The Chorion Proteome of Diaphorina citri, the Vector of Huanglongbing Disease in Citrus

Simple Summary Huanglongbing is currently the most devastating disease of citrus worldwide. It is believed that controlling the vector, Diaphorina citri, is the most important and effective method of disease management. To achieve this goal, we have employed RNA interference (RNAi), a promising technique for controlling D. citri. Our present strategy is to target D. citri egg hatching. By reducing the rate of egg hatching, we will reduce the vector population and subsequently reduce the spread of disease. In order to interfere with the egg hatching mechanism, we studied the chorion proteome to identify potential gene candidates for RNAi. Abstract Nowadays, the Asian citrus psyllid, Diaphorina citri (Kuwayama) (Hemiptera: Liviidae) is considered the most devastating pest of citrus because it transmits “Candidatus Liberibacter asiaticus”, the putative causal agent of huanglongbing (HLB) or citrus greening. Controlling the vector is the main strategy used to mitigate HLB. Targeting D. citri at the very early stages of its development may offer an effective control strategy. Identifying chorion proteins will contribute to a better understanding of embryo development and egg hatching and thus could lead to valuable targets to better control psyllid populations. Herein, we analyze the chorion proteins of D. citri. Mass spectrometry-based bottom-up/shotgun proteomics and databases were queried to achieve protein identification. Fifty-one proteins were identified in D. citri chorion. The D. citri chorion proteins were divided into eight categories according to their biological or molecular function: i—enzymes (25%); ii—binding proteins (10%); iii—structural proteins (8%); iv—homeostasis-related proteins, mostly vitellogenins (8%); v—proteins related to gene expression (6%); vi—immune system proteins (6%); vii—other proteins (16%); and viii—uncharacterized proteins (21%). The composition of the chorion proteome suggested that the hatching rate could be reduced by silencing chorion-related genes. The proteomic analysis of D. citri chorion tissue allowed us to identify its proteins, providing promising new targets for D. citri control through RNA interference technology.


Introduction
The Asian citrus psyllid, Diaphorina citri (Kuwayama) (Hemiptera: Liviidae), is a sapsucking hemipteran that serves as the main vector for "Candidatus Liberibacter asiaticus", the putative causal agent of citrus huanglongbing (HLB) [1,2]. Once inoculated into the host tree, the bacterium multiplies and initiates a cascade of host plant responses that leads to callose deposition in leaves [3,4], starch accumulation, asymmetrical leaf mottling [5], and root loss [6]. HLB is rapidly transmitted by D. citri as it feeds on the phloem sap of Rutaceae species, which include Citrus and its relatives [1]. D. citri is the insect vector of "Ca. L. asiaticus" in Asia, Brazil, and the USA [7,8]. Severe economic losses result from the infestations with D. citri because it colonizes the new shoots, transmitting "Ca. L. asiaticus" Insects 2021, 12, 959 2 of 14 to trees and causing damage to foliage. Trees infected with "Ca. L. asiaticus" are short-lived, have reduced yields, and produce smaller, lopsided fruit with poor quality juice [9][10][11][12].
Mitigation of HLB has been achieved primarily through the chemical control of D. citri, but it has failed to contain the spread of the disease in the US. Chemical pesticide use for D. citri control has led to reports of insecticide resistance [13,14] and is non-specific, and offtarget effects may harm beneficial insects such as honeybees. Furthermore, most insecticides have negative environmental impacts, and applications are difficult to coordinate among growers [15]. Numerous non-chemical control strategies have been attempted with some degree of success, including kaolin clay treatments [16], heat treatments [17], enhanced nutritional programs [17][18][19], and biological control [20] (see reviews [21,22]), but nearly 100% of Florida citrus groves are currently affected by HLB disease.
A relatively modern biotechnological approach known as RNA interference (RNAi) has shown promising results for "silencing" genes in D. citri to study functional genomics. The sequencing of the D. citri genome and the resulting transcriptome has allowed the discovery of many diverse predicted proteins [23][24][25]. However, the new challenge is to identify the functions and roles these genes play in the lifecycle of D. citri and find those that can be exploited against it. RNAi techniques have been utilized extensively to study potential control targets. Double-stranded RNA (dsRNA) can be applied to adult psyllids topically [26], and through an artificial diet [27][28][29], while nymphs can be treated easily through topical feeding [30] or soaking in dsRNA solutions [31]. RNAi technologies were recently reviewed [32]. We targeted genes implicated in the development and metamorphosis of D. citri (abnormal wing disc, muscle protein 20) [31,33,34], gender ratios (boule and transformer-2 homologues) [27,28], metabolism (sucrose hydrolase) [35], and insecticide resistance (cytochrome P450, acetylcholinesterases, glutathione S-transferase) [26,29,30,36]. So far, RNAi treatments to D. citri have been restricted to the laboratory.
The order Hemiptera, which includes phloem-feeding aphids, psyllids, whiteflies and leafhoppers, pass through several life stages, from egg to nymphs to adult, without a pupal stage. D. citri females lay 400-600 eggs in the soft new flush of citrus or other host plants, which incubate at ambient temperatures for about 4 d [37]. Nymphs pass through five instar stages before molting into the adult form 12-13 d later [37]. During incubation, the chorion (eggshell) protects insect embryos from the environment [38]. During the later stages of oogenesis, the chorion matrix is secreted between the oocyte and overlaying somatic follicle cells [39]. Its multilayered structure performs a variety of specialized functions such as providing substrate attachment, resistance to acid or alkalis, preventing water loss, and allowing gas exchange throughout development [39,40]. The differences in ecological environment and, specifically, oviposition substrates have determined through evolution the chorion morphology, organization, and composition among different species [41]. Chorion proteins are the components that play a pivotal role in chorion assembling and stabilization and ensure the success of embryo development after oviposition [42].
To our knowledge, the very early developmental stages, including egg hatching and embryo development, have not been fully exploited to control D. citri, with only one report of embryo suppression using CRISPR gene knock-out [43,44]. To target egg production by RNAi, information about the proteins that function in chorion formation would be needed. Therefore, we used proteomic approaches to aid in the identification of D. citri chorion proteins to provide new targets to control psyllids from their earliest stages. To achieve this goal, we employed mass spectrometry-based bottom-up/shotgun proteomics in conjunction with the Uniprot database to identify the D. citri chorion proteins.

Insect Colonies
The laboratory colony of D. citri was reared continuously on healthy Citrus macrophylla seedlings 3-6 months old. The seedlings and D. citri were maintained in a controlledtemperature growth room with a 16:8 h L:D photoperiod, 27 ± 2 • C, and 60 ± 5% relative

Chorion Collection and Sample Preparation
Egg collection was carried out in an artificial medium created specifically for egg hatching from D. citri in our laboratory [45] ( Figure 1A). Briefly, the medium was comprised of 25% of sucrose, 1% yeast, sterilized water (10 mL), and 0.56 g of unflavored gelatin. The leaves of Citrus macrophylla plants containing psyllid eggs were collected and washed for 5 min in 0.03% bleach (1% of a commercial product), followed by three rinses in sterile water. Afterwards, the eggs were carefully removed from the leaves using a modified dissecting needle (bent slightly at the tip), immediately placed on the artificial medium, and kept at ambient temperature (25 • C). Following the hatching of the first instar D. citri nymphs, approximately 420 choria were recovered. The choria were homogenized with a pestle in a solution of 0.1 M of ammonium acetate (NH 4 Ac) and were briefly centrifuged to discard debris. The sample was transferred to a new microcentrifuge tube, and the determination of protein concentration was performed using the Bicinchoninic acid method (Pierce TM BCA Protein Assay Kit, ThermoFisher Scientific, Waltham, MA, USA). The sample was stored at −80 • C until analysis.

Insect Colonies
The laboratory colony of D. citri was reared continuously on healthy Citrus macrophylla seedlings 3-6 months old. The seedlings and D. citri were maintained in a controlled-temperature growth room with a 16:8 h L:D photoperiod, 27 ± 2 °C, and 60 ± 5% relative humidity. This facility is located at the Citrus Research and Education Center in Lake Alfred (28.092783°N, 81.72307°E), Florida.

Chorion Collection and Sample Preparation
Egg collection was carried out in an artificial medium created specifically for egg hatching from D. citri in our laboratory [45] ( Figure 1A). Briefly, the medium was comprised of 25% of sucrose, 1% yeast, sterilized water (10 mL), and 0.56 g of unflavored gelatin. The leaves of Citrus macrophylla plants containing psyllid eggs were collected and washed for 5 min in 0.03% bleach (1% of a commercial product), followed by three rinses in sterile water. Afterwards, the eggs were carefully removed from the leaves using a modified dissecting needle (bent slightly at the tip), immediately placed on the artificial medium, and kept at ambient temperature (25 °C). Following the hatching of the first instar D. citri nymphs, approximately 420 choria were recovered. The choria were homogenized with a pestle in a solution of 0.1 M of ammonium acetate (NH4Ac) and were briefly centrifuged to discard debris. The sample was transferred to a new microcentrifuge tube, and the determination of protein concentration was performed using the Bicinchoninic acid method (Pierce TM BCA Protein Assay Kit, ThermoFisher Scientific, Waltham, MA, USA). The sample was stored at −80 °C until analysis.

Reduction/Alkylation and In-Solution Digestion of Chorion Proteins
The chorion sample (

Reduction/Alkylation and In-Solution Digestion of Chorion Proteins
The chorion sample (25 µL initial volume) in 0.1 M of NH 4 Ac was reduced with 5 µL

LC-MS/MS Mass Spectrometry Analysis
LC-MS/MS was carried out at the Interdisciplinary Center for Biotechnological Research, University of Florida, Gainesville, FL. The digested sample was resuspended in 0.1% formic acid, and mass spectrometry was performed on an EASY-nLC TM 1200 ultrahigh-performance liquid chromatography system (Thermo Fisher Scientific, Waltham, MA, USA) connected to an Orbitrap Fusion TM Tribrid TM instrument equipped with a nanoelectrospray source (Thermo Fisher Scientific, Waltham, MA, USA). The digested sample was loaded into a C18 trapping column (Acclaim TM PepMap TM 100, 75 µm inner diameter × 2 cm length, 3 µm particle size, and 100 Å pore size) and eluted with a C18 analytical column (Acclaim TM PepMap TM 100, 75 µm inter diameter × 15 cm length, 2 µm particle size, and 100 Å pore size) at a flow rate of 250 nL/min. The separation was using solvent A (0.1% formic acid in water) and solvent B (0.1% formic acid and 80% acetonitrile) as the mobile phases, increasing the gradient as follows: 2-35% of solvent B over 0-140 min; 35-80% of solvent B over 40-45 min, 80-98% of solvent B over 45-46 min, and kept at 98% of solvent B until 60 min [47]. The full MS1 scan (m/z 350-2000) was performed on the Orbitrap Fusion with a resolution of 120,000 at m/z 200 [48]. The automatic gain control (AGC) target was 2 × 10 5 , with 50 ms as the maximum injection time. Monoisotopic precursor selection (MIPS) was enforced to filter for peptides. Peptides bearing + 2-6 charges were selected with an intensity threshold of 1 × 10 4 . Dynamic exclusion of 15 s was used to prevent resampling the high abundance peptides. The top speed method was used for data-dependent acquisition within a cycle of 3 s. The MS/MS was carried out in the ion trap, with a quadrupole isolation window of 1.3 Da. Fragmentation of the selected peptides by collision-induced dissociation (CID) was done at 35% of normalized collision energy. MS 2 spectra were detected in the linear ion trap with the AGC target as 1e4 and the maximum injection time as 35 ms.

Database Searches
MS/MS data were analyzed using Mascot version 2.7.0.1 (Matrix Science, London, UK) by searching against the UnitProt-Diaphorina_citri_20200316 database (unknown version, 22,073 entries) (https://www.uniprot.org/proteomes/UP000079169/ accessed on 20 September 2021), assuming digestion with trypsin. Fragment ion mass tolerance was set at 1.00 Da and parent ion tolerance at 10 ppm 0 +18 of pyrrolysine and carbamidomethyl of cysteine, specified in Mascot as fixed modifications [46,47]. Gln->pyro-Gln of the Nterminus, deamidate of asparagine and glutamine, and oxidation of methionine were specified in Mascot as variable modifications. Peptide identifications were accepted if they could be established at greater than 95.0% probability by the Scaffold Local FDR algorithm. Protein identifications were accepted if they could be established at greater than 99.0% probability and contained at least two identified peptides. Protein probabilities were assigned by the Protein Prophet algorithm [49]. Further, MS/MS-based peptide and protein identification was validated using Scaffold version 4.10.0 (Proteome Software Inc., Portland, OR, USA).

Results and Discussion
The chorion covers the whole surface area of the insect eggs to impair water loss, allow gas exchange during development, and protect the eggs from the maternal body [50]. The chorion is a highly specialized, multilayered barrier that allows for the fertilization of the oocyte and adjusts its permeability to allow optimal conditions for embryo development. These conditions vary dramatically depending on the insect species and its ecology, i.e., whether eggs are laid on plant leaves, in soil, above or below ground, or in water. Chorion composition, structure, and physical properties determine its permeability, restricting the egg's development to specific environments [51]. Chorion is composed of proteinaceous and organic molecules [52] that can be identified, in part, through proteomics analysis [51,53,54]. In the current study, we identified 51 proteins (Table 1) in D. citri chorion. The proteins were categorized by their biological or molecular function, as indicated in Figure 1B. The D. citri chorion proteins putatively identified were divided into eight categories: (i) enzymes such as hydrolases, transferase, and enzymes related to GTPase and glycosidase activity; (ii) binding proteins; (iii) structural proteins; (iv) homeostasis-related proteins (mostly vitellogenins); (v) proteins related to gene expression; (vi) immune system proteins; (vii) other proteins; and (viii) uncharacterized proteins.

Enzymes
The largest group found in the chorion proteins were enzymes (25%), and these consisted largely of hydrolases such as the probable chitinase 10 GH18 family (306 kDa), the endochitinase GH18 family (68 kDa), chitooligosaccharidolytic β-N-acetylglucosaminidaselike (38 kDa), and alpha galactosidase (17 kDa). Several glycoside hydrolases were identified in D. citri chorion (chitinase or endo-N-acetyl-β-D-glucosaminidase (ENGase), chitinase-like lectins (chi-lectins/proteins (CLPs), alpha mannosidase, and alpha-glucosidase). Although the function of these enzymes in insect chorion is not completely understood, in general, they are involved in chitin biosynthesis and the degradation of insect anatomical structures, such as the trachea, mouthparts, and cuticle [55]. On the other hand, the presence of carbohydrates in chorion has been involved in fertilization as a putative ligand for sperm enzymes [56].
Three proteins were characterized as peptidases, including carboxypeptidase D-like (145 kDa), aminopeptidase (92 kDa), and an uncharacterized 91 kDa protein. We previously detected both a carboxypeptidase and a hypothetical aminopeptidase in the cytosolic protein fraction of adult D. citri [46] that are likely involved in general proteolysis. Similarly, in the saliva proteins of D. citri, Yu and Killiny [47] detected a 472.7 kDa protein with histone acetyltransferase activity similar to histone acetyltransferase p300, but its small size in the chorion fraction (30 kDa) suggests a different homology. The remaining enzymes identified included epoxide hydrolase 4-like (46 kDa) and apoptosis-inducing factor-1-mitochondrial (81 kDa), which was assigned as an oxyreductase.

Binding Proteins
Three types of binding proteins (10% of total proteins) were identified in the D. citri chorion-nucleic-acid-binding, odorant-binding, and chitin-binding proteins. The chitin binding proteins included protein obstructor-E-like (27 kDa) and an uncharacterized protein (51 kDa). Protein obstructor-E-like has been reported to be critical in Drosophila larval development, and mutants lacking this gene failed to metamorphose correctly [57]. Protein obstructor-E directs cuticle formation during the pupation of holometabolous insects, shifting the body shape from a longitudinal (larval) arrangement to a more lateral (adult) orientation [57]. Whether this gene applies to insects without a larval stage remains to be demonstrated but offers an interesting challenge.
A ubiquitous nucleic acid-binding protein identified as zinc finger homeobox protein 3-like (85 kDa) has been found in the chorion of D. citri, as was odorant-binding protein-1 (16 kDa), which is common to many insects. The identification of odorant-binding proteins (OBP) in our sample, which is considered a chemoattractant for sperm in Anopheles gambiae and Aedes aegypti [51,54], suggests that the chorion of D. citri is important for fertilization and, therefore, is an excellent silencing target to prevent gamete fertilization.

Structural and Protection Proteins
Two proteins, vegetative cell wall protein gp1-like (52 kDa) and a 39 kDa uncharacterized protein, were assigned as structural proteins (8% of total proteins). The gp1-like cell wall protein is the product of a gene primarily found in plants, but it was also identified in five hemipterans. Cuticle protein 7-like (protection function) was identified in the chorion, and interestingly, more than 800 cuticle proteins have been identified in insects as a major component of their main body covering [58]. Although we found no literature existing specifically about cuticle protein 7-like, it and the gp1-like protein present opportunities for further study.

Vitellogenins
Several proteins identified as homeostasis-related proteins (8%) in the chorion could be possible targets to control D. citri by RNAi. We identified two vitellogenins that functioned as a nutritional source of eggs (170 kDa), as well as for lipid transport (64 kDa), and two vitellogenins associated with the Von Willebrand factor domain (VWFD) that are required for normal homeostasis. Vitellogenin (Vg) is a precursor protein that produces the major storage protein, vitellin, in insect eggs [59]. Several Vg genes have been silenced by RNAi in Bemisia tabaci [60], Rhodnius prolixus [39], and Nilaparvata lugens [61], causing a significant decrease in survival rate, increased mortality, reduction in egg count, and distorted egglaying patterns, suggesting that Vg genes are promising targets for pest control. Vg protein was also found in the body and head of white-backed planthoppers, and its gene expression was highest in mature females, suggesting an important role in reproduction [62].

Gene Expression Regulation Proteins
Two proteins identified as Golgin subfamily A member 4-like (180 and 192 kDA) were detected by LC-MS/MS of D. citri choria. Miao et al. [62] reported the presence of golgin subfamily genes in the salivary proteome of the plant hopper, Sogatella furcifera. Its biological function was assigned as a Golgi transporter. A third low-quality cytoskeletal protein, myosin-II-like (non-muscle) (151 kDa), was found in the sample and is thought to be active during the mitosis and invagination of eggs during Drosophila embryogenesis [63].

Immune Response
Two proteins related to immune response (6% of total proteins) were identified, including leucine-rich repeats and immunoglobulin-like domains protein 1 (LRIG1) (60 kDa) and a 10 kDa uncharacterized protein. LRIG1 is thought to interact with epidermal growth factors during oogenesis in mammals, but its study in insects is incomplete [64]. However, in mammals, its immune response relates to the wound healing of the epidermis and suppresses cell proliferation in embryonic tissues [65].

Other Proteins and Uncharacterized Proteins
Two cellular process proteins (16% of total proteins), ubiquilin-1 (39 kDa) and the RNA polymerase-associated factor (Paf 1) CTR9 homolog (158 kDa), were identified and classified in the "others" category of Figure 1B. A similar ubiquilin protein was identified in B. tabaci fed a high phenylpropanoid/flavonoid diet. Up-regulation of this gene indicated an adaptation in B. tabaci for the detoxification of plant phenolics [66]. The RNA polymerase II-CTR9 homolog is present ubiquitously in all organisms and regulates DNA transcription to mRNA at the SH2 domain. It is essential for embryonic development, particularly the histone H3 trimethylation of lysine [67]. In Drosophila, the Paf1 homolog CTR9 is localized to the germ cells and maturing eggs, and knock-out results in aberrant polyploid morphology of the nuclei [67]. These defects eventually became lethal to embryos and early-stage larvae.
Interestingly, 21% of identified proteins were uncharacterized but were assigned a predicted function. Two of the predicted functions included environmental challenges and the calcium-dependent carbohydrate recognition domain (CRD) ( Table 1). The uncharac-terized genes with CRD-predicted function could be involved in plant lectin recognition as a part of the innate immune system of D. citri [68]. Although there were a large number of hypothetical proteins, the genes encoding these proteins could be subjected to silencing using RNAi in order to identify their biological function in D. citri.

Conclusions
Eggs are highly vulnerable when they are deposited on leaves because they are exposed to environmental conditions, predators, and infections [69]. In addition, Ammar et al. [70] showed that nymphs of D. citri acquire the bacterial pathogen of HLB at a higher rate than adult D. citri. Therefore, interrupting the lifecycle at the earliest stages should have the greatest impact on the transmission of the HLB pathogen. Silencing these chorionrelated genes in the early stages of choriogenesis would be advantageous to reduce the viability of the eggs after oviposition, likely reducing the hatching rate. The proteomic analysis of recovered D. citri choria allowed us to identify its constituent proteins and provide promising new targets for further investigations of D. citri control through RNAi.  Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.
Data Availability Statement: Data will be shared upon request to the corresponding authors.