DOI: 10.1002/arch.
21451
ARTICLE
Parasitism and venom of ectoparasitoid
Scleroderma guani impairs host cellular immunity
Li-Fang Li1 Zhi-Wen Xu1
Xue-Min Ren1 Jia-Ying Zhu1
1 Key Laboratory of Forest Disaster Warning and
Control of Yunnan Province, Southwest Forestry
University, Kunming, China
2 College of Plant Protection, Yunnan Agricultural
University, Kunming, China
Correspondence
Jia-Ying Zhu, Key Laboratory of Forest Disas-
ter Warning and Control of Yunnan Province,
Southwest Forestry University, Kunming 650224,
China.
Email: jyzhu001@gmail.com
Funding information
Grant sponsor: Key Project of Applied Basic
Research of Yunnan Province; Grant number:
2017FA014; Grant sponsor: National Natural
Science Foundation of China; Grant number:
31660629; Grant sponsor: Fund of Reserve
Talents for Young and Middle-Aged Academic and
Technological Leaders of Yunnan Province; Grant
number: 2013HB077.
Arch. Insect Biochem. Physiol. 2018;e21451.
https://doi.org/10.1002/arch.21451
Nai-Yong Liu1 Guo-Xing Wu2
Abstract
Venom is a prominently maternal virulent factor utilized by para-
sitoids to overcome hosts immune defense. With respect to roles
of this toxic mixture involved in manipulating hosts immunity, great
interest has been mostly restricted to Ichneumonoidea parasitoids
associated with polydnavirus (PDV), of which venom is usually con-
sidered as a helper component to enhance the role of PDV, and
limited Chalcidoidea species. In contrast, little information is avail-
able in other parasitoids, especially ectoparasitic species not carry-
ing PDV. The ectoparasitoid Scleroderma guani injects venom into its
host, Tenebrio molitor, implying its venom was involved in suppression
of hosts immune response for successful parasitism. Thus, we investi-
gated the effects of parasitism and venom of this parasitoid on coun-
teracting the cellular immunity of its host by examining changes of
hemocyte counts, and hemocyte spreading and encapsulation abil-
ity. Total hemocyte counts were elevated in parasitized and venom-
injected pupae. The spreading behavior of both granulocytes and
plasmatocytes was impaired by parasitization and venom. High con-
centration of venom led to more severely increased hemocyte counts
and suppression of hemocyte spreading. The ability of hemocyte
encapsulation was inhibited by venom in vitro. In addition to immedi-
ate effects observed, venom showed persistent interference in hosts
cellular immunity. These results indicate that venom alone from
S. guani plays a pivotal role in blocking hosts cellular immune
response, serving as a regulator that guarantees the successful
development of its progenies. The findings provide a foundation
for further investigation of the underlying mechanisms in immune
inhibitory action of S. guani venom.
KEYWORDS
hemocyte, immunity, parasitization, parasitoid, venom
wileyonlinelibrary.com/journal/arch 
c 2018 Wiley Periodicals, Inc. 1 of 13
2 of 13 LI ET AL .

1 INTRODUCTION

To combat the infection of parasites and pathogens, insects have evolved efficient and powerful innate immune sys-
tems composed of humoral and cellular immune responses to protect themselves (Richards & Dani, 2008). Humoral
response involves the synthesis and activity of immune-related molecules (e.g., antimicrobial peptides, lysozyme,
and phenoloxidase), while cellular immunity depends on hemocyte participation in spreading, encapsulation, phago-
cytosis, nodulation, lysis, and apoptosis (Hillyer, 2016; Strand 2008; Urbański, Czarniewska, Baraniak, & Rosiński,
2014). In general, humoral and cellular immune response is only an artificial border, since humoral response is often
the result of hemocytes activity. Two types of immune responses were simultaneously used against the invaders by
insects.
In order to overcome hosts immune responses for ensuring the successful parasitism and development of their
progenies, parasitoids in turn lead to the development of effective strategies that allow them to evade, sup-
press, or modulate hosts immune responses. It is clear that polydnavirus (PDV) present in the wasp calyx fluid
as the most studied maternal parasitoid factor can manipulate hosts immunity following parasitization (Strand
& Burke, 2014, 2015). Especially, these genetic symbionts of parasitoid wasps can trigger host immunosuppres-
sion, so that host hemocytes are prevented from encapsulating (primary defense against parasitoids) the para-
sitoid's eggs and/or larvae (Beckage, 1998; Rodríguez-Pérez & Beckage, 2006). As another important virulent fac-
tor injected into the hemocoel of the host during parasitization, venoms composed of a complex cocktail of pep-
tides and proteins play important roles in suppressing the mounting host immune responses (Beckage & Gel-
man, 2004; Moreau & Asgari, 2015). In contrast to the well-documented physiological roles of PDVs acted by
inactivation of conserved and divergent hosts immune signaling pathways that amplify the immune response, the
roles of venom in inducing alterations of hosts immune responses are sparely studied (Beckage & Gelman, 2004).
Particularly, a great part of information concerning the functions of venoms is derived from the investigations
of PDV-carrying parasitoids that parasitize lepidopteran hosts, belonging to the families Ichneumonidae and Bra-
conidae (Asgari, 2006; Becchimanzi et al., 2017; Magdaraog, Tanaka, & Harvey, 2016; Malva et al., 2004; Pre-
vost, Eslin, Doury, Moreau, & Guillot, 2005). In these species, venoms are often not essential and recognized to
act synergistically with PDV to enhance its effect (Lanzrein, Pfister-Wilhelm, Wyler, Trenczek, & Stettler, 1998).
To our knowledge, parasitoids that are obliged to use venom to overcome the hosts immune response have been sub-
jected only to limited numbers of Chalcidoidea and Ichneumonoidea species including Leptopilina boulardi, Leptopilina
heterotoma, Nasonia vitripennis, Pteromalus puparum, and Pimpla hypochondriaca, most of which are endoparasitic wasps
(Cai, Ye and Hu, 2004; Lee et al., 2009; Rivers, Hink and Denlinger, 1993; Rivers Ruggiero, & Hayes, 2002; Richards &
Parkinson, 2000; Small, Paddibhatla, Rajwani and Govind, 2012; Zhang, Ye, Cai, & Hu, 2005). In addition, the roles of
venoms are variable among different parasitoid–host systems, to some extent due to the variability in venom com-
ponents responsible for variation in parasitoid virulence (Asgari & Rivers, 2011; Colinet, Mathé-Hubert, Allemand,
Gatti, & Poirié, 2013; Yu et al., 2007). More studies are required in diverse species to systemically evaluate func-
tions of their venoms involved in interfering hosts immune response, especially in ectoparasitoids not associated with
PDVs.
The ant-like bethylid wasp Scleroderma guani (Hymenoptera: Bethylidae) is a larval or pupal ectoparasitoid indige-
nous to China, which can polyphagously parasitize more than 50 species of insects belonging to 22 families of
Coleoptera, Lepidoptera, and Hymenoptera, and generally uses wood-boring pests as hosts (Chen & Cheng, 2000; Zhu,
Yang, Zhang, Wu, & Yang, 2013). Due to its high natural parasitism rates and easy mass rearing using Tenebrio molitor
(Coleoptera: Tenebrionidae) pupae as the preferred host, this parasitoid is effectively used as the biological control
agent of economical important long-horned beetles including those serving as the vector to disseminate the notorious
pine wilt nematode, a quarantine organism at the top of the list of the pathogenic species in many countries (Li, Lu, Liu,
Zhang, & Zhou, 2011).
Regarding S. guani, it is belonging to Chrysidoidea thought to not carry PDV, representing a unique evolution
between Ichneumonoidea and Vespoidea (Peters et al., 2017). Information on the role of venoms from Ichneumonoidea
and Vespoidea (social or solitary species) has been relatively well documented (Asgari & Rivers, 2011; Moreau & Asgari,
LI ET AL . 3 of 13

2015), whereas no physiological roles of venoms from Chrysidoidea has yet been characterized. Several investigations
revealed that venom of S. guani appears to be involved in inducing differential expression of host genes related to immu-
nity (Zhu et al., 2013; Zhu, Wu, Ze, Stanley, & Yang, 2014a; Zhu, Wu and Zhang, 2014b), while its effects on host immune
response have not been determined. In view of these, we here aimed to unravel the cellular immune defenses of T. moli-
tor pupae influenced by its parasitization, and in particular by its venom.

2 MATERIALS AND METHODS

2.1 Insects rearing and parasitization

The colony of S. guani was maintained in the laboratory using T. molitor pupae as host as described by Zhu et al. (2013).
After emergence, both male and female adults originated from one same host were kept together in one glass tube
for mating, which were fed with 20% honey solution absorbent on cotton. As most of the male adults will die after
5 days of emergence at this condition, and this period is enough for the mating of females with them, one 6-day-old
female wasp was used to parasitize one T. molitor becoming to pupae after 6 h. Once parasitizaiton was recorded, female
wasp was removed and parasitized pupa was kept in a Petri dish at 25 ± 1◦ С with relative humidity of 75% prior to
use.

2.2 Venom preparation and injection

Venom reservoir was dissected from 5- or 6-day-old female adults following the method of Parkinson and Weaver
(1999). After several wash to remove any other tissue debris and hemolymph, 60 intact reservoirs were transferred to
30 𝜇l sterile phosphate buffered saline (PBS, pH 7.4) in a 1.5-ml Eppendorf tube. Reservoir was broken by putting the
tube into the liquid nitrogen. Then, it was centrifuged at 12,000 g for 10 min at 4 ◦ С to collect the supernatant. Prior
to formal tests, the concentration of crude venom was adjusted to 0.05 and 0.1 venom reservoir equivalent (VRE) (one
VRE being defined as the supernatant from one torn venom reservoir in 1 𝜇l PBS) as described by Zhang et al. (2005).
Newly pupated T. molitor as described above was used in the experiments. Totally, 2 𝜇l of venom sample was microin-
jected into each pupal body cavity. Then, venom-injected pupae were put on Petri dish, and kept at the conditions as
described above. Control pupae were treated identically with the same volume of PBS.

2.3 Hemocyte identification, counts, spreading, and encapsulation

Hemocytes of T. molitor pupae were classified into different types according to the criteria of Brehelin, Zachary, and
Hoffman (1978) and Strand and Noda (1991). Influence of parasitization and venom on host pupal hemocyte counts
and spreading were measured at 1, 4, 6, 12, 24, 48, and 96 h after treatment in vivo. Pupae were surface sterilized with
75% ethanol (v/v), dried and then bled onto concavity slide by piercing the elytrum. Hemolymph collected into 2 𝜇l anti-
coagulant solution as a pool from at least four pupae was set as one biological replicate. After being gently mixed, 10 𝜇l
of hemolymph was pipetted into a disposable hemocytometer and allowed hemocytes to settle for 3 min at 25◦ С. The
number of hemocytes was counted under an inverted phase contrast microscope (Nikon). Their concentrations in the
hemolymph were calculated following the manufacture's instruction of hemocytometer. Similarly, spreading behav-
ior was measured by recording the morphology of plasmatocytes and granulocytes according to previously described
criteria (Ribeiro & Brehélin, 2006). During observation of hemocyte counts and spreading, at least three randomly
selected fields of the view were evaluated with three biological replicates. In addition to total hemocyte counts, prohe-
mocytes and oenocytoids among four different hemocyte types were not recorded during spreading assays, because
they were small parts of the total hemocyte population, and had no very noticeable morphology after spreading. For
encapsulation assay in vitro, 10 𝜇l of hemolymph was suspended in 180 𝜇l Grace's insect medium (Invitrogen) in a PCR
tube. Then, 2 𝜇l of venom sample prepared, as described in section 2.2, was added. After the addition of Sephadex
4 of 13 LI ET AL .

F I G U R E 1 Representative micrograph of different haemocyte types and their proportion from the pupae of Tenebrio
molitor. (A) Representative microscopic image of different hemocyte types. (B) Percentage number of different hemo-
cyte types. GR, granulocyte; PL, plasmatocyte; PH, prohemocyte; OC, oenocytoid; S, spread; NS, not spread

A-50 beads (Pharmacia), the tube was incubated for 6 h at room temperature with a rotation at a low speed of circles
per minute to keep the beads in contact with hemocytes. As described by Wu, Ye, Zhu, Chen, and Hu (2008), beads
assigned to five classes (1–5) according to the thickness of the capsule were observed and recorded under the phase
contrast microscope. Ratios of spreading and encapsulation were calculated following the methods of Zhang et al.
(2005) and Wu et al. (2008).

2.4 Data analysis

Data were collected from three biological replicates. The analysis was performed by SPSS software for Windows using
a one-way analysis of variance and Fisher's least significant difference test for significance. All percentage data were
arcsine transformed prior to analysis. Results were shown as mean ± standard deviation (SD). An acceptance level of
statistical significance was set as P < 0.05.

3 RESULTS

3.1 Hemocyte types

Four main hemocyte types including granulocytes, plasmatocytes, prohemocytes, and oenocytoids were identified in
the pupae of T. molitor (Figure 1A). In the population of hemocytes, granulocytes and plasmatocytes were dominant,
comprising almost all hemocytes (Figure 1B). The number of granulocytes was higher than that of plasmatocytes, which
is the major type. It was much easier to distinguish plasmatocytes and granulocytes from two other types once spread-
ing had occurred. In comparison with the morphology of rounded shape of nonspreading hemocytes, spreading plas-
matocytes showed a flat elliptoid or angular shape and longer filopodia at the surface of the slide plate, and spreading
granulocytes with retractile granules in the cytoplasm had extended pseudopods and shorter filopodia from the outer
surface of the cells (Figure 1A).

3.2 Hemocyte counts

In both nonparasitized and parasitized pupae, total hemocyte counts comprising all four hemocyte types were similar
to each other within 4 h (Figure 2A). But they were significantly higher in parasitized pupae than those of nonpar-
asitized pupae from 6 to 96 h. During this time interval, total hemocyte counts significantly increased after venom
LI ET AL . 5 of 13

F I G U R E 2 Changes of hemocyte count from Tenebrio molitor pupae after parasitization by Scleroderma guani. (A) Total
hemocytes, (B) granulocytes, and (C) plasmatocytes. Error bars show data range (n = 3). Different letters on the same
sampling time indicate significant differences between treatments at P < 0.05 level

injection as well. Amounts of cell debris in the hemolymph tended to increase with the increase in collected time point.
High hemocyte load was observed to be associated with the injection of venom after 12 h at the concentration of 0.1
VRE and 4 h at the concentration of 0.2 VRE (Figure 3A). When the pupae were injected with more amount of venom,
more hemocytes were induced to be produced with a time-related response. Regarding to granulocytes and plasmato-
cytes, the number of both of them displayed similar changing patterns to total hemocyte counts following parasitization
and venom injection (Figures 2 and 3).
6 of 13 LI ET AL .

F I G U R E 3 Changes of hemocyte count from Tenebrio molitor pupae after injection with Scleroderma guani venom.
(A) Total hemocytes, (B) granulocytes, and (C) plasmatocytes. VRE, one venom reservoir equivalent. Error bars show
data range (n = 3). Different letters on the same sampling time indicate significant differences between treatments at
P < 0.05 level

3.3 Hemocyte spreading

Both parasitizaiton and venom injection displayed time-dependent inhibitory effects on the spreading of granulocytes
and plasmatocytes. Significant spreading effects became visible in all treatments after 4 h. In nonparasitized pupae,
over 80% of granulocytes and plasmatocytes were extensively spread, but the spreading ratios of them were lower than
45% after 96 h parasitization (Figure 4). In venom-injected pupae, a venom dose-dependent inhibition of cell spreading
LI ET AL . 7 of 13

F I G U R E 4 Changes of hemocytes spreading of Tenebrio molitor pupae after parasitization by Scleroderma guani. (A)
Granulocytes and plasmatocytes, (B) granulocytes, and (C) plasmatocytes. Error bars show data range (n = 3). Different
letters on the same sampling time indicate significant differences between treatments at P < 0.05 level

was observed. After 96 h, 0.2 VRE venom resulted in a reduced cell (granulocytes and plasmatocytes) spreading per-
centage of 31.67%, compared to PBS (81.38%) and 0.1 VRE venom (49.91%) (Figure 5).

3.4 Hemocyte encapsulation

Since the injected beads were difficult to obtain from the pupal hemocoel, the effect of venom on the encapsulation
of hemocytes was only assessed in vitro. Five grades of encapsulated beads were classified based on the amounts of
hemocytes and thickness of hemocyte layer on the surface of the beads (Figure 6A). Comparing the encapsulation index
8 of 13 LI ET AL .

F I G U R E 5 Changes of hemocytes spreading of Tenebrio molitor pupae after injection with Scleroderma guani venom.
(A) Granulocytes and plasmatocytes, (B) granulocytes, and (C) plasmatocytes. VRE, one venom reservoir equivalent.
Error bars show data range (n = 3). Different letters on the same sampling time indicate significant differences between
treatments at P < 0.05 level

of PBS control to that of venom, it was found that venom can inhibit the encapsulation ability of hosts hemocytes to
beads (Figure 6B). The encapsulation index had decreased from 64.17% in the control to 47.33% in the 0.2 VRE venom.
But exposure of hemocytes to venom did not show a dose-dependent response on encapsulation at the current two
evaluated doses.
LI ET AL . 9 of 13

F I G U R E 6 Encapsulation response of hemocytes from Tenebrio molitor pupae inhibited by Scleroderma guani venom.
(A) Five grades (I–V) of encapsulated beads by hemocytes are shown. (B) Inhibition of encapsulation ability of hemo-
cytes by venom. Error bars show data range (n = 3). Different letters indicate significant differences between treat-
ments at P < 0.05 level

4 DISCUSSION

According to our results, it was obviously seen that parasitism of S. guani and its venom plays a role in modulating
hosts immune system by triggering the elevation of hemocyte counts, which were evidenced by the alterations in
total hemocytes, and granulocytes and plasmatocytes composed of the majority of the hemocyte population. Simi-
lar results were reported in other parasitoid–host systems, of which these parasitoids are not associated with PDVs
and use venom alone as the maternal virulent factor. For example, parasitism by P. puparum resulted in a significant
increase in the total number of hemocytes in Pieris rapae up to 5 days after parasitization (Cai et al., 2004). High con-
stitutive production of hemocytes in Drosophila melanogaster is a panacea against the infection of Leptopilina (Kacsoh
& Schlenke, 2012; Sorrentino, Melk, & Govind, 2004). However, it is often reported that parasitism of the species
from Ichneumonoidea carrying PDVs has the effects on raising hosts hemocyte counts, while venom alone of species
from this superfamily does not have such ability. For instance, total hemocyte counts of Chilo suppressalis larvae in the
late stages increased after parasitism by Cotesia chilonis, but venom alone did not cause alteration in the hemocyte
counts (Teng et al., 2016). In contrast, a reduction or no change in hemocyte population by parasitizaiton or venom
10 of 13 LI ET AL .

of parasitoids regardless of PDV association was documented (Nishikawa, Yoshimura, & Iwabuchi, 2013; Rivers, Rug-
giero, & Hayes, 2002; Teramoto & Tanaka, 2004; Yu et al., 2007). These indicate that there is diverse way of hosts
hemocyte population change induced by parasitism and venom of parasitoids from different evolutionary classes,
which depends on the specific evolutionary relationship between host and parasitoid. Additionally, venomous pro-
teins involved in modulation hosts hemocyte population might be diverse in different parasitoids. To date, they have
been not yet isolated. High hemocytes loading was supposed to reflect a response to wound plugging and healing,
hemolymph coagulation, and hemocyte clumping (Lackie, 1988), whereas this hypothesis is intriguing in those hosts
with the reduction or no change in hemocytes after parasitization or injection of parasitic factors. Elevated hemo-
cytes in P. rapae pupae parasitized by P. puparum was evidenced not to result by wounding the cuticle, but rather by
venom (Cai et al., 2004). It has been demonstrated that reduction in the number of circulating hemocytes caused by
PDV is resulted by the breakdown of the hematopoietic organ (Teramoto & Tanaka, 2004). Due to the supply of cir-
culating hemocytes from hematopoietic organs, venom of S. guani could promote them to release more numbers of
hemocytes under the condition that host is not rapidly killed by this wasp. As suggested by Strand and Noda (1991),
since the S. guani offspring needs to feed the hemolymph releasing from the wound of parasitized hosts after egg
hatching, the higher hemocyte counts in parasitized or venom-injected pupae may reflect the absence of developing
parasitoids.
Our findings significantly reveal that parasitism by S. guani and venom from this parasitoid wasp has immediately
adverse effects on hemocytes spreading in vivo. With respect to spreading, hemocytes from envenomated pupae were
altered in nearly identical fashion to that observed for natural parasitism. Immediate short-term effect of venom on
hemocytes encapsulation was also detected in vitro. Such phenomenon is consistent with other studies that venom can
generally suppress the spreading and encapsulation markedly at the early phase of incubation (Furihata, Matsumoto,
Kimura, & Hayakawa, 2013; Rivers et al., 2002; Yu et al., 2007; Zhang et al., 2005). During the late stages of obser-
vation, no recovery of spreading and encapsulation was recorded, in agreement with previous reports in parasitoids
devoid of PDVs including P. hypochondriaca, P. puparum, and N. vitripennis (Cai et al., 2004; Richards & Parkinson, 2000;
Rivers et al., 2002). It appears that venom from these species acts a persistently inactive cellular immune response.
This is different from few studies related to parasitoids taking along PDVs where parasitization and venom can only
inhibit spreading and encapsulation at the early stages, but hemocytes recovered at the late stages (Lavine & Beckage,
1996; Teng et al., 2016; Webb & Luckhart, 1994; Yu et al., 2007). In these cases, venom might lose the functions after
expression of PDV genes, but PDVs play an important role in long-term of immune competence, suggesting that venom
and viral genes may be evolutionary exchanged to perform related functions. Since encapsulation requires the active
participation of granulocytes and plasmatocytes spreading, the spreading of them greatly inhibited in some measure
led to be deprived of their ability of encapsulation. Compared to parasitism and venoms of other endoparasitoids, it
seems that S. guani venom has a less inhibitive effect on encapsulation (Cai et al., 2004; Richards & Parkinson, 2000).
This might be due to that eggs of S. guani do not develop within cavity of host, which leads to the active venom pro-
teins involved in inhibiting hemocyte encapsulation that may need not to be evolved by this parasitoid to some extent.
Recently, calreticulin involved in preventing encapsulation by inhibiting hemocyte spreading behavior has been iden-
tified as one of the relative main venom constituents from most of the available parasitoids used to decipher venom
components (Laurino et al., 2016; Manzoor, UlAbdin, Webb, Arif, & Jamil, 2016; Poirié, Colinet, & Gatti, 2014; Shaina,
UlAbdin, Webb, Arif, & Jamil, 2016; Zhang, Schmidt, & Asgari, 2006). However, this protein does not present as main
component in venom of S. guani (Zhu, 2016).
Since ectoparasitoids develop on the body surface of host where they are not fully exposed to circulating host
hemolymph-associated immune responses like endoparasitoids, venoms of them often are believed to cause perma-
nent host paralysis and developmental arrest other than the suppression of the hosts immune response for favoring
successful development of their offsprings (Hu et al., 2014; Moreau & Asgari, 2015; Wang & Yang, 2008). Interestingly,
it was notably found in this study that S. guani venom has subtly virulent effects on cellular defense of T. molitor. Since S.
guani injects poisonous venom into the host hemocoel prior to lay the eggs on its body surface during oviposition, and
its larvae will contact with hosts hemolymph released from the feeding hole, it indicates that venom of this parasitoid
is essential for the survival of its larvae to avoid hosts cellular immune attack.
LI ET AL . 11 of 13

ORCID
Jia-Ying Zhu http://orcid.org/0000-0002-4533-8203

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How to cite this article: Li L-F, Xu Z-W, Liu N-Y, Wu G-X, Ren X-M, Zhu J-Y. Parasitism and venom of ectopar-
asitoid Scleroderma guani impairs host cellular immunity. Arch Insect Biochem Physiol. 2018;e21451. https://doi.
org/10.1002/arch.21451