spp.
In the Negev region, where plantings start in January, E. funiculi penetrated the crop at the end of
April, as natural vegetation, including V. encelioides, started to wilt. In the Arava region, where
plantings start in August, first egg-parasitism was recorded in October. Once the parasitoid entered the
plots, parasitism rates increased to an average of 94% within 6 weeks, corresponding to 2-3 E. funiculi
generations. Implications on biological control and strategies to manage O. laevigatus egg-parasitism
are discussed.

Key words: Orius laevigatus, Erythmelus funiculi, egg parasitism, sweet pepper.

Introduction

The minute pirate bug Orius laevigatus (Fieber) (Hemiptera: Anthocoridae) is a key
component of the IPM strategy in protected sweet pepper in temperate Europe as well as the
Mediterranean region. The predatory bug consumes the pollen in sweet pepper flowers,
allowing its establishment in the crop even in the absence of its key prey, the Western flower
thrips Frankliniella occidentalis. In recent years, the availability of suitable alternative food
(high quality Artemia sp. cysts, BioArtFeed, BioBee), has made it possible to introduce O.
laevigatus into the pepper crop even before flowering (Sade et al. 2018), and has paved the
way to its application also in ornamentals such as gerbera and chrysanthemum (Gerben
Messelink pers. communication).
Despite the vast experience gained with O. laevigatus, a puzzling phenomenon has
been observed in sweet pepper under protection , in particular at the Western Negev region,
south-west Israel, since ca. 2010: a few months after their successful establishment and

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increase, O. laevigatus populations start declining, in many cases until a full collapse. The
population consists mainly of adults and eggs, with relatively few nymphs. Despite of
extensive oviposition, the populations does not increase, and many eggs remain unhatched for
longer than normal. Repeated predator releases, supported by alternative food, did not prevent
population decline. Extensive residue analysis of plant samples from commercial plots,
combined with semi-field trials, have shown that some chemical compounds indeed reduce O.
laevigatus populations (unpublished data), but could not explain the sharp population decline
observed on the plants. Survival, fecundity and fertility of O. laevigatus collected from
commercial plots was normal.
In June 2018, during a routine scouting visit of a sweet pepper cultivation in walking
tunnels at the settlement Bnei Nezarim south-west Israel, (31°08'56.7"N 34°19'22.4"E), a tiny
parasitic wasp was observed actively parasitizing O. laevigatus egg. Typically, O. laevigatus
inserts its eggs singly into the plant’s tissue. In the case of the sweet pepper plant, eggs are
laid in young and fresh shoots, young fruits and often in the leaf veins. The whole egg is
embedded in the plant’s tissue except the operculum which protrudes out. The parasitoid
exhibited a typical oviposition behavior: the wings were elevated in a right angle to the
wasp’s body, the antennae were folded downwards parallel to the underside of the parasitoid’s
body and the ovipositor was inserted through the operculum of the Orius egg. This behavior
lasted about 15 minutes and could be easily followed by X10 magnifier. Eggs of O. laevigatus
were collected from the plot and adult parasitoids that emerged from them have been
identified as the parasitic wasp Erythmelus funiculi (Hymenoptera: Mymaridae).
Following the discovery of the egg parasitoid, a bioassay was carried out in the laboratory to
verify that E. funiculi can indeed complete its development on O. laevigatus eggs. Also, an
extensive monitoring program was launched to study the phenology and distribution of E.
funiculi in Israel, the abundance of egg parasitism in commercial pepper plots and their
immediate surroundings and to study in-plot rates of parasitism and population dynamics of
O. laevigatus. Here we report on our findings and discuss approaches for the management of
this phenomenon under commercial production conditions.

Material and methods

Identification
Parasitic wasps were collected either by sticky traps or by incubating O. laevigatus eggs
which were sampled from pepper crops in 2018-2019 (see `Field monitoring program`). More
than 40 individuals were inspected and identified as belonging to the species Erythmelus
funiculi (Annecke & Doutt, 1961) (Mymaridae). For detailed description see Results and
discussion.

Bioassay
Trials were conducted to verify that E. funiculi readily parasitizes and completes its
development in O. laevigatus eggs. Live E. funiculi adults were collected by incubating plant
samples containing O. laevigatus eggs, taken from sweet pepper plots. Honey was provided
to the adult parasitoids from their emergence until the bioassay has ended.
To produce O. laevigatus eggs, sweet pepper seedlings (variety: Canon; n=4) were
exposed to adult predatory bugs. Each seedling was incubated (25°C, 70% RH and 16:8 L:D)
in a sterilization pouch (30X44 cm, YIPAK) containing 30 O. laevigatus adults and 0.1g of
decapsulated Artemia sp. cysts (BioArtFeed, BioBee) as feed. After 24 hours, the adult bugs
were removed. At the end of this procedure, each seedling inhabited 28-48 eggs. The

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seedlings were then planted individually in a 1L pot. A ventilated plastic container (1L) was
tightly fitted on the pot, to cover the seedling. Eight unsexed E. funiculi adults (0-48 hours
post-emergence) were inserted into the containers in the treatment group, while no parasitoids
were placed in the control group. The seedlings were incubated for 30 days (21°C, 70% RH
and 16:8 L:D). Under these conditions, development time of O. laevigatus eggs is less than 7
days. On days 7 and 30 the seedlings were removed from the containers and the number of O.
laevigatus eggs that were unhatched, hatched with operculum present and hatched with
operculum absent were scored. Starting at incubation day 7, a blue sticky card (10X10cm)
was fitted in each container above the seedlings. The sticky cards were inspected for the
presence of adult wasps and were replaced once in 2-3 days until no more wasps were
captured. The numbers of E. funiculi adults per sticky card were recorded. Both O. laevigatus
and BioArtFeed are produced by BioBee Sde Eliyahu LTD.

Field monitoring program

Sampling started in June 2018 and continued until July 2019. To study the geographic
distribution of E. funiculi, 132 samples were taken from 41 sweet pepper plots, originating
from 19 farms, distributed over 15 agricultural settlements in the Negev and Arava, arid
regions in the south of Israel, where most of the country's pepper production takes place.
Some plots were sampled repeatedly, to follow phenology and population dynamics of O.
laevigatus and E. funiculi. Plant samples (n = 7) containing Orius spp. eggs were also taken
from an alternative wild host plant, Verbesina encelioides (Asteraceae), bordering sweet
pepper farms.
During samplings in sweet pepper plots, typical oviposition sites of O. laevigatus, i.e.,
young tissue on the apices, fruit-buds and flowers, were searched on 20-30 plants. Plant parts
containing O. laevigatus eggs were cut off, leaving a stem length of at least 5 cm. Each
sample consisted of 15-25 stems and contained an average of 61.3 ± 7.2 (mean ± se) O.
laevigatus eggs. The stem tips were immediately inserted into an agar-filled (1%) plastic pot
(120ml). The pots were packed in a cooled polystyrene box and were transported in a
refrigerated vehicle to the laboratory, where they were stored (12°C) until incubation start.
Once in the laboratory, each stem was inserted into an agar-filled 50ml plastic tube
(Figure 1a). The tubes were attached to a blue sticky card and were inserted into a sterilization
pouch (30X44 cm, YIPAK, Figure 1b). Envelops were sealed with an impulse sealer and were
incubated in the laboratory for at least 22 days (21°C, 70% RH and 16:8 L:D). The sticky
cards were replaced on the 12th incubation day to avoid mold growth on the captured insects.
Eryhtmelus funiculi wasps and O. laevigatus nymphs on the sticky cards were counted.
Parasitism rate was calculated as the percentage of E. funiculi adults from the total number of
O. laevigatus nymphs and parasitoids counted. Samples with less than five emerged insects
were omitted from the calculation of parasitism rates.
Population dynamics of O. laevigatus was measured in several of the sampled plots. In
each plot, 60 plants were randomly examined once a week, for the presence of Orius mobile
stages. In each sampled plant, the total number of predatory bugs per plant was counted
during a 30 second scan with a X10 magnifying lens over the strata of the plant in which O.
laevigatus is typically found. Orius laevigatus frequency was defined as the percentage of
plants out of the 60 sampled plants with the predatory bug present.

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 a b

Figure 1: incubation of plant samples containing O. laevigatus eggs. Samples were collected
in protected sweet pepper plots in Israel. Sampled stems were inserted into agar containing
tubes (a), fitted on blue sticky-cards and inserted into a sterilization pouch for incubation (b).
Orius laevigatus nymphs and E. funiculi adults were captured and counted on the sticky cards
after an incubation period of at least 22 days (21°C, 70% RH and 16:8 L:D).

Results and discussion

Identification and taxonomy

Parasitic wasps were collected and identified as Erythmelus funiculi (Annecke & Doutt,
1961). Identification of E. funiculi was based on the original description of the species by
Annecke and Doutt (1961). The females of this species are well recognized by the unique
proportions of the funicular segments (Figure 2a), F3 is almost the length of F5 and distinctly
longer than F2 and F4. Male antenna has a short second flagellar segment (Figure 2b). Fore
and hind wings are typical for the genus (Figure 2c). Erythmelus funiculi is brown in color
(Figure 2d), with pale legs and parts of the antenna (for detailed description see Annecke &
Doutt, 1961).This species was originally described from Pretoria, South Africa and was later
found also in Uganda (Trjapitzin 1993). In 2000 the species was found in the Hawaiian
Islands by Beardsley and Huber and was believed to be a recent accidental introduction. In
Israel it was first found in 2013 via suction sampling (Miriam Kishinevsky, unpublished
data). This is the first host record for this species.

Bioassay
In the non-parasitoid exposed treatment (control), 95% of the eggs have hatched by trial day
7, but none of the eggs have hatched in the parasitoid exposed plants. In the control, the
operculum was present in 79% of the eggs (Figure 3a), while it was missing in 16% of the
eggs (Figure 3b). The operculum was attached by a thin membrane to the egg interior (Figure
3a). That membrane was also present in eggs with operculum absent (Figure 3b). In the
parasitoid exposed treatment, wasps emergence occurred at trial day 19-21, and 85% of the
exposed eggs have yielded adult wasps, assuming all emerging wasps have been captured on
the sticky cards. The appearance of parasitized eggs differed from non-parasitized eggs: the
operculum was absent in most cases (Figure 3c) and the membrane attaching the operculum to
the egg was always absent (Figures 3c and 3d).

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 a

c d

Figure 2. The wasp Erythmelus funiculi (Annecke and Doutt, 1961) collected from incubated
plant samples containing O. laevigatus eggs taken from sweet pepper crops in Israel in
August 2018. E. funiculi female antenna (a); Male antenna (b); Fore and hind wings (c);
Habitus, male (d).

a b

c d

Figure 3. Eggs of Orius laevigatus after hatching of nymphs (a, b) or the emergence of E.
funiculi adults (c, d). In unparasitized eggs, the operculum was present in most cases (a) and
was connected to the egg by a membrane, which was also visible in eggs without operculum
(a, b). In the majority of parasitized eggs, the operculum was absent after parasitoid
emergence (c) and the membrane attaching the operculum to the egg was always absent (c, d).

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Field monitoring program
Throughout the entire sampling period, parasitism of O. laevigatus eggs was found in 17 of
the 19 farms sampled with an average parasitism rate of 52 ± 4% (mean ± se, n = 119, Figure
4). Erythmelus funiculi was present both in the Negev (Figure 4a) and the Arava (Figure 4b)
regions.
In the Negev, field monitoring started in July 2018, in plots that were planted 2-6
months before. In this season, the Western flower thrips activity is normally at its peak and O.
laevigatus populations are well established. Egg parasitism was found in 23 out of the 24
sampled pepper plots (Figure 4a) and average parasitism rate was 70.8 ± 4% per sample
(mean ± se, n = 37) . In 2019, newly planted plots were monitored immediately after O.
laevigatus was introduced, from January onwards. No parasitism was measured on these
crops until 30th of April (Figure 4a). However, on V. encelioides plants neighboring some of
the sampled plots, Orius spp. eggs were parasitized (49 ± 13%, mean ± se, n=6) by E. funiculi
in samples taken between 27/12/2018 and 12/01/2019. Hence, although parasitoids were
active on the vegetation in the immediate vicinity of the plots and despite the presence of O.
laevigatus eggs on sweet pepper plants, parasitism did not occur on the crops until the end of
April, when the rainfall declined and natural vegetation started drying and wilting.
Apparently, E. funiculi is active also in the winter, but it is the reduction in availability of
hosts on natural vegetation during spring that drives it to search for alternative hosts in the
crop environment. During the spring and summer of 2019, parasitism occurred in 17 out of 19
sampled plots at the Negev region.
In the Arava region, with crop cycles from mid-July until May, first egg-parasitism
was recorded in October (Figure 4b). Throughout the season, E. funiculi emerged in 12 out of
the 22 samples taken and parasitism rate was 31 ± 9% (mean ± se, n= 19). Samples taken at
the end of April, have shown that Orius spp. eggs were also parasitized on V. encelioides in
the Arava region . The Arava is a very arid area and no pepper cultivation takes place between
May and July. Erythmelus funiculi may utilize Orius hosts on alternative plants, such as V.
encelioides, in the period between crop cycles. It is still surprising, however, that these host
plants can support E. funiculi populations in such an arid region during the summer.
Figure 5 shows the dynamics of O. laevigatus populations and the corresponding egg
parasitism rates in four selected plots at Farhi-farm, Sde Nitzan, the Negev region
(31°13'18.3"N 34°24'42.2"E). Following BioBee's release protocol, alternative food
(decapsulated Artemia sp. cysts, BioArtFeed, Biobee) was applied on seedlings prior to trans-
planting and O. laevigatus adults were released immediately after the transplant. Orius
laevigatus populations gradually increased following introduction, reaching an average
frequency of 76% and an average density 1.34 mobile stages per flower by the time first egg
parasitism was detected. Within a period of 6 weeks, corresponding to the length of 2-3 E.
funiculi generations (see Bioassay results), parasitism rate increased from 0 to 91% on
average (Figure 5). Parallel to the increase in parasitism rate, Orius populations declined,
reaching an average frequency of 13% immediately after the peak parasitism rate (Figure 5).
Despite of repeated additional O. laevigatus releases, population decline continued. Analysis
of chemical residues on leaf samples have shown that no incompatible insecticides were
applied in the plots, that could explain the trend. It is hence evident that E. funiculi was
responsible for the collapse of O. laevigatus populations in these plots

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 100%
80%
60%
40%
20%
0%
a
100%
80%
60%
40%
20%
0%
b
01/07/2018 01/10/2018 01/01/2019 01/04/2019 01/07/2019

Figure 4. Parasitism rate of O. laevigatus eggs on sweet pepper crops in Israel. Samples of
plant material containing O. laevigatus eggs originate from greenhouse sweet pepper farms in
(a) the Negev and (b) the Arava regions. Following sample-incubation, hatching O. laevigatus
nymphs and emerging E. funiculi wasps were counted. Parasitism rate is calculated as the
percentage of E. funiculi adults from the total number of O. laevigatus nymphs and
parasitoids in the sample.

.
a Parasitism rate
O. laevigatus frequency
Parasitism rate
O. laevigatus frequency
b

01/02/2019 01/04/2019 01/06/2019 01/08/2019 01/10/2019

Figure 5. Parasitism rates and population dynamics of O. laevigatus in four sweet pepper plots
at Farhi-farm, Sde Nitzan, the Negev region, Israel (a, b, c, d correspond to plots 1, 6, 7, 9
respectively). Parasitism rate was calculated as described in Figure 4. Orius laevigatus
frequency is defined as the percentage of plants out of 60 sampled plants with the predatory
bug present.

In the years since this survey, some experience was gained in managing this
phenomenon. First, we have found that the adult parasitoid is easily detectable on yellow
sticky cards used for monitoring other pests (thrips, whiteflies). There was, however, no
strong correlation between parasitism rate and numbers of wasps captured on cards (data not

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shown). Hence, the cards are mainly useful as a monitoring tool to detect presence of wasps
on the crop, but not to estimate parasitism rates.
In several field trials (unpublished data), O. laevigatus compatible chemical
treatments, dusting of sulfur or Spinosad application, were applied preventively to combat the
egg parasitoid. The treatments were applied after O. laevigatus had established but before
parasitoids were detected. Spinosad was also tested as a curative tool, after the collapse of O.
laevigatus populations, to clean the crop from parasitoids before re-introduction of the
predatory bug. No consistent differences in parasitism levels were found between chemically
treated and untreated plots. Given the risk of colatteral damage to the biological system, we
have stopped applying chemicals to control E. funiculi.
In other trials, a curative approach of repeated O. laevigatus releases combined with
alternative food application (Artemia sp. cysts), has proven to be uneffective (unpublished
data). It is likely that adding alternative food and adult predatory bugs to the system, simply
results in higher availability of host eggs, thereby increasing the parasitoid population rather
than reducing it. Thus far, our experience shows that the most effective strategy is 'zero
intervention', i.e: stopping O. laevigatus introductions once parasitoids are detected, allowing
predator populations to collapse, thereby speeding up the subsequent collapse of the parasitoid
population. This is the approach we are practicing in greenhouse sweet pepper in Israel
nowadays.
Given BioBee's protocol of alternative food based early Orius introduction, there was
a concern that the early presence of hosts on the crop would attract egg parasitoids earlier in
the season. However, we could not find support for this hypothesis: as mentioned above, the
egg parasitoids invaded the crops only when natural vegetation surrounding the greenhouses
wilted, irrespective of the presence of hosts on the sweet pepper crop.
Following results from the current study, various other questions are yet to be
answered. Has E. funiculi recently colonized crops in Israel or was it „under the radar“ for
decades? Is parasitism of Orius eggs by E. funiculi limited to Israel or a widespread
phenomenon? Is it an aspect that has been overlooked in our understanding of Orius
dynamics on crops? These questions and others should be addressed in future research.

Acknowledgements

Shira Gal and Achiad Sviri for their help with photographing E. funiculi adults.

References

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Chalcidoidea. Entomological Memoirs (Department of Agricultural Technical Services,
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Beardsley, J.W. and Huber, J.T., 2000. Key to genera of Mymaridae in the Hawaiian Islands,
with notes on some of the species (Hymenoptera: Chalcidoidea).
Sade A, Steinberg S, Salinger-Bubnov M, Gilboa E, Klempert G, Roitman N, Grosman A
(2019) Improved western flower thrips control through artemia-based early introduction
of Orius laevigatus in commercial pepper greenhouses. IOBC/WPRS Bull 147:39–46
Trjapitzin, S.V. 1993. A new Erythmelus (Hymenoptera: Mymaridae) from central Asia, an
egg parasitoid of Circulifer spp. (Homoptera: Cicadellidae). – Entomological News
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