Differential cellular and humoral immune responses of
the Asian citrus psyllid Diaphorina citri to its ecto- and
endoparasitoids

Zhuo Yu, Liwen Feng, Mohao Xiong, Wenfeng Hu, Jun Zhong, Cong Zhang, Marylène Poirié, Jean-Luc Gatti, Bin Xia, Bin Wan

<figure><img src="image_1.png" /></figure>

To cite this version:

Zhuo Yu, Liwen Feng, Mohao Xiong, Wenfeng Hu, Jun Zhong, et al.. Differential cellular and humoral immune responses of the Asian citrus psyllid Diaphorina citri to its ecto- and endoparasitoids. Journal of Pest Science, 2025, 99 (1), pp.26. 10.1007/s10340-025-01998-9. hal-05407396

HAL Id: hal-05407396

https://hal.inrae.fr/hal-05407396v1

Submitted on 17 Dec 2025

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1 **Differential cellular and humoral immune responses of the Asian citrus psyllid**

2 *Diaphorina citri* to its ecto- and endo-parasitoids

3

4 Zhuo Yu¹†, Liwen Feng¹†, Mohao Xiong¹, Wenfeng Hu¹, Jun Zhong¹, Cong Zhang¹,

5 Marylène Poirié², Jean-Luc Gatti², Bin Xia¹, Bin Wan¹*

6

7 ¹School of Life Sciences, Nanchang University, Nanchang, China 330,031.

8 ²Université Côte d'Azur, INRAE, ISA, Sophia Antipolis, France

9 * Author of correspondence: Bin Wan, bwan@ncu.edu.cn

10 †These authors contributed equally to this work

11

12 **Abstract**

13 The Asian citrus psyllid *Diaphorina citri* is the vector of the plant pathogen *Candidatus*
14 Liberibacter asiaticus (CLas), the causal agent of the Huanglongbing disease which is
15 devastating for the citrus industry. Here, we investigated the cellular and humoral immune
16 responses of *D. citri* against the ectoparasitoid *Tamarixia radiata* and the endoparasitoid
17 *Diaphorencyrtus aligarhensis*, which are used for its biological control. We first described the
18 hemocytes of *D. citri* nymphs and classified them into five major classes based on morphology
19 and function: prohemocytes, plasmatocytes (the most abundant phagocytic cells), granulocytes,
20 adipohemocytes and oenocytoids. Parasitism by *T. radiata* resulted in a significant large
21 reduction (~50%) in the number of all host-adherent hemocytes (prohemocytes, plasmatocytes,
22 granulocytes), with also a transient decrease in prohemocytes followed by an increase in
23 granulocytes. In contrast, parasitism by *D. aligarhensis* only alters slightly the hemocytes
24 repartition. Surprisingly, we were unable to induce a clear melanization response in *D. citri*
25 hemolymph or adherent cells using classical activation tests. After parasitism we tested the
26 expression of some immunity related genes in the *D. citri* Imd, JAK/STAT, Toll and PO
27 pathways, and observed that there were all significantly affected but with different amplitude
28 and temporality by the two parasitoids. 24h and 48h after *T. radiata* parasitism, the effect on
29 immune genes remained important, whereas for *D. aligarhensis*, the genes returned to basal
30 levels or were slightly downregulated. These results complement our knowledge of *D. citri* of
31 cellular and humoral immunity, and contribute to a deeper understanding of host-parasitoid
32 immune interactions.
33 **Key words**

34 Parasitoid wasps, Asian citrus psyllid, Immune pathways, Hemocytes, Melanization

35 **Introduction**

36 Citrus greening disease, also known as Huanglongbing (HLB), is a destructive bacterial disease
37 of citrus trees causing severe losses to the citrus industry (Coates et al. 2020). Fruit from
38 infected trees develop poorly and remain green and bitter, and the trees will eventually die due
39 to the obstruction of the sap flow. The Asian citrus psyllid *Diaphorina citri* Kuwayama
40 (Hemiptera: Psyllidae), a sap feeding insect, is the main insect vector of *Candidatus*
41 Liberibacter asiaticus (CLas), the bacteria causal agent of HLB (Vyas et al. 2015). CLas is
42 transmitted in a circulative-propagative manner by *D. citri*; CLas is acquired by the psyllid from
43 the phloem of infected citrus, which then replicates in the insect hemolymph and colonized its
44 various organs (Arp et al. 2017). As there is no cure for HLB, effectively controlling psyllid
45 vectors has been imperative for reducing the HLB’s spread (Lei et al. 2022). Many countries
46 mostly used chemical control to eliminate *D. citri*. However, the frequent use of insecticides
47 has resulted in developing resistance to pesticides, and has increased the risk of environmental
48 pollution and food safety (Corallo et al. 2021). Thus, increasing efforts have been made toward
49 developing biological control solutions to *D. citri*. This biocontrol has been done mainly by the
50 use of specific parasitoids *Diaphorencyrtus aligarhensis* (Hymenoptera: Encyrtidae) and
51 *Tamarixia radiata* (Hymenoptera: Eulophidae). *Diaphorencyrtus aligarhensis* is a koinobiont
52 and solitary endoparasitoid that can parasitize the 2nd through 4th *D. citri* instar, but four instar
53 nymphs seem to be preferred. Its eggs are laid inside the psyllid nymph and develop there.
54 Depending on the geographical origin, *D. aligarhensis* can be either arrhenotokous (e.g.,
55 populations from Pakistan) or thelytokous, which consists of only females due to male-killing
56 infection with *Wolbachia* (e.g., populations from China) (Chien et al. 1989; Hoddle 2012).
57 *Tamarixia radiata* is an idiobiont and solitary ectoparasitoid that can parasitize all nymphal
58 stages of *D. citri*, but four instar nymphs seem to be preferred (Vankosky and Hoddle 2019).
59 Its egg and early larvae develop outside the host. While *T. radiata* is also infected by *Wolbachia*,
60 the parasitoid offspring sex ratio is only slightly biased towards females (Ashraf et al. 2021).
61 When tested for host range, both parasitoids exhibited a high specificity for *D. citri* (ref)
62 Studies of insect immunity provide valuable insights that contribute to a better understanding
63 of the immune system in other organisms, such as mammals (Lemaître and Hoffmann 2007;
64 Silva et al. 2024; Sheehan et al. 2018). In this regard, the host-parasitoid interaction is an ideal
experimental system to study the evolution between parasite virulence and host resistance, an
arm race falling within the “Red Queen” framework (Brockhurst et al. 2014), which is mainly
related to the host immune response. In the parasitoid-host system, parasitoids must evade or
suppress host immune responses in order to successfully parasitize the host. To do this they
have evolved a variety of strategies that can be divided into active and/or passive mechanisms
(Yang et al. 2021). Passive mechanisms include developing in locations that protect the
parasitoid from encapsulation or possessing surface features that prevent the host from
recognizing the parasitoid as non-self (Schmidt et al. 2001). Active mechanisms involve
suppression of host immunity through various virulence factors such as venoms that are injected
by female wasps at oviposition (Poirié et al. 2014). In turn, hosts can also evolve corresponding
resistance mechanisms under selection pressures imposed by parasitoids, such as increased
numbers of circulating hemocytes, enhanced encapsulation capacity, and even the evolution of
specialized hemocytes (i.e. lamellocytes in *Drosophila melanogaster*) against parasitoids
(Wertheim 2022; Carton et al. 2008). In general, endoparasitoid eggs/larvae are directly in
contact with the host's immune system. While ectoparasitoid eggs/larvae do not directly in
contact with the host's immune system, they may be affected by the host through changes in
hemolymph and melanization and clotting of the feeding site. It is speculated that
ectoparasitoids may secrete factors into the wound site that inhibit clotting and wound healing
(Strand and Pech 1995). Previous studies have reported that two *D. citri* parasitoid species have
more than 70% emergence success of offspring, implying that both parasitoids are able to
successfully evade/suppress the host's immune system, although they have different parasitism
strategies and that the effect of their venom is different; the venom of *T. radiata* being more
efficient to subdue the host than that of *D. aligharensis* (Wan et al. 2024). To our knowledge
there are no reports of these parasitoids parasitism effect on the immune response of *D. citri*.
The insect innate immune system consists of cellular and humoral defenses (Lemaître and
Hoffmann 2007). Cellular defenses include several mechanisms that are mediated by
hemocytes, such as phagocytosis, nodulation, and encapsulation (Dolezal 2023; Nappi et al.
2009). Humoral defenses include Toll, immune deficiency (Imd), Janus kinase/signal
transducer and activator of transcription (JAK/STAT) pathways that regulate, for example,
antimicrobial peptides (AMPs) expression in the fat body, and enzymatic cascades that regulate
melanization and clotting (Lemaître and Hoffmann 2007). In general, the Toll pathway is
activated by fungal and Gram-positive infections while the Imd pathway is activated by Gram-
negative bacterial infections (Tanji et al. 2007). The Jak/STAT signaling pathway coordinates
immune responses from cytokines and regulates multiple homeostasis mechanisms in the host
(Bang 2019; Agaisse and Perrimon 2004).

The current understanding of the *D. citri* immune system is largely based on the 'in silico'
description of its immune defense genes (Arp et al. 2017), while the components of the cellular
immunity are largely unknown. Annotation of the genome suggests that *D. citri* have a reduced
immune system, lacking the *Imd* gene, most antimicrobial peptides (AMPs), 1,3-β-glucan
recognition proteins (GNBPs), and complete peptidoglycan recognition proteins (PGRPs), but
Toll and JAK/STAT pathways, and Dual oxidase, Nitric oxide synthase, prophenoloxidase, and
cellular immune defense genes were present (Arp et al. 2016). This is similar to the immune
systems of many Hemiptera now characterized, where incompleteness of the Imd pathway has
been hypothesized to facilitate the establishment of symbiotic bacterial partners (Gerardo et al.
2020; Ma et al. 2022).

Insect innate immune responses involve complex mechanisms. For instance, insects rely on
their immune defense mechanisms to quickly identify and destroy invading pathogens, parasites,
and parasitoids, and these organisms have also evolved ways to evade or interfere with the host
immune response (Vinson 1990). In *D. citri*, our understanding of these relationships is largely
lacking. Here, we characterized the *D. citri* hemocytes and assessed *D. citri* humoral immunity
response at the gene level in their interactions with each of the two parasitoids *T. radiata* or *D.
aligarhensis*. We classified the *D. citri* hemocytes on morphological and functional basis, and
studied the change in the different hemocyte classes upon parasitism. We coupled this with
qPCR analysis of the expression of chosen immune genes of different immune pathways in *D.
citri*. Our results showed that both the cellular and humoral responses to parasitization by the
two parasitoid species differ.

121 **Materials and methods**

122 **Biological material**

Colonies of the thelytokous endoparasitoid *D. aligarhensis* (due to the presence of *Wolbachia*)
and the arrhenotokous ectoparasitoid *T. radiata* were obtained from the Ganzhou Citrus Science
Institute (Ganzhou, China). They were maintained separately on third to fifth instar *D.citri*
nymphs grown on potted orange jasmine (*Murraya exotica*) plants. Plants were grown under
controlled temperature and humidity (27°C, 60 ± 5% RH) in greenhouses with natural light,
before being used for psyllid rearing. Plants were caged prior to oviposition by 200-300 *D. citri*
females and the subsequent nymphal development occurred in the greenhouses. Parasitoids
130

were mass-produced as needed by introducing 50 females into the psyllid cage, and offspring
were collected daily from first emergence. Both parasitoids have been maintained in these
laboratory conditions for the past two years.

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## Parasitization procedure

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To optimize the parasitization rate, the ratio of parasitoids to hosts was increased, and the volume of the parasitization cage was reduced. An aerated plastic container containing an 8 cm high seedling of *M. exotica* was used as a parasitism unit, in which twenty 4th instar *D. citri* nymphs were exposed 24 h to four 6- to 10-day-old female wasps (*D. aligarhensis* or *T. radiata*). For each parasitism experiment, hosts were divided to do three time points: collected at 0h, 24h, and 48h after parasitoid removal (24, 48, 72 h after the host was initially exposed to the parasitoid). For the endoparasitoid *D. aligarhensis*, *D. citri* nymphs were dissected to determine parasitism. Prior to *D. aligarhensis*-parasitized host collection, 5 out of 20 alive hosts were randomly selected from each container and dissected under a microscope to ensure effective parasitism. Only when at least four of the five selected nymphs contained parasitoid eggs (i.e., parasitism rate > 80%), the remaining alive nymphs in the container were considered effectively parasitized and could be used for subsequent treatments. For the ectoparasitoid *T. radiata*, alive hosts carrying non-shriveled parasitoid eggs were collected for subsequent treatments.

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## Hemolymph collection and adherent hemocyte preparation

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*D. citri* nymphs (4th instar) were rinsed with PBS and immersed in a 30 µl drop of Grace's insect cell culture medium (Invitrogen, California, USA) on a Petri dish. Under a stereomicroscope, the ventral cuticle was carefully opened with dissecting tweezers, and the fluid escaping from the nymph, hereafter referred to as hemolymph, was collected and transferred to glass coverslips at the bottom of the well of a 24-well plate and then placed in a humid chamber. Hemocytes were allowed to settle down and adhere for 45 minutes. Non-adherent hemocytes and plasma were then removed and transferred to another well for further observation. The adherent hemocytes were washed gently three times with PBS.

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## F-actin and nuclei staining

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The washed adherent hemocytes were fixed with 4% paraformaldehyde solution (Sangon, Shanghai, China) for 15 minutes and then washed three times for 10 minutes with PBS. The hemocytes were then permeabilized with 0.1% Triton X-100 in PBS (Sangon) for 15 min and incubated with 1:1000 phalloidin-iFluor 488 (Abcam, Cambridge, United Kingdom) overnight at 4°C. Hemocytes were then washed three times in PBS for 10 minutes each. Coverslips were
mounted on slides with mounting medium containing DAPI (Abcam) to label the nuclei.

## Characterization of hemocyte types

For the determination of the total adherent hemocyte counts (THC), hemolymph from individual *D. citri* nymph (4th instar) was collected and stained as described above, and all labeled hemocytes (actin labeling + DNA labeling) on the slide were counted with a 10x objective lens under a fluorescence microscope (Leica DM4B, Leica, Wetzlar, Germany). The experiment was performed in eleven biological replicates, and each replicate contains the hemolymph of an individual nymph.

For the proportion of different adherent hemocyte types, hemolymph from 5 *D. citri* nymphs (4th instar) was collected on the same drop and hemocytes stained as described above. The surface distribution of hemocytes and the percent of different hemocyte types were estimated from 15 randomly selected fields (20x objective under fluorescent microscope). The classification and *nomenclature* of hemocytes was referenced to different studies related to insect hemocyte morphology and, in particular, those published by Gupta (1979), which summarizes the hemocyte types in Hemiptera. The experiment was performed in twelve biological replicates of five nymphs.

For the above hemocyte counting, the method used was adapted from Schmitz et al. (2012). Multiple fields were captured using a microscope (Leica DM4B) with consistent exposure time and magnification settings, and the images were exported. The raw images were processed using the FIJI image processing software (REF), with thresholds manually determined, and the software used to automatically quantified the number of fluorescent cells. The number of identified hemocyte in the different fields from one slide were then summed. The surface distribution of hemocytes can also be calculated using this method.

## Phagocytosis activity

For the determination of the *in vivo* phagocytosis ability of hemocytes, GFP-expressing *Escherichia coli* and fluorescent latex beads (2 µm-diameter carboxylate-modified polystyrene latex beads, red fluorescent; Sigma) were injected into the *D. citri* nymphs. Prior to microinjection, *E.coli*-GFP were washed twice with PBS. Bacteria were then resuspended in PBS at a concentration of 3.8 × 10⁸ CFU/ml. Fluorescent latex beads were collected by centrifugation at 8,000 rpm for 5 min, the supernatant was removed and the beads were resuspended in PBS and then washed twice by centrifugation. A volume of 23 nl of latex beads or *E.coli*-GFP was injected between the thorax and abdomen of the 4th instar nymph. 50 nymphs
were used for each injected group. Injected nymphs were returned to the plant container for 24h to allow phagocytosis by hemocytes. Hemolymph from five injected nymphs was pooled and used for adherent hemocytes preparation. Phagocytosis was examined by fluorescence microscopy, and the surface distribution, type, and percentage of hemocytes that phagocytized fluorescent latex beads or *E. coli*-GFP were estimated from 20 random fields on the slide (40x objective under fluorescent microscope). The experiment was performed in three biological replicates, and each replicate contains fifteen nymphs (randomly collected from surviving psyllids 24h after injection).

## Effect of parasitism on the differential hemocyte population

To investigate the effects of parasitism on the number of the different hemocyte types, hemolymph was pooled from five 4th instars parasitized and unparasitized nymphs of the same age at 0 h, 24 h, and 48 h post-parasitism period. To exclude unwanted side effects caused by the ovipositor puncture, the hemolymph of five nymphs punctured with a sterile needle was also collected as a control. To estimate the variation, the number of hemocytes and their types were counted on 5 random fields on the slide (20x objective under fluorescent microscope). The experiment was performed in four biological replicates.

## Effect of parasitism on the immune pathways

Based on the annotation of *D. citri* immune genes (Arp et al. 2017), we selected six key genes from different pathways for time series studies by q-PCR : the pattern recognition receptor genes *Dscam2*, the Toll pathway associated genes *Spaetzle 3-like*, the Imd and JNK associated gene *Tank-binding kinase 1 (TBK1)*, the JAK/STAT pathway associated genes *Jak2*, and two immunity effector genes, the melanization related gene *Phenoloxidase 1-like* and *Dual oxidase* involved in the production of reactive oxygen species (ROS) (Table S1). The *beta-actin* gene was used as the reference gene to normalize the expression of target genes and correct variation.

To analyze the expression patterns of immune-related genes in the immature stage of *D. citri*, *D. citri* eggs (n=100), nymphs from the 1st to 3rd instar (n=60 per instar stage), nymphs from the 4th and 5th instars (n=30 per instar stage), were collected. To investigate the effects of parasitism on the expression of immune-related genes in *D. citri* nymph, both parasitized and unparasitized nymph (n=15 per time point) at 0 h, 24 h, and 48 h post-parasitism period were collected. The collected samples were subsequently frozen in a 1.5 ml centrifuge tube at-80°C.

Total RNA was extracted using Trizol reagent (Sangon), concentration and purity was assessed spectrophotometrically (NanoPhotometer N60 IMPLEN, Munich, Germany). Extracted RNA
226 was then used for cDNA synthesized by using the PrimeScript™ RT Master Mix Reagent Kit
227 (Takara, Beijing, China). Quantitative RT-PCR (qPCR) was carried out using the TB Green™
228 Premix Ex Taq™II (Takara) and run on a PCR System (StepOnePlus Real-Time, Thermo
229 Fisher Scientific, Waltham, USA). PCR mixture (20 µl) contained 2µL of each primer 10 µM,
230 2 µL cDNA, 6µL ddH₂O and 10 µL SYBR Green Master Mix. Amplification using the
231 following cycling parameters: 95 °C for 3 min, followed by 40 cycles of 95 °C for 10 sec, 60 °C
232 for 20 s and 72°C for 25 s. The gene-specific primers used were designed with Primer Premier
233 5.0 software (Premier Biosoft International, CA, USA) and are listed in Table S1 and Fig. S1.
234 The relative expression analysis of target genes was calculated by the 2⁻ΔΔCT method as
235 previously described by Livak and Schmittgen (2001). All samples were run in technical
236 duplicates, and all experiments were performed three times.

237 Hemocyte phenoloxidase (PO) activity

238 Prophenoloxidase (PPO) may be an inactive zymogen that need to be activated before assessing
239 PO activity. Three protocols were used for detecting PO activity in hemocytes of *D. citri* nymph
240 (4^th^ instar). In the first method, according to Rizki and Rizki (1959), nymphs were placed in
241 either in microtubes with 70% ethanol to activate PPO, or control tubes with 70% ethanol and
242 phenylthiourea (PTU, 2 mg/ml in 70% ethanol, sigma) which specifically inhibits PPO, and
243 then let for 48h. In the second method, according to Bruno et al. (2023), each nymph was
244 individually placed into a microtube with PBS, and the tubes were heated at 65°C for 22 min
245 to induce spontaneous activation of PPO. For these two methods, after the indicated time,
246 nymphs were removed from the tubes and observed for melanization, and black cells
247 underneath the cuticle were searched under a microscope. Ten nymphs were used for each
248 experiment and three replicates were performed. In the third method, hemolymph and/or
249 hemocyte samples were mixed with dopamine (DOPA) as a substrate (Nappi et al. 1987) as
250 described in Laughton and Siva-Jothy (2011) and Schmitz et al. (2012). Collected hemolymph
251 was incubated PBS alone as a control or PBS with DOPA (Sangon, 1mg/ml or 5mg/ml) and
252 photographed after 0.5, 1, and 2 h. Hemolymph from five naive nymphs were used in each
253 experiment and three replicates were performed. Five nymphs parasitized by *T. radiata* at 72 h
254 post-parasitism and two nymphs parasitized by *D. aligarhensis* at 48 h post-parasitism were
255 used in each experiment and two replicates were performed.

256 For adherent hemocytes, after PBS washing, they were fixed with either a DOPA solution at 1
257 mg/ml in 35% ethanol to reveal the ethanol-activated PPO or 35% ethanol alone as a control.
258 After 30 min hemocytes were then washed three times in PBS. Coverslips were mounted on
slides using the Mounting Medium with DAPI (Abcam) and observed. Cells containing PO should appear brown-dark under bright field microscopy (20x objective under fluorescent microscope). Cells from five naive nymphs were used in each experiment and twelve replicates were performed. Cells from two nymphs parasitized by *T. radiata* at 48 h post-parasitism period and one nymph parasitized by *D. aligarhensis* at 24 h post-parasitism period were treated separately the same way and five replicates were performed.

## Data Analysis

All data were tested using the Shapiro-Wilk normality test before analysis of the homogeneity of variances (homoscedasticity) using Levene's test. The surface distribution of hemocytes in the hemolymph of *D. citri* and the percent of different hemocyte types were analyzed using two-tailed unpaired Student's t-tests (normal distribution and homogeneity of variance), Welch's t-test (normal distribution and heterogeneity of variance), or Mann-Whitney U test (non-normal distribution or strong heterogeneity of variance). The effect of parasitism on the total hemocyte counts was analyzed using two-tailed unpaired Student's t-tests. The effect of parasitism on the expression of immune-related genes and the determination of hemocyte PO activity was analyzed using the Mann-Whitney U test. The expression patterns of immune-related genes in the immature stage of *D. citri* were analyzed using One-way ANOVA with Tukey's multiple comparisons test. Details of the statistical analysis are provided in the figure legends and all data can be found in Table S2 to S7. For each data set, the mean ± SEM was used to summarize the data. Data analysis was performed using SPSS 24.0 software and graphs were generated using GraphPad Prism 8.0 software.

## Results

### Outcome of *Diaphorina citri* parasitism

During parasitism, the female *T. radiata* (ectoparasitoid) probes the ventral surface of the *D. citri* nymph and deposits an egg on the host abdomen near one of its hind legs (Fig. 1A-C). Less than 24 hours after, at the site of oviposition a local immune response of *D. citri* was observed, within an average of six melanized spots appearing around the egg deposition site (Fig. 1B-C, Fig. S2A). We observed that 12 days after parasitization by *T. radiata*, 27 % of psyllid nymphs did not produce either adult parasitoid wasps or adult psyllids, and both later died (Wan et al. 2024).
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The female *D. aligarhensis* (endoparasitoid) usually probes the dorsal surface of the *D. citri* nymph's abdomen before ovipositing (Fig. 1D). At day 16 after parasitization, from 73% of the *D. citri* submitted to parasitism an adult parasitoid wasp emerged, while only 6% of adult psyllids emerged (Wan et al. 2024). Surprisingly, most of the remaining psyllids (21%) were still alive but blocked at the final nymph stage, and when parasitized, the parasitoid inside did not develop either. In this case, parasitoid larvae showed no obvious signs of melanization or encapsulation even if they can be surrounded by some cells (Fig. 1E, Fig. S2B).

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<figure><img src="image_2.png" /></figure>

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<caption>**Fig. 1** Parasitism of *Diaphorina citri* nymph by *Tamarixia radiata* or *Diaphorencyrtus aligarhensis*. (A) *T. radiata* female laying an egg underneath a *D. citri* nymph. (B) The egg of *T. radiata* (black arrow in enlargement) attached to the host abdomen. (C) after egg removal, multiple melanization spots are visible on host abdomen (white arrow in enlargement). (D) *D. aligarhensis* female ovipositing an egg inside a *D. citri* nymph. (E) Larvae of the *D. aligarhensis* at 16 days post-parasitism that had not developed.</caption>

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**D. citri hemocytes: characterization and function**

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*D. citri* nymph hemolymph circulating hemocytes were divided into five types based on morphological and functional classification of insect hemocytes (Devauchelle 1971; Gupta 1979) : prohemocytes, plasmatocytes, granulocytes, adipohemocytes, and oenocytoids (Fig. 2).
Adipohemocytes are ovoid with large globular inclusions (Fig. 2D), and oenocytoids were large and round cells with a low nucleus/cytoplasm ratio and no structures extruding from the cell membrane (Fig. 2E). Adipohemocytes and oenocytoids were unable to adhere to foreign
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surfaces, but prohemocytes, plasmatocytes, and granulocytes were strongly adherent on glass slide. We thus focus the rest of the study on these adherent hemocytes. The total number of adherent hemocytes was 903 ± 87 for an individual *D. citri* 4th instar nymph, among which plasmatocytes represented the most abundant population, accounting for ~ 86% of cells (Fig. 3D-E). Plasmatocytes were variable in shape, showing oval, stellate, or irregular shapes, generally expanding long filopodia and large lamellipodia (Fig. 2B; Fig. 3B). As a result, their measured surface ranged from 15 to 350 µm². The proportion of prohemocytes and granulocytes was much lower, about 7% each (Fig. 3E). Prohemocytes were small round cells (surface from 15 to 80 µm²) with a smooth membrane without protrusions and a very high nucleus/cytoplasm ratio (Fig. 2A; Fig. 3A). Granulocytes were larger cells, from round to oval shape, and contained many cytoplasmic granules and vesicles (Fig. 2C; Fig. 3C). Their surface ranged from 48 to 600 µm². They can sometimes form small pseudopodia or filopodia, but these are much shorter than those of plasmatocytes. A few other adherent cell types that did not fit into the three main morphotypes were not detailed because they represented less than 1% of the cells.

The phagocytic function of the different adherent hemocyte types was tested by injecting latex beads or fluorescent *E. coli*-GFP into the body cavity of *D. citri* 4th instar nymphs. 24 h after injection, all adherent hemocyte showed an ability to engulf latex beads or bacteria (Fig. S3 and Fig. S4), but the predominant ones were the plasmatocytes. Indeed, ~16% of plasmatocytes but only ~0.8% of prohemocytes and ~0.7% of granulocytes, showed some latex bead phagocytosis (Fig. S3, Fig. S5).

<figure><img src="image_3.png" /></figure>

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**Fig. 2 Hemocytes in citrus psyllid hemolymph.** (A) Prohemocytes. (B) Three plasmatocytes beginning to adhere to the glass slide showing extended filopodia (black arrows). (C) Adherent granulocyte with extending lamellipodia. (D) Adipohemocytes with large globular (white arrows) inclusions. (E) Oenocytoids. These two last types lack adhesive properties. Scale bar = 20 µm for all pictures.

<figure><img src="image_4.png" /></figure>

**Fig. 3 Characteristics of different the adherent hemocyte types.** Fluorescent picture showing the actin cytoskeleton (stained in green by phalloidin-GFP) and the nucleus (in blue by DAPI) of representative prohemocytes (panel A), plasmatocytes (panel B), and granulocytes (panel C). (D) Total adherent hemocyte count from a single *Diaphorina citri* nymph. Mean ± SEM, n=11. (E) Proportion of the different adherent hemocyte types in *D. citri* hemolymph. Mean ± SEM, n=12. Scale bar = 10 µm, same for all pictures.

## Effect of parasitism on the hemocyte population and composition

To determine whether parasitism by *T. radiata* or *D. aligarhensis* altered the adherent hemocyte populations of *D. citri* nymphs, hemolymph of both parasitized and unparasitized nymphs were collected and analyzed at different times after parasitism (Fig. 4). Compared to the control, the hemocyte count (HC) was unchanged at 0h post-parasitism period. The HC was significantly decreased at 24h post-parasitism period by *T. radiata* parasitism and remained significantly lower at 48h (24h, *t*=2.754, *P* < 0.05; 48h, *t*=2.747, *P* < 0.05), while no significant difference in HC was observed after *D. aligarhensis* parasitism during the same time period.
<figure><img src="image_5.png" /><figcaption>**Fig. 4.** Effects of parasitism on total adherent hemocyte counts of *Diaphorina citri* nymphs. Adherent hemocyte counts in the hemolymph of *D.citri* nymphs that were unparasitized (green bars), parasitized by *Tamarixia radiata* (blue bars) or parasitized by *Diaphorencyrtus aligarhensis* (yellow bars) at different post-parasitism times. For each experiment, the slide contained pooled hemolymph from 5 nymphs and cells were counted from 5 randomly selected microscopic fields. Mean ± SEM, n= 4. Two-tailed unpaired Student's t-test (*P* < 0.05; ns, not significant).</figcaption></figure>

In more details, compared to the control, the percentage of prohemocytes in *D. citri* parasitized by *T. radiata* significantly decreased at 0h post-parasitism and returned to the control level at 24h (Fig. 5) (t = 2.640, *P* < 0.05). On the contrary, prohemocytes significantly increased at 0h and 24h post-parasitism by *D. aligarhensis*, and seemed more elevated, but not significantly, than control at 48h (0h, t = 2.708, *P* < 0.05; 24h, t = 3.091, *P* < 0.05). Prohemocytes were not affected by parasitism while granulocytes were affected significantly by *T. radiata* parasitism only at 24h, although their average number was higher than control also at 0h and 48h. Compared to the control, the percentage of plasmatocytes in *D. citri* parasitized by *T. radiata* and by *D. aligarhensis* slightly decreased at 0h, 24h, and 48h, but not significantly. When we analyzed the hemocyte surface distribution of parasitized and unparasitized *D. citri* (Fig. S6), we observed that both parasitism more subtly modified the distribution of cells during the time frame of the following 48h. At 0h post-parasitism period by *T. radiata*, a decrease, although not significant, occurs in cells with less than 50 µm² of surface, while an increase in those over 130-190 µm² becomes significant, suggesting may be a relationship between these two populations (130-150, t = -3.779, *P* < 0.01; 150-170, t = -4.090, *P* < 0.01; 170-190, *Z* = -2.323,
P < 0.05). On the contrary, after *D. aligarhensis* parasitism, cells with a surface of less than 50 µm² increase, an increase that becomes significant at 24h and also 48h (24h: 10-30, Z = -2.309, P < 0.05; 24h: 30-50, t = -3.491, P < 0.05; 48h: 10-30, Z = -2.309, P < 0.05). However, this parallels a significant decrease in 70-150 µm² cells, visible at 24h, but that were replenished at 48h, suggesting may be a relationship between these two populations (24h: 70-90, t = 4.959, P < 0.01; 24h: 90-110, t = 2.597, P < 0.05; 24h: 110-130, t = 3.602, P < 0.05; 24h: 130-150, t = 3.388, P < 0.05).

Since oviposition induced some wounding to the host, we examined whether the mechanical wounding alone was able to induce a response in the hemocyte composition. The percentages of different hemocyte types of control *D. citri* and mechanically injured *D. citri* 0h, 24h, and 48h after wounding did not significantly change (Fig. S7).

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<figure><img src="image_6.png" /></figure>

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Fig. 5. Changes in the different hemocyte types after parasitism. The hemocyte types of unparasitised *Diaphorina citri* nymphs and nymphs parasitized by *Tamarixia radiata* or *Diaphorencyrtus aligarhensis* were estimated at different times after parasitism. For each experiment, the slide contained pooled hemolymph from 5 nymphs and all cells from 5 different random fields on the slide were analyzed. Mean ± SEM, n=4. Significance was determined by two-tailed unpaired Student's t-test, Mann-Whitney U-test or Welch's t-test (*P* < 0.05).

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403 **Effect of parasitism on the immune pathways**

To investigate the effects of *T. radiata* and *D. aligarhensis* parasitization on different *D. citri* immune pathways, we analyzed the mRNA expression levels of the *Dscam2*, *Spaetzle 3-like*, *TBK1*, *Jak2*, *Phenoloxidase 1-like*, and *Dual oxidase* genes. We first examined the expression during *D. citri* development patterns from egg to the 5th instar nymph stages (Fig. S8). We observed that the expression levels of *Jak2*, *Phenoloxidase 1-like*, and *Dual oxidase* did not
409 significantly change during development, whereas the expression levels of *TBK1*, *Dscam2* and
410 *Spaetzle 3-like* decreased significantly between the egg and the second instar nymph stages and
411 remained low in the third to fifth instar stages (*Dscam2*, *F* = 31.55, *df* = 5, *P* < 0.0001; *Spaetzle
412 3-like*, *F* = 6.445, *df* = 5, *P* < 0.01; *TBK1*, *F* = 10.77, *df* = 5, *P* < 0.001; *Jak2*, *F* = 3.425, *df=
413 5*, *P* < 0.05).

414 After parasitization of the 4th instar nymphs by *T. radiata*, the expression levels of *Dscam2*,
415 *TBK1*, and *Jak2* were significantly increased, followed by these of *Spaetzle 3-like* and
416 *Phenoloxidase 1-like* 24h later. Thereafter the expression of *Dscam2*, *TBK1*, *Jak2*,
417 *Phenoloxidase1-like* remained significantly upregulated at 48h post-parasitism period while
418 this of *Spaetzle 3-like* was downregulated. The expression pattern of *Dual oxidase* was always
419 significantly reduced after parasitism (Fig. 6) (*Dscam2*: 0h, *Z* = -2.558, *P* < 0.01; *Dscam2*: 48h,
420 *Z* = -2.882, *P* < 0.01; *Spaetzle 3-like*: 24h, *Z* = -2.882, *P* < 0.01; *Spaetzle 3-like*: 48h, *Z*
421 = -2.558, *P* < 0.01; *TBK1*: 0h, *Z* = -2.324, *P* < 0.05; *TBK1*: 24h, *Z* = -2.739, *P* < 0.01; *TBK1*: 48h,
422 *Z* = -2.739, *P* < 0.01; *Jak2*: 0h, *Z* = -2.558, *P* < 0.01; *Jak2*: 24h, *Z* = -2.739, *P* < 0.01; *Jak2*:
423 48h, *Z* = -2.739, *P* < 0.01; *Phenoloxidase 1-like*: 24h, *Z* = -2.558, *P* < 0.01; *Phenoloxidase 1-
424 like*: 48h, *Z* = -2.739, *P* < 0.01; *Dual oxidase*: 0h, *Z* = -2.324, *P* < 0.05; *Dual oxidase*: 48h, *Z
425 = -2.739, *P* < 0.01). Parasitization by *D. aligarhensis*, increased significantly the expression of
426 *TBK1* and *Jak2*, albeit slightly, at 0h post-parasitism period (Fig. 7). Then the expression levels
427 of *Dscam2*, *Spaetzle 3-like*, *Phenoloxidase 1-like* were increased significantly at 24h post-
428 parasitism period (Fig. 7). 48 hours after a period of parasitism, the expression of *Dscam2*,
429 *Spaetzle 3-like*, and *Jak2* were significantly decreased, while the expression levels of *TBK1* and
430 *Phenoloxidase 1-like* returned to their control level. The expression pattern of *Dual oxidase*
431 was significantly downregulated at 24h post-parasitism period and then upregulated at 48h post-
432 parasitism period (Fig. 7) (*Dscam2*: 0h, *Z* = -2.739, *P* < 0.01; *Dscam2*: 24h, *Z* = -2.739, *P* <
433 *Dscam2*: 48h, *Z* = -2.882, *P* < 0.01; *Spaetzle 3-like*: 24h, *Z* = -2.739, *P* < 0.01; *Spaetzle
434 3-like*: 48h, *Z* = -2.739, *P* < 0.01; *TBK1*: 0h, *Z* = -2.882, *P* < 0.01; *Jak2*: 0h, *Z* = -2.556, *P* <
435 *Jak2*: 48h, *Z* = -2.739, *P* < 0.01; *Phenoloxidase 1-like*: 24h, *Z* = -2.739, *P* < 0.01; *Dual
436 oxidase*: 24h, *Z* = -2.882, *P* < 0.01; *Dual oxidase*: 48h, *Z* = -2.739, *P* < 0.01).
<figure><img src="image_7.png" /><figcaption>B Spaetzle 3-like</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>C</figcaption></figure>

<figure><img src="image_9.png" /><figcaption>D Jak2</figcaption></figure>

<figure><img src="image_10.png" /><figcaption>E Phenoloxidase 1-like</figcaption></figure>

<figure><img src="image_11.png" /><figcaption>F Dual oxidase</figcaption></figure>

<figure><img src="image_12.png" /></figure>

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Fig. 6. Effect of *Tamarixia radiata* parasitism on the immune pathways of *Diaphorina citri* nymphs. The mRNA levels measured by RT-qPCR for *Dscam2* (A), *Spaetzle 3-like* (B), *TBK1* (C), *Jak2* (D), *Phenoloxidase 1-like* (E), *Dual oxidase* (F) genes at different times after parasitism were compared to expression in unparasitized *D. citri* of the same age. Mean ± SEM, n=6. Significance was determined by a two-tailed Mann-Whitney U test (*P* < 0.05; **P** < 0.01; ns, not significant).

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<figure><img src="image_13.png" /><figcaption>B Spaetzle 3-like</figcaption></figure>

<figure><img src="image_14.png" /><figcaption>C TBK1</figcaption></figure>

<figure><img src="image_15.png" /><figcaption>D Jak2</figcaption></figure>

<figure><img src="image_16.png" /><figcaption>E Phenoloxidase 1-like</figcaption></figure>

<figure><img src="image_17.png" /><figcaption>F Dual oxidase</figcaption></figure>

<figure><img src="image_18.png" /></figure>

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Fig. 7. Effect of *Diaphorencyrtus aligarhensis* parasitism on the immune pathways of *Diaphorina citri* nymphs. The mRNA levels measured by RT-qPCR for *Dscam2* (A), *Spaetzle 3-like* (B), *TBK1* (C), *Jak2* (D), *Phenoloxidase 1-like* (E), *Dual oxidase* (F) genes at different times after parasitism were compared to expression in unparasitized *D. citri* of the same age. Mean ± SEM, n=6. Significance was determined by a two-tailed Mann-Whitney U test (*P* < 0.05; **P** < 0.01; ns, not significant).

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454 Phenoloxidase (PO) activity

455 All the different attempts made to activate the PO in *D. citri* were unsuccessful. Hemolymph
456 alone or incubated for 2 hours with L-DOPA showed no melanization (Fig. S9A). Immersing
457 intact nymphs in ethanol for 48 hours or heating them for 22 minutes at 65°C did not produce
458 the black spots that indicate cell melanization observed in other insects (Fig. S10A-B). After
459 incubation of *D. citri* adherent hemocytes with L-DOPA in ethanol, no hemocytes were
460 melanized, only small-sized adherent vesicles without labeled DNA showed some melanization
461 (Fig. S9B-D). Based on the temporal dynamics of the *Phenoloxidase 1-like* transcription level
462 of parasitized *D. citri* nymphs, (at 48h, 72h post-parasitism period for *T. radiata* and 48h for *D.
463 aligarhensis*), hemolymph and hemocytes of parasitized nymphs were treated as described
464 above but no significant melanin production was detected and only a few small-sized adherent
465 vesicles showed some melanization similar to unparasitized nymphs (Fig. S11).

466 Discussion

467 Effect of parasitism on hemocytes

468 The cellular immune response of insects relies on different hemocyte types with different
469 functions, particularly to fight pathogens and parasitoids (Eleftherianos et al. 2021). In the
470 hemolymph of *D. citri* nymphs, five main types of hemocytes were found, three of which had
471 adhesive and phagocytic properties: the prohemocytes and the plasmatocytes, which are
472 common and widely conserved among insects, and the granulocytes, another basic type of
473 insect hemocyte (Eleftherianos et al. 2021). Plasmatocytes certainly play a major role in
474 phagocytosis since a large proportion of them showed this function and they are also the
475 majoritarian cells. In Diptera such as *Hermetia illucens* and *Drosophila melanogaster*,
476 plasmatocytes are the only phagocytosis competent hemocyte, while in other insect orders such
477 as Lepidopterans, both plasmatocytes and granulocytes perform phagocytosis (Meister and
478 Lagueux 2003; Lavine and Strand 2002; Bruno et al. 2023). In the hemipteran *Acyrthosiphon
479 pisum*, plasmatocytes and granulocytes were also shown to phagocyte bacteria and beads
480 (Schmitz et al. 2012). Our results showing that some of the prohemocytes may phagocytize
481 beads and bacteria is quite different from these previous reports. Prohemocytes are viewed as
482 progenitor cells, able to differentiate into other hemocyte categories (Lavine and Strand 2002)
483 and those demonstrating phagocytic function here were may be in their way to differentiate in
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plasmatocytes or granulocytes. The interplay between these different types of cells has yet to be established in *D. citri*.

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The two non-adherent types were classified as oenocytes and adipohemocytes only on the basis of their morphology (Hillyer et al. 2003; Ribeiro and Brehélin 2006; Schmitz et al. 2012). They have been less observed in Hemiptera, partly because they are non-adherent and partly because they are fragile cells (Hillyer et al. 2003; Ribeiro and Brehélin 2006; Schmitz et al. 2012). In insects, oenocytes are of ectodermal origin and their size, number, and anatomical locations are species dependent (Makki et al. 2014). Among their different physiological functions, they have been involved in fatty acid and hydrocarbon metabolism, In *D. melanogaster*, larval oenocytes synthesize very-long-chain fatty acids required for tracheal water-proofing and in adult they produce cuticular hydrocarbons (Makki et al. 2014). They have also been involved in immunity by releasing a prophenoloxidase (Shrestha and Kim 2009; Essawy et al. 1985). Adipohemocytes contain lipid-like vesicles, sometimes large enough to deform the cell. They may look similar to spherulocytes although their size appears smaller (Cook et al. 1985; Schmitz et al. 2012). The function of these cells is suggested to be energy storage in lipids and glycogen form; however, they may also play other role such as the formation of extracellular trap (NETosis) and participate to hemolymph coagulation when they lyse upon stimulation (Eleftherianos et al. 2021; Schmitz et al. 2012).

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The total number of *D. citri* nymph adherent hemocytes was approximately 900, with 86% being plasmatocytes, while prohemocytes and granulocytes represent each 7%. We have not quantified the two other types of hemocytes but from our observation they may represent only a few percent of the total hemocytes in hemolymph. By comparison, the *Drosophila* 3rd instar larva can have more than 3000 circulating hemocytes, 95% of which are plasmatocytes (Petraki et al. 2015), while Hemiptera such as *Acyrthosiphon pisum* contain about 600 adherent hemocytes (Schmitz et al. 2012) and among them 50 to 70% are plasmatocytes, the rest being mainly granulocytes. Overall, these data suggest that *D. citri* nymphs possess a full spectrum of hemocytes, similar to that found in many insect species, capable of contributing to different physiological functions, including immune functions.

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Because parasitoids need to avoid the host immune response for the survival of their eggs, particularly the host egg encapsulation by hemocytes, female often suppress the host hemocyte by injecting specific venom components (Eleftherianos et al. 2021; Wan et al. 2020; Nappi et al. 2009). *D. citri* nymph parasitized by *T. radiata* showed a strong reduction in the hemocyte count post-parasitism. This reduction did not affect strongly the hemocyte type repartition although a decrease of prohemocytes and an increase in granulocytes occurred, suggesting may
be a differentiation of prohemocytes into granulocytes. Thus, this ectoparasitoid destroys rapidly a large number of hemocytes whatever their types. Since *T. radiata* inject a deadly venom at oviposition (Wan et al. 2024), some of its components may be active against the host cells including the hemocytes. This allow also to avoid a possible host immune response, in particular the healing of the wound made for the feeding site of the external parasitoid larva. In comparison, *D. citri* nymph parasitized by *D. aligarhensis* showed no clear change in hemocyte count nor important change in the cell types proportion. This suggests that the host hemocytes did not recognize the parasitoid egg and the female wasp did not inject components against to these cells. Indeed, a very low probability of encapsulation (1%) of this endoparasitoid was previously reported by Rohrig et al. (2011), and we rarely observed the formation of a sort of cellular aggregation around parasitoid larvae blocked in their development at the third or fourth instar and unable to reach their pupal stage. Endoparasitoids have developed different ways to avoid the host cellular response, some hide their egg in host tissues, decreasing their accessibility while other uses egg surface components that mimic the host tissue (Godfray 1994). Since *D. aligarhensis* egg and larva are not hidden in *D. citri* tissues, but are free floating in the haemocoel Rohrig et al. (2011), the second hypothesis is the more probable. Alternatively, *D. citri* did not invest in defense against *D. aligarhensis* thus having no reaction and lacking specific immune cells like the drosophila lamellocytes specialized in the capsule formation (Rizki and Rizki 1992). However, we cannot exclude that the production of such cells is blocked by the parasitoid venom components without affecting the other hemocyte types.

One striking result is the difficulty to observe a phenoloxidase activity in *D.citri* hemolymph or in hemocytes. Phenoloxidase (PO) is a key enzyme in the formation of melanin involved in immunity and different other physiological processes(Nappi and Christensen 2005; Lu et al. 2014). One phenoloxidase (PO) gene *Phenoloxidase 1-like* is reported in *D. citri* genome and this gene expression was significantly increased after parasitization by the two parasitoids (see also further). We also observed that melanization occurred around the site of the *T. radiata* egg, suggesting the wound-healing of the female probing sites. It is generally accepted that PO are produced as prophenoloxidase (PPO), an inactive zymogen, that can be activated either by a conformational change or by a proteolytic cleavage (Lu et al. 2014). Parasitoids have evolved several mechanisms to inhibit host PPO activation particularly by blocking the proteases involved in the activation cascades with venom serpin for example (Yan et al. 2023; Colinet et al. 2009). *D. citri* PPO did not autoactivate like PPO of other insects (such as in *Drosophila* hemolymph), and none of the methods used here (solvent, T°) allowed for its activation. Since
these methods were mainly based on inducing conformational changes, we can hypothesize that it is difficult to change the *D. citri* pro-enzyme conformation. May be a more specific action of protease is needed but, apparently, they were not present or active in hemolymph at the collection time. However, although none of the three adherent types were clearly reacting, we observed some darken small adherent vesicles after activation on our cell preparations. They may be small reacting cells and their nucleus labeling made invisible by the melanization that block the light (Brenner and Hearing 2008) or large vesicles/blebs circulating in the hemolymph, either coming from lysis of another type of hemocytes, such as oenocytes that may have some phenoloxidase (Shrestha et al. 2011).

## 561 Effect of parasitism on the immune pathways

Like some other Hemiptera that live in symbiosis with an obligate bacterium, *D. citri* shows a reduced innate immune system, in particular it lacks genes in the Imd pathway (*Imd*, *Dredd* and *relish*), most antimicrobial peptides, 1,3-β-glucan recognition proteins (GNBPs), and complete peptidoglycan recognition proteins (Arp et al. 2016). However, that did not preclude the existence of an immune response to parasitoids involving the Toll, the Imd, the JAK/STAT and the PO pathways. These pathways are launched by pattern recognition receptors (PRRs) which recognize pathogen-associated molecular patterns associated to the invaders (Wang et al. 2019). In *D. melanogaster* it was previously shown that *Lectin-24A* was upregulated after parasitization by *Leptopilina boulardi* and by *Asobara tabida* (Keebaugh and Schlenke 2012). On the contrary, several PRRs in *Pieris rapae* were shown to be downregulated after parasitization by *Pteromalus puparum* (Zhu et al. 2015). In our study, the expression of *Dscam2*, a receptor involved in pathogens recognition (Ng and Kurtz 2019), was significantly increased post-parasitism by *T. radiata* and by *D. aligarhensis*, although with a different timing. This is true also for all the other genes tested, except for the DUOX genes that was mainly repressed after *T. radiata* parasitism. One general observation is also that the increase expression seemed higher and lasted longer after *T. radiata* than *D. aligarhensis* parasitism.

The Toll and Imd pathways synergistically activate the innate immune system of insects in response against microorganisms (Wang and Ligoxygakis 2006), while the JAK/STAT pathway is implicated in response to bacteria, fungi, viruses and parasitoids, and is also involved in development, cell proliferation, differentiation, and apoptosis including the differentiation and proliferation of hemocytes (Zeidler and Bausek 2013). In *D. citri*, part of the Imd pathway was absent, although *TBK1* and the downstream JNK pathway were identified, and thus may be activated by an alternative signal (Tanji et al. 2007; Larabi et al. 2013; Arp et
al. 2016). Previous studies reported that parasitism activates the Toll, Imd and JNK, JAK/STAT pathways of the host. For instance, genome-wide analysis of *Drosophila* indicated that several immune-related genes of these pathways exhibited significantly increased expression levels following parasitization by endoparasitoid *Asobara tabida* (Wertheim et al. 2005). The ectoparasitoid *P. vindemmiae* infection also activated these pathways in *Drosophila* (Yang et al. 2019). In our study, the expression of *TBK1*, *Jak2*, and *spaetzle 3-like* genes in *D. citri* was significantly increased post-parasitism. However, the two parasitoid species induced different spatio-temporal patterns of immune gene expression post-parasitism: *TBK1* and *Jak2* genes were kept upregulated after parasitization by *T. radiata* while *Spaetzle 3-like* and *Jak2* genes were significantly downregulated after parasitization by *D. aligarhensis*, suggesting a lower immune response of *D. citri* to *D. aligarhensis*.

Our results also show a down regulation of the *Dual oxidase (Duox)* expression after parasitism, which is maintained in the case of *T. radiata* but not *D. aligarhensis*. *Duox* mediates the production of reactive oxygen species (ROS) and is a major immune mechanism regulating insect gut-microbe homeostasis and also the gut functions (Jang et al. 2021). This enzyme is also involved in the wound healing mechanisms by producing $H_2O_2$ that attracts hemocytes to the wound site (Moreira et al. 2010). The production of ROS by the host is also a means to eliminate the parasitoid egg (Nappi and Christensen 2005). This suggests that the parasitoid need to decrease, at least temporarily, the ROS response of the host, particularly in the case of the ectoparasitoid that needs to feed through the cuticle and the tissues of the host during a certain period. While PO may be linked to the ROS production (Nappi and Christensen 2005) and although we observed an increase in the *Phenoloxidase 1-like* expression, as described before, we were unable to demonstrate an activity of this enzyme in the hemolymph before or after parasitism, nor clearly determine which cells may produce this enzyme. Thus, its role in *D. citri* response against parasitoid remain to be explored.

## Conclusion

Our studies characterized, for the first time, the morphology and function of *D. citri* hemocytes. We also demonstrate for the first time that the ectoparasitoid affects the cellular immunity of *D. citri* and that humoral immune response occurred in reaction to the two biocontrol parasitoid species. Although this reaction may be too low to resist to the parasitoid it can help some of the psyllids to escape parasitism. This provides an effective model to gain insight into the mechanisms by which different parasitoids evade/suppress the host's immune response, and contributes to the subsequent use of parasitoids for management and biocontrol. They will help
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in the future to study the immune response in a tripartite host-pathogen-parasitoid interactions
such as in the *D. citri* infected by CLas.

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**Supplementary Information** The online version contains supplementary material available at
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**Author Contributions** Z.Y., J.-L.G., M.P., B.X. and B.W. conceived and designed the experiments. Z.Y., L.F., M.X., W.H., J.Z. and C.Z. conducted the experiments. Z.Y., W.H. and B.W. analyzed the data. Z.Y., J.-L.G. and B.W. wrote the manuscript. All authors read, corrected, and approved the manuscript.

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**Funding** This work was supported by National Natural Science Foundation of China (Grant No. 32460702 and No. 32102204); Natural Science Foundation of Jiangxi Province, China (Grant No. 20224BAB215021); the French Government (National Research Agency, ANR) through the "Investments for the Future" programs LABEX SIGNALIFE ANR-11-LABX-0028-01 and IDEX UCAJedi ANR-15-IDEX-01.

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**Data availability** Available on request.

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**Declarations**

638

**Conflict of interest** The authors declare no competing interests.

639
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Journal of Pest Science

*Supplementary Information*

**Differential cellular and humoral immune responses of the Asian citrus psyllid *Diaphorina citri* to its ecto- and endo-parasitoids**

Zhuo Yu<fnref n="1" />†, Liwen Feng<fnref n="1" />†, Mohao Xiong<fnref n="1" />, Wenfeng Hu<fnref n="1" />, Jun Zhong<fnref n="1" />, Cong Zhang<fnref n="1" />,
Marylène Poirié<fnref n="2" />, Jean-Luc Gatti<fnref n="2" />, Bin Xia<fnref n="1" />, Bin Wan<fnref n="1" />*

**Contents**

**Fig. S1** PCR-amplification products of immune-related genes from *Diaphorina citri*.

**Fig. S2** Observable response of *Diaphorina citri* nymph to parasitism.

**Fig. S3** *In vivo* phagocytosis of fluorescence latex beads.

**Fig. S4** *In vivo* phagocytosis of fluorescent *Escherichia coli*-GFP.

**Fig. S5** Surface distribution of hemocytes after injection of fluorescent latex beads.

**Fig. S6** Surface distribution of hemocytes after parasitism.

**Fig. S7** Percentage of different hemocyte types in *Diaphorina citri* nymphs at different times after
mechanical injury.

**Fig. S8** RT-qPCR of the six immune-related genes expression during *Diaphorina citri* development.

**Fig. S9** Phenoloxidase activity in hemocytes of *Diaphorina citri* nymphs.

**Fig. S10** Phenoloxidase activity in *Diaphorina citri* nymphs.

**Fig. S11** Phenoloxidase activity in hemocytes of parasitized *Diaphorina citri* nymphs

**Table S1** All primers used for qPCR in this study.

**Table S2** Results from Student's t-tests for comparing hemocyte counts in the hemolymph of *Diaphorina citri* that were unparasitized and parasitized by parasitoids.

**Table S3** Results from Student's t-tests, Mann-Whitney U test, or Welch's t-test for comparing the percentages of the hemocyte with different areas in the hemolymph of *Diaphorina citri* that were

<footnote n="1">¹ School of Life Sciences, Nanchang University, Nanchang, China 330,031.</footnote>

<footnote n="2">² Université Côte d'Azur, INRAE, ISA, Sophia Antipolis, France</footnote>

<footnote>* Author of correspondence: Bin Wan, bwan@ncu.edu.cn</footnote>

<footnote>† These authors contributed equally to this work</footnote>
unparasitized and parasitized by parasitoids.

**Table S4** Results from Student's t-tests, Mann-Whitney U test, or Welch's t-test for comparing the percentages of the different hemocyte types in the hemolymph of *Diaphorina citri* that were unparasitized and parasitized by parasitoids.

**Table S5** Results from One-way ANOVA for comparing the mRNA expression levels of immune-related genes in immature stages of *Diaphorina citri* development.

**Table S6** Results from Mann-Whitney U test for comparing the mRNA expression levels of immune-related genes at 0h, 24h, 48h post-parasitism period both in parasitized and unparasitized *Diaphorina citri*.

**Table S7** Results from Mann-Whitney U test for comparing undefined melanized hemocytes from hemolymph of *Diaphorina citri* treated with ethanol (EtOH) or L-DOPA with ethanol (EtOH+L-DOPA).
<figure><img src="image_19.png" /><figcaption>**Fig. S1** PCR-amplification products of immune-related genes from *Diaphorina citri*. Picture of a 1% agarose gel electrophoresis showing the unique band obtained for each amplified gene sequence at the awaited molecular weight (Table S1). The amplicon lengths awaited for the genes from *β-Actin* to *Jak2* were 170bp, 276bp, 254bp, 349bp, 358bp, 205bp, 331bp, respectively. Molecular Marker in Bp.</figcaption></figure>
A

<figure><img src="image_20.png" /></figure>

B

<figure><img src="image_21.png" /></figure>

<figure><img src="image_22.png" /></figure>

**Fig. S2 Observable response of *Diaphorina citri* nymph to parasitism.** (A) Number of melanization spots on the abdomen of *D. citri* nymphs parasitized by *Tamarixia radiata* observed 24 h after parasitism. 20 nymphs were analyzed. (B) Developmental outcome of *D. citri* nymphs 16 days after parasitization by *Diaphorencyrtus aligarhensis* female wasps, showing that 21% of the parasitized psyllids remained arrested at the final nymphal stage, from 73% of the parasitized nymphs a wasp emerged, while from the remaining nymphs, parasitism failed or no parasitism occurred and an adult psyllid emerged. Development of 19 nymphs was followed during the course of 3 experiments.
<figure><img src="image_23.png" /><figcaption>**Fig. S3** *In vivo* phagocytosis of fluorescence latex beads. Fluorescent micrograph of prohemocytes (panel A1-3), plasmatocytes (panel B1-3), and granulocytes (panel C1-3) with phagocytosed fluorescence latex beads (yellow spots). The actin cytoskeleton is stained in green by phalloidin-GFP and the nucleus in blue by DAPI. Scale bar = 10 µm. (D) Bar plot showing the percentage of each hemocyte type containing latex beads; brown bar, hemocytes with latex beads; green bar, hemocytes without latex beads. For each experiment, 3 slides contained pooled hemolymph of 5 nymphs, in which over 300 cells/slide were analyzed. Data represent the mean ± SEM of 3 experiments.</figcaption></figure>
<figure><img src="image_24.png" /><figcaption>**Fig S4** *In vivo phagocytosis of fluorescent *Escherichia coli*-GFP.* 24 h after injection of *E. coli*-GFP into *Diaphorina citri* nymphs, adherent hemocytes were observed by fluorescence microscopy. The images show an example of prohemocytes (panel A), plasmatocytes (panel B) and granulocytes (panel C) that have taken up numerous *E. coli*-GFP. Phalloidin-labeled actin in red, DAPI-labeled nucleus in blue, and fluorescent *E. coli*-GFP in green. Picture 4 is an overlay of pictures 1 to 3 for each panel.</figcaption></figure>
<figure><img src="image_25.png" /></figure>

**Fig S5** Surface distribution of hemocytes after injection of fluorescent latex beads. 24 h after injection of fluorescent latex beads, *Diaphorina citri* nymphs were bled and adherent hemocytes were observed by fluorescence microscopy, and classified into 11 groups based on cell surface area from 10 to >210 µm². The number of cells with (brown bar) or without (green bar) beads was counted. For each experiment, 3 slides containing each the hemolymph pool of 5 nymphs, in which over 300 cells/slide were analyzed. Data represent the mean ± SEM of 3 experiments.
<figure><img src="image_26.png" /><figcaption>**Fig. S6 Surface distribution of hemocytes after parasitism.** Hemolymph of *Diaphorina citri* nymphs that were unparasitized, parasitized by *Tamarixia radiata*, parasitized by *Diaphorencyrtus aligarhensis* was obtained at different times after parasitism. Adherent hemocytes were classified based on cell surface area into 11 groups from 10 to >210 µm². For each experiment, the slide contained pooled hemolymph from 5 nymphs in which over 50 cells were analyzed. Data represent the mean ± SEM of 4 experiments. Significance was determined by a two-tailed unpaired Student's t-test or Mann-Whitney U test (*=P <</figcaption></figure>
0.05; **=P < 0.01).
<figure><img src="image_27.png" /><figcaption>**Fig. S7** Percentage of different hemocyte types in *Diaphorina citri* nymphs at different times after mechanical injury. For each experiment, the slide contained pooled hemolymph from 5 nymphs in which over 50 cells were analyzed. Data represent the mean ± SEM of 4 experiments. Significance was determined by a two-tailed unpaired Student's t-test (ns, not significant).</figcaption></figure>
<figure><img src="image_28.png" /></figure>

**Fig. S8 RT-qPCR of the six immune-related genes expression during *Diaphorina citri* development.** Gene expression was measured on eggs and different nymph instars of *D. citri* (1N, 1st instar; 2N, 2nd instar; 3N, 3rd instar; 4N, 4th instar; 5N, 5th instar). Relative expression level to the expression level of the *β-actin* gene used as an internal control. Data represent the mean ± SEM of 4 experiments. Significance was determined by one-way ANOVA with Tukey's multiple comparison test. Different letters on the bars indicate significant differences (*p*<0.05) between different samples.
<figure><img src="image_29.png" /><figcaption>**Fig. S9 Phenoloxidase activity in hemocytes of *Diaphorina citri* nymphs.** (A) Hemolymph were incubated with either 1mg/ml L-DOPA or 5mg/ml L-DOPA, or PBS as control. No melanin formation could be observed 0.5h, 1h, 2h after treatment. (B) Adherent hemocytes were incubated with ethanol alone (EtOH) or ethanol and L-DOPA (EtOH+L-DOPA) to reveal the PO activity. Almost all of the total hemocytes appeared colorless by bright field microscopy and showed a fluorescent nucleus when stained with DAPI. (C) Only a few small round cell-like vesicle appeared darker by bright field microscopy, but none showed a fluorescent nucleus when stained with DAPI. (D) No difference in the number of undefined melanized small round cell-like vesicle was observed between the two treatments. For each experiment, the slide contained pooled hemolymph from 5 nymphs in which over 120 cells were analyzed. Data represent mean ± SEM of 12 experiments. Significance was determined by a two-tailed unpaired Student's t-test (ns, not significant).</figcaption></figure>
<figure><img src="image_30.png" /><figcaption>**Fig. S10 Phenoloxidase activity in *Diaphorina citri* nymphs.** (A) The same *D. citri* 4th instar nymph was microphotographed before (upper panel) and after (lower panel) heating at 65°C to look for melanization spots, but no spots could be observed after heat treatment. (B) *D. citri* were immersed in centrifuge tubes filled with 70% ethanol (EtOH 70%) or PTU with ethanol (EtOH 70%+PTU) for 48h to activate or inhibit PO activity, no spots could be observed in the two treatments.</figcaption></figure>
<figure><img src="image_31.png" /><figcaption>Fig. S11 Phenoloxidase activity in hemocytes of parasitized *Diaphorina citri* nymphs.</figcaption></figure>

(A) Hemolymph of *D. citri* nymphs 48 hours after parasitism by *Tamarixia radiata* or (B) after parasitism by *Diaphorencyrtus aligarhensis* was incubated with either 1mg/ml L-DOPA or 5mg/ml L-DOPA, or PBS as control. No melanin formation could be observed at 0.5h, 1h, and 2h after treatment. Adherent hemocytes after parasitism by *T. radiata* (C) or after parasitism by *D. aligarhensis* (D) were incubated with ethanol or L-DOPA (EtOH+L-DOPA) to reveal the PO activity. Only a few small round cell-like particles appeared darker by bright field microscopy, but none showed a fluorescent nucleus when stained with DAPI. (E) No difference was observed in the number of undefined melanized small round cell-like vesicle between the two treatments. For each hemolymph experiment, the drop contained pooled hemolymph from 2-5 nymphs. For each hemocyte experiment, the slide contained pooled hemolymph from 1-2 nymphs in which over 40 cells were analyzed. Data represent mean ± SEM of 5 experiments.
<table><caption>**Table S1** Primers used for qPCR in the study.</caption><thead><tr><th>GenBank ID</th><th>gene name</th><th>Forward primer</th><th>Reverse primer</th></tr></thead><tbody><tr><td>XM_026829329.1</td><td>Down syndrome cell adhesion molecule-like protein Dscam2</td><td>AACTCCTCCGCCGTGTCCAT</td><td>CCTTCCAGCGTAAGCGT AGTGA</td></tr><tr><td>XM_017442724.2</td><td>Dual oxidase</td><td>TCTGTCCGTCTCACAA GCAC</td><td>TTGCGACCCTTGTTGAC CTC</td></tr><tr><td>XM_008470434.2</td><td>Spaetzle 3-like</td><td>TTCATCCGGGGTCCTCC ATGT</td><td>AGTCCTCCGCTTCCCAG TCT</td></tr><tr><td>XM_008472659.3</td><td>Phenoloxidase 1-like</td><td>AACCTAGTTCGGCTAC TCCAA</td><td>CTCACATACACCTCGTC CATAC</td></tr><tr><td>XM_026820054.1</td><td>Tyrosine-protein kinase Jak2</td><td>ATGGATACGCTGCTGA GTCG</td><td>AGAGGTCCTCAATGCTG CAC</td></tr><tr><td>XM_008477381.3</td><td>Serine/threonine-protein kinase TBK1</td><td>ACGCCTATGATCAGCTG CAA</td><td>TCAATGTCAGAGGGGAG GGT</td></tr><tr><td>DQ675553.1</td><td>β-Actin</td><td>CCCTGGACTTTGAACA GGAA</td><td>CTCGTGGATACCGCAAG ATT</td></tr></tbody></table>

<table><caption>**Table S2.** Results from Student's t-tests for comparing hemocyte counts in the hemolymph of *Diaphorina citri* that were unparasitized or parasitized by parasitoids (*Diaphorencyrtus aligarhensis* or *Tamarixia radiata*).</caption><thead><tr><th rowspan="2">Source of variation</th><th rowspan="2">Fig.</th><th colspan="3">Statistical analysis</th></tr><tr><th>df</th><th>Test statistic</th><th><i>p</i> value</th></tr></thead><tbody><tr><td colspan="5"><strong>Control vs T. radiata</strong></td></tr><tr><td>At 0 h post-parasitism period</td><td>4</td><td>6</td><td><i>t</i> = 0.554</td><td>0.600</td></tr><tr><td>At 24 h post-parasitism period</td><td></td><td>6</td><td><i>t</i> = 2.754</td><td><strong>0.033</strong></td></tr><tr><td>At 48 h post-parasitism period</td><td></td><td>6</td><td><i>t</i> = 2.747</td><td><strong>0.033</strong></td></tr><tr><td colspan="5"><strong>Control vs D. aligarhensis</strong></td></tr><tr><td>At 0 h post-parasitism period</td><td>4</td><td>6</td><td><i>t</i> = 0.091</td><td>0.930</td></tr><tr><td>At 24 h post-parasitism period</td><td></td><td>6</td><td><i>t</i> = 0.077</td><td>0.941</td></tr><tr><td>At 48 h post-parasitism period</td><td></td><td>6</td><td><i>t</i> = 0.049</td><td>0.963</td></tr></tbody></table>
<table><caption>**Table S3.** Results from Student's t-tests, Mann-Whitney U test, or Welch's t-test for comparing the percentages of the hemocyte with different areas in the hemolymph of *Diaphorina citri* that were unparasitized or parasitized by parasitoids (*Diaphorencyrtus aligarhensis* or *Tamarixia radiata*).</caption><thead><tr><th rowspan="2">Source of variation</th><th rowspan="2"></th><th rowspan="2">Fig.</th><th colspan="3">Statistical analysis</th></tr><tr><th>df</th><th>Test statistic<br>(t, Z)</th><th>p value</th></tr></thead><tbody><tr><td colspan="6"><strong>Control vs T. radiata</strong></td></tr><tr><td rowspan="12">At 0 h post-parasitism period</td><td>Cell area:10-30µm²</td><td>S6</td><td>6</td><td>t = 2.090</td><td>0.082</td></tr><tr><td>30-50µm²</td><td></td><td>6</td><td>t = 2.206</td><td>0.070</td></tr><tr><td>50-70µm²</td><td></td><td>6</td><td>t = 0.181</td><td>0.862</td></tr><tr><td>70-90µm²</td><td></td><td>6</td><td>t = -0.467</td><td>0.657</td></tr><tr><td>90-110µm²</td><td></td><td>6</td><td>t = -0.460</td><td>0.662</td></tr><tr><td>110-130µm²</td><td></td><td>6</td><td>t = -1.169</td><td>0.287</td></tr><tr><td>130-150µm²</td><td></td><td>6</td><td>t = -3.779</td><td><strong>0.009</strong></td></tr><tr><td>150-170µm²</td><td></td><td>6</td><td>t = -4.090</td><td><strong>0.006</strong></td></tr><tr><td>170-190µm²</td><td></td><td>-</td><td>Z = -2.323</td><td><strong>0.029</strong></td></tr><tr><td>190-210µm²</td><td></td><td>6</td><td>t = -0.990</td><td>0.360</td></tr><tr><td>&gt; 210µm²</td><td></td><td>3.928</td><td>t = -1.133</td><td>0.322</td></tr><tr><td rowspan="12">At 24 h post-parasitism period</td><td>Cell area:10-30µm²</td><td></td><td>6</td><td>t = 3.311</td><td><strong>0.016</strong></td></tr><tr><td>30-50µm²</td><td></td><td>6</td><td>t = 3.002</td><td><strong>0.024</strong></td></tr><tr><td>50-70µm²</td><td></td><td>6</td><td>t = 1.892</td><td>0.107</td></tr><tr><td>70-90µm²</td><td></td><td>6</td><td>t = 3.424</td><td><strong>0.021</strong></td></tr><tr><td>90-110µm²</td><td></td><td>6</td><td>t = -1.035</td><td>0.340</td></tr><tr><td>110-130µm²</td><td></td><td>6</td><td>t = -2.350</td><td>0.057</td></tr><tr><td>130-150µm²</td><td></td><td>6</td><td>t = -3.771</td><td><strong>0.009</strong></td></tr><tr><td>150-170µm²</td><td></td><td>6</td><td>t = -3.583</td><td><strong>0.012</strong></td></tr><tr><td>170-190µm²</td><td></td><td>3.655</td><td>t = -1.579</td><td>0.196</td></tr><tr><td>190-210µm²</td><td></td><td>-</td><td>Z = -0.887</td><td>0.486</td></tr><tr><td>&gt; 210µm²</td><td></td><td>3.015</td><td>t = -1.320</td><td>0.278</td></tr></tbody></table>
<table><tbody><tr><td rowspan="12">At 48 h post-parasitism period</td><td>Cell area:10-30µm²</td><td>-</td><td>Z = -1.443</td><td>0.200</td></tr><tr><td>30-50µm²</td><td>6</td><td>t = 2.307</td><td>0.061</td></tr><tr><td>50-70µm²</td><td>6</td><td>t = 3.258</td><td><strong>0.017</strong></td></tr><tr><td>70-90µm²</td><td>6</td><td>t = 4.017</td><td><strong>0.007</strong></td></tr><tr><td>90-110µm²</td><td>3.033</td><td>t = 2.904</td><td>0.061</td></tr><tr><td>110-130µm²</td><td>6</td><td>t = -2.250</td><td>0.065</td></tr><tr><td>130-150µm²</td><td>6</td><td>t = -2.607</td><td><strong>0.040</strong></td></tr><tr><td>150-170µm²</td><td>6</td><td>t = -2.552</td><td><strong>0.043</strong></td></tr><tr><td>170-190µm²</td><td>3.609</td><td>t = -2.164</td><td>0.104</td></tr><tr><td>190-210µm²</td><td>6</td><td>t = -1.691</td><td>0.142</td></tr><tr><td>&gt; 210µm²</td><td>3.129</td><td>t = -2.721</td><td>0.069</td></tr></tbody></table>

Control vs D. aligarhensis

<table><tbody><tr><td rowspan="12">At 0 h post-parasitism period</td><td>Cell area:10-30µm²</td><td>S6</td><td>4.181</td><td>t = -1.700</td><td>0.161</td></tr><tr><td>30-50µm²</td><td>-</td><td>Z = -0.866</td><td>0.486</td></tr><tr><td>50-70µm²</td><td>6</td><td>t = -0.857</td><td>0.424</td></tr><tr><td>70-90µm²</td><td>-</td><td>Z = -1.156</td><td>0.343</td></tr><tr><td>90-110µm²</td><td>6</td><td>t = 2.938</td><td><strong>0.026</strong></td></tr><tr><td>110-130µm²</td><td>6</td><td>t = 2.089</td><td>0.082</td></tr><tr><td>130-150µm²</td><td>-</td><td>Z = -1.155</td><td>0.343</td></tr><tr><td>150-170µm²</td><td>-</td><td>Z = -1.452</td><td>0.200</td></tr><tr><td>170-190µm²</td><td>-</td><td>Z = -0.887</td><td>0.486</td></tr><tr><td>190-210µm²</td><td>-</td><td>Z = -1.548</td><td>0.200</td></tr><tr><td>&gt; 210µm²</td><td>6</td><td>t = 2.004</td><td>0.092</td></tr></tbody></table>

<table><tbody><tr><td rowspan="4">At 24 h post-parasitism period</td><td>Cell area:10-30µm²</td><td>-</td><td>Z = -2.309</td><td><strong>0.029</strong></td></tr><tr><td>30-50µm²</td><td>6</td><td>t = -3.491</td><td><strong>0.013</strong></td></tr><tr><td>50-70µm²</td><td>4.020</td><td>t = 1.733</td><td>0.158</td></tr><tr><td>70-90µm²</td><td>6</td><td>t = 4.959</td><td><strong>0.003</strong></td></tr><tr><td>90-110µm²</td><td>6</td><td>t = 2.597</td><td><strong>0.041</strong></td></tr></tbody></table>
<table><tbody><tr><td>110-130µm²</td><td>3.579</td><td>t = 3.602</td><td><strong>0.027</strong></td></tr><tr><td>130-150µm²</td><td>6</td><td>t = 3.388</td><td><strong>0.015</strong></td></tr><tr><td>150-170µm²</td><td>-</td><td>Z = -1.452</td><td>0.200</td></tr><tr><td>170-190µm²</td><td>6</td><td>t = 0.820</td><td>0.444</td></tr><tr><td>190-210µm²</td><td>-</td><td>Z = -1.512</td><td>0.343</td></tr><tr><td>&gt; 210µm²</td><td>-</td><td>Z = -1.183</td><td>0.343</td></tr><tr><td>At 48 h post-parasitism period</td><td>Cell area:10-30µm²</td><td>-</td><td>Z = -2.309</td><td><strong>0.029</strong></td></tr><tr><td>30-50µm²</td><td>6</td><td>t = 0.266</td><td>0.799</td></tr><tr><td>50-70µm²</td><td>6</td><td>t = 2.233</td><td>0.067</td></tr><tr><td>70-90µm²</td><td>3.024</td><td>t = 1.343</td><td>0.271</td></tr><tr><td>90-110µm²</td><td>3.018</td><td>t = 1.312</td><td>0.281</td></tr><tr><td>110-130µm²</td><td>6</td><td>t = -0.555</td><td>0.599</td></tr><tr><td>130-150µm²</td><td>6</td><td>t = -0.014</td><td>0.989</td></tr><tr><td>150-170µm²</td><td>6</td><td>t = 0.102</td><td>0.922</td></tr><tr><td>170-190µm²</td><td>6</td><td>t = 0.571</td><td>0.589</td></tr><tr><td>190-210µm²</td><td>-</td><td>Z = -1.076</td><td>0.343</td></tr><tr><td>&gt; 210µm²</td><td>6</td><td>t = -0.189</td><td>0.856</td></tr></tbody></table>
<table><caption>**Table S4.** Results from Student's t-tests, Mann-Whitney U test, or Welch's t-test for comparing the percentages of the different hemocyte types in the hemolymph of *Diaphorina citri* that were unparasitized or parasitized by parasitoids (*Diaphorencyrtus aligarhensis* or *Tamarixia radiata*).</caption><thead><tr><th rowspan="2">Source of variation</th><th rowspan="2"></th><th rowspan="2">Fig.</th><th colspan="3">Statistical analysis</th></tr><tr><th>df</th><th>Test statistic (t, Z)</th><th>p value</th></tr></thead><tbody><tr><td colspan="6"><strong>Control vs T. radiata</strong></td></tr><tr><td rowspan="3">Prohemocytes</td><td>At 0 h post-parasitism period</td><td rowspan="3">5</td><td>6</td><td>t = 2.640</td><td><strong>0.039</strong></td></tr><tr><td>At 24 h post-parasitism period</td><td>6</td><td>t = 0.377</td><td>0.719</td></tr><tr><td>At 48 h post-parasitism period</td><td>6</td><td>t = 0.638</td><td>0.547</td></tr><tr><td rowspan="3">Plasmatocytes</td><td>At 0 h post-parasitism period</td><td rowspan="3"></td><td>6</td><td>t = 0.753</td><td>0.480</td></tr><tr><td>At 24 h post-parasitism period</td><td>6</td><td>t = 2.064</td><td>0.085</td></tr><tr><td>At 48 h post-parasitism period</td><td>6</td><td>t = 1.649</td><td>0.150</td></tr><tr><td rowspan="3">Granulocytes</td><td>At 0 h post-parasitism period</td><td rowspan="3"></td><td>3.132</td><td>t = 2.078</td><td>0.126</td></tr><tr><td>At 24 h post-parasitism period</td><td>-</td><td>Z = -2.309</td><td><strong>0.029</strong></td></tr><tr><td>At 48 h post-parasitism period</td><td>6</td><td>t = 2.309</td><td>0.060</td></tr><tr><td colspan="6"><strong>Control vs D. aligarhensis</strong></td></tr><tr><td rowspan="3">Prohemocytes</td><td>At 0 h post-parasitism period</td><td rowspan="3">5</td><td>6</td><td>t = 2.708</td><td><strong>0.035</strong></td></tr><tr><td>At 24 h post-parasitism period</td><td>6</td><td>t = 3.091</td><td><strong>0.021</strong></td></tr><tr><td>At 48 h post-parasitism period</td><td>6</td><td>t = 2.065</td><td>0.085</td></tr><tr><td rowspan="3">Plasmatocytes</td><td>At 0 h post-parasitism period</td><td rowspan="3"></td><td>6</td><td>t = 1.774</td><td>0.126</td></tr><tr><td>At 24 h post-parasitism period</td><td>6</td><td>t = 1.513</td><td>0.181</td></tr><tr><td>At 48 h post-parasitism period</td><td>6</td><td>t = 1.183</td><td>0.281</td></tr><tr><td rowspan="3">Granulocytes</td><td>At 0 h post-parasitism period</td><td rowspan="3"></td><td>3.152</td><td>t = 0.567</td><td>0.609</td></tr><tr><td>At 24 h post-parasitism period</td><td>6</td><td>t = 1.675</td><td>0.145</td></tr><tr><td>At 48 h post-parasitism period</td><td>6</td><td>t = 1.553</td><td>0.171</td></tr><tr><td colspan="6"><strong>Control vs Mechanically injured</strong></td></tr><tr><td rowspan="3">Prohemocytes</td><td>At 0 h post-parasitism period</td><td rowspan="3">S7</td><td>8</td><td>t = 0.628</td><td>0.548</td></tr><tr><td>At 24 h post-parasitism period</td><td>8</td><td>t = 0.884</td><td>0.403</td></tr><tr><td>At 48 h post-parasitism period</td><td>8</td><td>t = 2.298</td><td>0.051</td></tr><tr><td rowspan="2">Plasmatocytes</td><td>At 0 h post-parasitism period</td><td rowspan="2"></td><td>8</td><td>t = 1.221</td><td>0.257</td></tr><tr><td>At 24 h post-parasitism period</td><td>8</td><td>t = 1.256</td><td>0.245</td></tr></tbody></table>
<table><tr><td></td><td>At 48 h post-parasitism period</td><td>8</td><td>t = 0.315</td><td>0.761</td></tr><tr><td rowspan="3">Granulocytes</td><td>At 0 h post-parasitism period</td><td>8</td><td>t = 1.485</td><td>0.176</td></tr><tr><td>At 24 h post-parasitism period</td><td>8</td><td>t = 2.121</td><td>0.067</td></tr><tr><td>At 48 h post-parasitism period</td><td>8</td><td>t = 2.221</td><td>0.057</td></tr></table>
<table><caption>**Table S5.** Results from One-way ANOVA for comparing the mRNA expression levels of immune-related genes in immature stages of *Diaphorina citri* development.</caption><thead><tr><th rowspan="2">Source of variation</th><th rowspan="2">Fig.</th><th colspan="3">Statistical analysis</th></tr><tr><th>df</th><th>F</th><th>p value</th></tr></thead><tbody><tr><td colspan="5"><strong>Comparisons at each development stage</strong></td></tr><tr><td>Dscam2</td><td>S8</td><td>5</td><td>31.55</td><td><strong>&lt;0.0001</strong></td></tr><tr><td>Spaetzle 3-like</td><td></td><td>5</td><td>6.445</td><td><strong>0.0018</strong></td></tr><tr><td>TBK1</td><td></td><td>5</td><td>10.77</td><td><strong>0.0002</strong></td></tr><tr><td>Jak2</td><td></td><td>5</td><td>3.425</td><td><strong>0.0271</strong></td></tr><tr><td>Phenoloxidase 1-like</td><td></td><td>5</td><td>1.563</td><td>0.2300</td></tr><tr><td>Dual oxidase</td><td></td><td>5</td><td>3.028</td><td>0.0795</td></tr></tbody></table>
<table><caption>**Table S6.** Results from Mann-Whitney U test for comparing the mRNA expression levels of immune-related genes at 0h, 24h, 48h post-parasitism period both in parasitized and unparasitized *Diaphorina citri*.</caption><thead><tr><th rowspan="2">Source of variation</th><th rowspan="2"></th><th rowspan="2">Fig.</th><th colspan="2">Statistical analysis</th></tr><tr><th>Test statistic (Z)</th><th>p value</th></tr></thead><tbody><tr><td colspan="5"><strong>Control vs Tamarixia radiata</strong></td></tr><tr><td rowspan="3">Dscam2</td><td>At 0 h post-parasitism period</td><td rowspan="3">6</td><td>-2.558</td><td><strong>0.0095</strong></td></tr><tr><td>At 24 h post-parasitism period</td><td>0</td><td>&gt;0.9999</td></tr><tr><td>At 48 h post-parasitism period</td><td>-2.882</td><td><strong>0.002</strong></td></tr><tr><td rowspan="3">Spaetzle 3-like</td><td>At 0 h post-parasitism period</td><td rowspan="3"></td><td>-1.279</td><td>0.257</td></tr><tr><td>At 24 h post-parasitism period</td><td>-2.882</td><td><strong>0.002</strong></td></tr><tr><td>At 48 h post-parasitism period</td><td>-2.558</td><td><strong>0.0095</strong></td></tr><tr><td rowspan="3">TBK1</td><td>At 0 h post-parasitism period</td><td rowspan="3"></td><td>-2.324</td><td><strong>0.024</strong></td></tr><tr><td>At 24 h post-parasitism period</td><td>-2.739</td><td><strong>0.004</strong></td></tr><tr><td>At 48 h post-parasitism period</td><td>-2.739</td><td><strong>0.004</strong></td></tr><tr><td rowspan="3">Jak2</td><td>At 0 h post-parasitism period</td><td rowspan="3"></td><td>-2.558</td><td><strong>0.0095</strong></td></tr><tr><td>At 24 h post-parasitism period</td><td>-2.739</td><td><strong>0.004</strong></td></tr><tr><td>At 48 h post-parasitism period</td><td>-2.739</td><td><strong>0.004</strong></td></tr><tr><td rowspan="3">Phenoloxidase 1-like</td><td>At 0 h post-parasitism period</td><td rowspan="3"></td><td>-0.548</td><td>0.662</td></tr><tr><td>At 24 h post-parasitism period</td><td>-2.558</td><td><strong>0.0095</strong></td></tr><tr><td>At 48 h post-parasitism period</td><td>-2.739</td><td><strong>0.004</strong></td></tr><tr><td rowspan="3">Dual oxidase</td><td>At 0 h post-parasitism period</td><td rowspan="3"></td><td>-2.324</td><td><strong>0.024</strong></td></tr><tr><td>At 24 h post-parasitism period</td><td>-1.279</td><td>0.257</td></tr><tr><td>At 48 h post-parasitism period</td><td>-2.739</td><td><strong>0.004</strong></td></tr><tr><td colspan="5"><strong>Control vs Diaphorencyrtus aligarhensis</strong></td></tr><tr><td rowspan="3">Dscam2</td><td>At 0 h post-parasitism period</td><td rowspan="3">7</td><td>-2.739</td><td><strong>0.004</strong></td></tr><tr><td>At 24 h post-parasitism period</td><td>-2.739</td><td><strong>0.004</strong></td></tr><tr><td>At 48 h post-parasitism period</td><td>-2.882</td><td><strong>0.002</strong></td></tr><tr><td rowspan="3">Spaetzle 3-like</td><td>At 0 h post-parasitism period</td><td rowspan="3"></td><td>-0.961</td><td>0.394</td></tr><tr><td>At 24 h post-parasitism period</td><td>-2.739</td><td><strong>0.004</strong></td></tr><tr><td>At 48 h post-parasitism period</td><td>-2.739</td><td><strong>0.004</strong></td></tr><tr><td rowspan="2">TBK1</td><td>At 0 h post-parasitism period</td><td rowspan="2"></td><td>-2.882</td><td><strong>0.002</strong></td></tr><tr><td>At 24 h post-parasitism period</td><td>-0.961</td><td>0.394</td></tr></tbody></table>
<table><caption>**Table S7.** Results from Mann-Whitney U test for comparing undefined small round cell-like vesicle from the hemolymph of *Diaphorina citri* treated with ethanol (EtOH) or L-DOPA with ethanol (EtOH+L-DOPA).</caption><tbody><tr><td></td><td>At 48 h post-parasitism period</td><td>-0.320</td><td>0.818</td></tr><tr><td rowspan="3">Jak2</td><td>At 0 h post-parasitism period</td><td>-2.556</td><td><strong>0.009</strong></td></tr><tr><td>At 24 h post-parasitism period</td><td>-0.961</td><td>0.394</td></tr><tr><td>At 48 h post-parasitism period</td><td>-2.739</td><td><strong>0.004</strong></td></tr><tr><td rowspan="3">Phenoloxidase 1-like</td><td>At 0 h post-parasitism period</td><td>0</td><td>&gt;0.9999</td></tr><tr><td>At 24 h post-parasitism period</td><td>-2.739</td><td><strong>0.004</strong></td></tr><tr><td>At 48 h post-parasitism period</td><td>-1.643</td><td>0.126</td></tr><tr><td rowspan="3">Dual oxidase</td><td>At 0 h post-parasitism period</td><td>0</td><td>&gt;0.9999</td></tr><tr><td>At 24 h post-parasitism period</td><td>-2.882</td><td><strong>0.002</strong></td></tr><tr><td>At 48 h post-parasitism period</td><td>-2.739</td><td><strong>0.004</strong></td></tr></tbody></table>

<table><thead><tr><th rowspan="2">Source of variation</th><th rowspan="2">Fig.</th><th colspan="2">Statistical analysis</th></tr><tr><th>Test statistic (Z)</th><th>p value</th></tr></thead><tbody><tr><td>EtOH vs EtOH+L-DOPA</td><td>S9</td><td>-0.866</td><td>0.410</td></tr></tbody></table>