Manual curation and phylogenetic analysis of chitinase family genes in the Asian citrus psyllid, Diaphorina citri

Abstract

Chitinases are enzymes that digest the polysaccharide polymer chitin. During insect development, breakdown of chitin is an essential step in molting of the exoskeleton. Knockdown of chitinases required for molting is lethal to insects, making chitinase genes an interesting target for RNAi-based pest control methods. The Asian citrus psyllid, Diaphorina citri, carries the bacterium causing Huanglongbing, or citrus greening disease, a devastating citrus disease. We identified and annotated 12 chitinase family genes from D. citri as part of a community effort to create high-quality gene models to aid the design of interdictory molecules for pest control. We categorized the D. citri chitinases according to an established classification scheme and re-evaluated the classification of chitinases in other hemipterans. In addition to chitinases from known groups, we identified a novel class of chitinases present in D. citri and several related hemipterans that appears to be the result of horizontal gene transfer.

Data description

During insect growth and development, the exoskeleton must be repeatedly shed and replaced. As part of this process, chitin, a polysaccharide polymer that is an important structural component of the cuticle, must be degraded [1]. Chitinases are enzymes that hydrolyze chitin into chitooligosaccharides, which can then be recycled to synthesize new chitin molecules [1, 2]. Restricting the degradation of chitin by inhibiting chitinases often results in lethality caused by molting defects (reviewed in [3]). Insect genomes usually contain 10–30 chitinase genes, with holometabolous insects generally having more than hemimetabolous insects [4]. These genes are often expressed in different stages and tissues, suggesting that they may have distinct roles during the life of the insect [2]. The various chitinase genes also encode proteins with different structures, particularly in the number of glycoside hydrolase 18 catalytic domains and chitin-binding domains (CBD). The most recent chitinase classification system, based on phylogenetic analysis and domain conservation of proteins from 20 species, divides chitinases into 10 groups (I–X) [5]. Most of these groups appear to be ancient, with all but groups V and X being present in the ancestor of insects and crustaceans. This classification system has recently been applied to the chitinases of two hemipteran insects [6, 7]. These studies concluded that almost all the chitinase groups are represented in at least some hemipterans. However, group IX chitinases seem to have been lost from the hemipteran lineage. Several hemipteran chitinase genes that could not be definitively classified have been tentatively assigned to group IV.

Context

We are part of a community that is manually curating genes from the genome of the Asian citrus psyllid, Diaphorina citri (Hemiptera: Liviidae; NCBI:txid121845), the vector of Candidatus Liberibacter asiaticus (CLas), the bacterium causing Huanglongbing (citrus greening disease) [8, 9]. The primary goal of this project is to create high-quality gene models of potential targets for gene-based pest control. The essential role of some chitinases during insect development makes them promising pest control targets. Several putative chitinase genes have previously been reported in D. citri, but these have not been manually curated [10]. Here, we report the annotation of the chitinase gene family in D. citri. We identified and annotated 11 chitinase genes, plus a gene encoding the related enzyme endo-beta-N-acetylglucosaminidase. We used phylogenetic and domain analyses to classify the chitinases according to the 10-group system established by Tetreau et al. [5]. Our results indicate that D. citri has a similar complement of chitinase genes to other hemipterans, but also has an unusual chitinase that seems to have arisen from a horizontal transfer event. Our phylogenetic analysis indicates that several hemipteran chitinases previously assigned to group IV are orthologous to this gene and should be reclassified.

Methods

Diaphorina citri chitinase genes were identified by BLAST analysis of D. citri sequences available on the Citrus Greening website [11] using orthologs from other insects as the query. To confirm orthology, we performed reciprocal BLASTs of the National Center for Biotechnology Information (NCBI) non-redundant protein database [12]. Genes were manually annotated in the D. citri v3 genome in Apollo (Apollo, RRID:SCR_001936; v2.1.0) using available evidence. A complete annotation workflow is available at protocols.io (Figure 1) [13].

Figure 1.

Protocols.io protocol for annotating genes in Diaphorina citri genome version 3. https://www.protocols.io/widgets/doi?uri=dx.doi.org/10.17504/protocols.io.bniimcce

Protein domains were identified using BLAST and InterPro (InterPro, RRID:SCR_006695) [14].

Phylogenetic analysis was performed with MEGA X (MEGA software, RRID:SCR_000667) [15]. Sequences were aligned with CLUSTALW (RRID:SCR_002909) [16] and trees were constructed by the neighbor-joining method with 1000 bootstrap replicates. Accession numbers for orthologs used in phylogenetic analysis are shown in Table 1. Counts for gene expression were obtained from the Citrus Greening Expression Network (CGEN) [17] and visualized using pheatmap (pheatmap, RRID:SCR_016418) [18] in R (R Project for Statistical Computing, RRID:SCR_001905) [19].

Table 1.

Accession numbers of proteins used in phylogenetic analysis.

Name in tree	Order	Species	Accession
TcCht1	Coleoptera	Tribolium castaneum	XP_971647.1
TcCht2	Coleoptera	Tribolium castaneum	XP_970191.2
TcCht3	Coleoptera	Tribolium castaneum	EFA08056.1
TcCht4	Coleoptera	Tribolium castaneum	NP_001073567.1
TcCht5	Coleoptera	Tribolium castaneum	NP_001034524.1
TcCht6	Coleoptera	Tribolium castaneum	XP_967813.1
TcCht7	Coleoptera	Tribolium castaneum	NP_001036035.1
TcCht8	Coleoptera	Tribolium castaneum	NP_001038094.1
TcCht9	Coleoptera	Tribolium castaneum	NP_001038096.1
TcCht10	Coleoptera	Tribolium castaneum	NP_001036067.1
TcCht11	Coleoptera	Tribolium castaneum	NP_001038095.1
TcCht12	Coleoptera	Tribolium castaneum	XP_972802.2
TcCht13	Coleoptera	Tribolium castaneum	NP_001036034.1
TcCht14	Coleoptera	Tribolium castaneum	XP_973005.1
TcCht15	Coleoptera	Tribolium castaneum	XP_973077.1
TcCht16	Coleoptera	Tribolium castaneum	NP_001034515.1
TcCht17	Coleoptera	Tribolium castaneum	XP_972719.1
TcCht18	Coleoptera	Tribolium castaneum	XP_973161.2
TcCht19	Coleoptera	Tribolium castaneum	XP_973119.2
TcCht20	Coleoptera	Tribolium castaneum	NP_001034516.3
TcCht21	Coleoptera	Tribolium castaneum	NP_001034517.1
TcIDGF2	Coleoptera	Tribolium castaneum	NP_001038092.1
TcIDGF4	Coleoptera	Tribolium castaneum	NP_001038091.1
TcENGase	Coleoptera	Tribolium castaneum	EFA09314.2
DmCht1	Diptera	Drosophila melanogaster	NP_609190.2
DmCht10	Diptera	Drosophila melanogaster	EAA46011.1
DmCht11	Diptera	Drosophila melanogaster	NP_572361.1
DmCht12	Diptera	Drosophila melanogaster	NP_726022.1
DmCht2	Diptera	Drosophila melanogaster	NP_477298.2
DmCht4	Diptera	Drosophila melanogaster	NP_524962.2
DmCht5	Diptera	Drosophila melanogaster	NP_650314.1
DmCht6	Diptera	Drosophila melanogaster	NP_572598.3
DmCht7	Diptera	Drosophila melanogaster	NP_647768.3
DmCht8	Diptera	Drosophila melanogaster	NP_611542.2
DmCht9	Diptera	Drosophila melanogaster	NP_611543.3
DmIDGF1	Diptera	Drosophila melanogaster	NP_477258.1
DmIDGF2	Diptera	Drosophila melanogaster	NP_477257.2
DmIDGF3	Diptera	Drosophila melanogaster	NP_723967.1
DmIDGF4	Diptera	Drosophila melanogaster	NP_727374.1
DmIDGF5	Diptera	Drosophila melanogaster	NP_611321.3
DmIDGF6	Diptera	Drosophila melanogaster	NP_477081.1
DcCht5	Hemiptera	Diaphorina citri	Dcitr06g10380.1.1
DcCht7	Hemiptera	Diaphorina citri	Dcitr07g07740.1.1
DcIDGF1	Hemiptera	Diaphorina citri	Dcitr02g06220.1.1
DcIDGF2	Hemiptera	Diaphorina citri	Dcitr02g06220.1.1
DcIDGF3	Hemiptera	Diaphorina citri	Dcitr02g06590.1.1
DcCht6	Hemiptera	Diaphorina citri	Dcitr10g04150.1.1
DcCht11	Hemiptera	Diaphorina citri	Dcitr01g03820.1.1
DcCht3	Hemiptera	Diaphorina citri	Dcitr07g08380.1.1
DcENGase	Hemiptera	Diaphorina citri	Dcitr01g14510.1.1
DcChtPE	Hemiptera	Diaphorina citri	Dcitr11g03190.1.1
DcCht10-1	Hemiptera	Diaphorina citri	Dcitr02g11110.1.1
DcCht10-2	Hemiptera	Diaphorina citri	Dcitr12g04430.1.1
ApCht1	Hemiptera	Acyrthosiphon pisum	NP_001162142.1
ApCht2	Hemiptera	Acyrthosiphon pisum	XP_001943038.2
ApCht3	Hemiptera	Acyrthosiphon pisum	XP_029343203.1
ApCht4	Hemiptera	Acyrthosiphon pisum	XP_001950380.1
ApCht5	Hemiptera	Acyrthosiphon pisum	XP_008181779.1
ApCht6	Hemiptera	Acyrthosiphon pisum	XP_008182857.1
ApCht7	Hemiptera	Acyrthosiphon pisum	XP_008183766.1
ApCht8	Hemiptera	Acyrthosiphon pisum	XP_001945470.2
ApENGase	Hemiptera	Acyrthosiphon pisum	XP_016658011.1
NlCht1(partial)	Hemiptera	Nilaparvata lugens	AJO25036.1
NlCht2	Hemiptera	Nilaparvata lugens	AJO25037.1
NlCht3	Hemiptera	Nilaparvata lugens	AJO25038.1
NlCht4	Hemiptera	Nilaparvata lugens	AJO25039.1
NlCht5	Hemiptera	Nilaparvata lugens	AJO25040.1
NlCht6	Hemiptera	Nilaparvata lugens	AJO25041.1
NlCht7	Hemiptera	Nilaparvata lugens	AJO25042.1
NlCht8	Hemiptera	Nilaparvata lugens	AJO25043.1
NlCht10	Hemiptera	Nilaparvata lugens	AJO25045.1
NlIDGF	Hemiptera	Nilaparvata lugens	AJO25056.1
NlENGase	Hemiptera	Nilaparvata lugens	AJO25057.1
BtCht2	Hemiptera	Bemisia tabaci	UDL18255.1
BtCht3	Hemiptera	Bemisia tabaci	UDL18256.1
BtCht4	Hemiptera	Bemisia tabaci	UDL18257.1
BtCht5	Hemiptera	Bemisia tabaci	UDL18258.1
BtCht6	Hemiptera	Bemisia tabaci	UDL18259.1
BtCht7	Hemiptera	Bemisia tabaci	UDL18260.1
BtCht8	Hemiptera	Bemisia tabaci	UDL18261.1
BtCht9	Hemiptera	Bemisia tabaci	UDL18262.1
BtCht10	Hemiptera	Bemisia tabaci	UDL18263.1
BtCht11	Hemiptera	Bemisia tabaci	XP_018912124.1
BtIDGF1	Hemiptera	Bemisia tabaci	UDL18264.1
BtIDGF2	Hemiptera	Bemisia tabaci	UDL18265.1
BtIDGF3	Hemiptera	Bemisia tabaci	UDL18266.1
BtENGase	Hemiptera	Bemisia tabaci	UDL18267.1
TuXP015788124.1	Trombidiformes	Tetranychus urticae	XP_015788124.1
SfXP025409901.1	Hemiptera	Sipha flava	XP_025409901.1
DnXP015372246.1	Hemiptera	Diuraphis noxia	XP_015372246.1
MpXP022167894.1	Hemiptera	Myzus persicae	XP_022167894.1
ArCAF1372083.1	Bdelloida	Adineta ricciae	CAF1372083.1
BcXP037026665.1	Diptera	Bradysia coprophila	XP_037026665.1
CnXP031616960.1	Diptera	Contarinia nasturtii	XP_031616960.1
AcCht-h	Lepidoptera	Agrius convolvuli	BAE16588.1
BmCht-h	Lepidoptera	Bombyx mori	XP_037867787.1
DpCht-h	Lepidoptera	Danaus plexippus plexippus	XP_032522474.1
PxCht-h	Lepidoptera	Papilio xuthus	KPJ01281.1
SlCht-h	Lepidoptera	Spodoptera litura	XP_022815620.1
OfCht-h	Lepidoptera	Ostrinia furnacalis	XP_028158980.1

Ortholog names used in the phylogenetic tree (Figure 3), taxonomic order, species name and accession number are shown.

Data validation and quality control

We identified and annotated chitinase genes in the chromosome-level D. citri v3 genome (Table 2). BLAST analysis, domain content and phylogenetic analysis were used to determine the orthology of annotated genes. We followed the established convention for naming chitinase genes, using the same name as the Drosophila melanogaster ortholog whenever possible [20].

Table 2.

Manually annotated chitinase family genes from Diaphorina citri.

Group	Gene/Isoform	OGSv3 ID	Evidence supporting annotation
			MCOT	Iso-Seq	RNA-Seq	Ortholog
I	Chitinase 5	Dcitr06g10380.1.1	MCOT12176.1.CO	X	X	X
II	Chitinase 10-1	Dcitr02g11110.1.1	MCOT12469.0.CO		X	X
II	Chitinase 10-2	Dcitr12g04430.1.1	MCOT05985.1.CT			X
III	Chitinase 7	Dcitr07g07740.1.1	MCOT01854.1.CT	X	X	X
V	Imaginal disc growth factor 1	Dcitr02g06220.1.1		X	X	X
V	Imaginal disc growth factor 2	Dcitr02g06210.1.1	MCOT17201.0.CT		X	X
V	Imaginal disc growth factor 3	Dcitr02g06590.1.1		X	X	X
VI	Chitinase 6	Dcitr10g04150.1.1	MCOT02473.0.CO	X		X
		Dcitr10g04150.1.2
VIII	Chitinase 11	Dcitr01g03820.1.1		X	X	X
X	Chitinase 3	Dcitr07g08380.1.1	MCOT14388.2.CO		X	X
ENGase	endo-beta-N-acetylglucosaminidase	Dcitr01g14510.1.1	MCOT20578.0.CT		X	X
ChtPE		Dcitr11g03190.1.1	MCOT00573.0.CT		X	X

The chitinase group, OGSv3 gene identifier and evidence types used during the annotation process are listed for each gene. MCOT identification numbers denote models from the Maker, Cufflinks, Oases and Trinity transcriptome [8].

Group I chitinases

Group I chitinases contain one catalytic domain and one C-terminal CBD (Figure 2) [2]. Most insects have a single group I chitinase (Table 3), which is typically named Chitinase 5 (Cht5) (Table 4). However, multiple group I chitinase genes have been found in mosquitoes [21], as well as in several hemimetabolous insects [4, 7, 22, 23]. Within the Hemiptera, Acyrthosiphon pisum and Bemisia tabaci have one Cht5 ortholog, while Nilaparvata lugens and Sogatella furcifera have two [4, 6, 7, 23]. We identified only one Cht5 gene in the D. citri genome (Tables 2 and 3, Figure 3). As expected, it encodes a protein with one catalytic domain and one CBD.

Figure 2.

Chitinase domain organization in Diaphorina citri. Chitinases are categorized by group based on phylogenetic analysis, sequence similarity, and domain organization. D. citri domain analysis was performed with InterPro. The two Group II proteins with different domain structures are both shown. Group V represents three proteins with the same domain structure: Idgf1, Idgf2 and Idgf3.

Table 3.

Estimated number of chitinase genes in various insect species.

Species	Chitinase groups
	I	II	III	IV	V	VI	VII	VIII	IX	X	ENGase	ChtPE	Total
D. melanogaster	1	1	1	4	6	1	1	1	1	0	1	0	18
A. gambiae	5	1	1	8	2	1	1	1	1	0	1	0	22
T. castaneum	1	1	1	14	2	1	1	1	1	1	1	0	25
S. furcifera	2	1	1	0	2	1	1	1	0	1	1	0	11
N. lugens	2	2	1	0	2	1	1	1	0	1	1	0	12
B. tabaci	1	2	1	0	3	1	1	1	0	1	1	2	14
A. pisum	1	1	1	0	1	1	0	1	0	1	1	1	9
D. citri	1	2	1	0	3	1	0	1	0	1	1	1	12

Table 4.

Insect chitinase orthologs.

	Dm	Ag	Ms	Tc	Sf	Nl	Bt	Ap	Dc
Group 1	Cht5	Cht5-1	Cht5	Cht5	Cht5	Cht5	Cht5	Cht5	Cht5
		Cht5-2
		Cht5-3
		Cht5-4
		Cht5-5
					Cht4	Cht4
Group 2	Cht10	Cht10	Cht10	Cht10	Cht10	Cht10		Cht2	Cht10-1
						Cht1	Cht10
							Cht4
									Cht10-2
Group 3	Cht7	Cht7	Cht7	Cht7	Cht7	Cht7	Cht7	Cht4	Cht7
Group 4	Cht4	Cht4	Cht8	Cht4
	Cht8	Cht8		Cht8
	Cht9	Cht9		Cht9
	Cht12	Cht12		Cht12
		Cht13		Cht13
				Cht14
				Cht15
		Cht16		Cht16
				Cht17
				Cht18
				Cht19
				Cht20
				Cht21
				Cht22
		Cht23
		Cht24
Group 5	IDGF1
	IDGF2
	IDGF3
	IDGF4		IDGF1		IDGF1		IDGF1
	IDGF5				IDGF2		IDGF2
	IDGF6						IDGF3
		IDGF2
		IDGF4
				IDGF2
				IDGF4
						IDGF
						Cht9
								Cht1
									IDGF1
									IDGF2
									IDGF3
Group 6	Cht6	Cht6	Cht6	Cht6	Cht6	Cht6	Cht6	Cht3	Cht6
							Cht2?
Group 7	Cht2	Cht2	Cht2	Cht2	Cht8	Cht8
Group 8	Cht11	Cht11	Cht11	Cht11	Cht2	Cht2	Cht11	Cht6	Cht11
Group 9	DmCht1		Cht1	Cht11
Group 10			Cht3	Cht3	Cht3	Cht3	Cht3	Cht8	Cht3
ENGase	CG5613	XP 310876.4		XP 008197368.1	EnGase	ENGase	ENGase	ENGase	ENGase
SI-Clp	CG8460	XP 317335.2		XP 971647.1
ChtPE								Cht7	PE
							Cht8
							Cht9

Revised group assignment of chitinase proteins from Drosophila melanogaster (Dm), Anopheles gambiae (Ag), Manduca sexta (Ms), Tribolium castaneum (Tc), Sogatella furcifera (Sf), Nilaparvata lugens (Nl), Bemisia tabaci (Bt), Acyrthosiphon pisum (Ap) and Diaphorina citri (Dc) based the analysis described in this work. A blank cell means no members of a particular group have been identified in that insect. Orthologs shown in italics indicate changes in group assignment based on our analysis. A question mark denotes uncertainty in the new classification.

Figure 3.

Phylogenetic tree of chitinase and chitinase-like family members. CLUSTALW was used to perform multiple sequence alignments. The tree was constructed with MEGA X software using neighbor-joining analysis (1000 bootstrap replicates). Bootstrap values below 50 are not shown. The final annotated tree graphic was created with BioRender.com [24]. Proteins used in tree construction are from Diptera: Drosophila melanogaster (Dm), Anopheles gambiae (Ag), Bradysia coprophila (Bc), Contarinia nasturtii (Cn); Lepidoptera: Agrius convolvuli (Ac), Bombyx mori (Bm), Danaus plexippus plexippus (Dp), Papilio xuthus (Px), Spodoptera litura (Sl), Ostrinia furnacalis (Of); Coleoptera: Tribolium castnaeum (Tc); Hemiptera: Nilaparvata lugens (Nl), Acyrthosiphon pisum (Ap), Bemisia tabaci (Bt), Diaphorina citri (Dc), Sipha flava (Sf), Myzus persicae (Mp), Diuraphis noxia (Dn), Arachnida: Tetranychus urticae (Tu); Rotifera: Adineta ricciae (Ar). D. citri proteins from genes annotated in this work are marked with black circles. Colors delineate chitinase groups which are also labeled. Genes that do not cluster well with any group are black.

Chitinase groups are based on the classification system established by Tetreau et al. [5], except for ChtPE, which is described in this work. D. citri gene numbers were determined based on our annotation of the D. citri v3 genome. Counts in other insects are based on the literature [4, 6, 7, 21, 23] and our phylogenetic analysis.

Group II chitinases

Group II chitinases are typically named Chitinase 10 (Cht10) in insects (Table 4) [2]. These chitinases are high-molecular-weight chitinases with multiple catalytic domains (some active and some inactive) and several CBDs [2]. Most previously studied insects have only one Cht10 gene (Table 3), although two were found in N. lugens (NlCht10 and NlCht1) [23]. Two of the chitinases we annotated in D. citri cluster with the Cht10 proteins during phylogenetic analysis. One of these, Cht10-1, is a typical Cht10 protein. It is a large, 21-exon gene that encodes a protein containing five catalytic domains and two CBDs. The second protein identified as a potential Cht10 in D. citri is much smaller and only contains a catalytic domain. Despite the difference in size and domain content, phylogenetic analysis indicates this protein is most closely related to the Cht10 proteins, so we have named it Cht10-2 (Figure 3). Interestingly, the B. tabaci Cht4 protein, which had been tentatively placed in group IV [6], also has only a catalytic domain and clusters with the group II chitinases in our tree. Thus, we suggest that this should be reassigned to group II (Tables 3 and 4). NlCht10, one of the N. lugens proteins classified as a group II chitinase [23], surprisingly clusters with the Drosophila and Tribolium group VI proteins in our tree (Figure 3). The high level of sequence identity between NlCht10 and NlCht1, however, indicates that NlCht10 should remain in group II. These conflicting phylogenetic results suggest that additional analysis of the N. lugens group II chitinases is warranted.

Group III chitinases

The group III chitinases are typically named Chitinase 7 (Cht7) in insects (Table 4) [2]. Most insects have one Cht7 that contains an N-terminal transmembrane domain, plus two catalytic domains followed by a CBD (Figure 2) [20]. In D. citri, we identified one Cht7 gene (Tables 2 and 3). As expected, the predicted protein contained two catalytic domains, followed by one CBD (Figure 2). Like the A. pisum and S. furcifera group III chitinases, DcCht7 has an N-terminal signal peptide [4, 7], suggesting that at least some hemipteran group III chitinases may be secreted and thus function differently than their orthologs in holometabolous insects that have an N-terminal transmembrane domain.

Group IV chitinases

In holometabolous insects, group IV is the largest and most diverse group of chitinases [2]. These chitinases have the greatest variation in domain organization and are found in clusters in some insect genomes, suggesting duplication events. In hemimetabolous insects, group IV has previously been used as a catch-all group for chitinases that could not be clearly assigned to a group [6, 23]. However, recently, several of the hemipteran chitinases previously assigned to group IV have been reclassified as group X chitinases [6]. Moreover, in our phylogenetic analysis (Figure 3), no D. citri chitinases clustered with group IV, and the other hemipteran chitinases that had previously been placed in group IV (B. tabaci Cht8 and Cht9) were part of a novel cluster discussed in more detail below. These observations suggest that hemipterans lack group IV chitinases.

Group V chitinases

The group V chitinases were first identified for their role in the growth of imaginal disc tissue in Drosophila and were named Imaginal disc growth factors (Idgf) [2, 25]. D. melanogaster has six Idgf genes, but most insects have fewer (Tables 3 and 4). Phylogenetic analysis suggests that there have been several independent duplications of Idgf genes in insect lineages [4]. In D. citri, we identified three Idgf genes (Tables 2 and 3), which we have named Idgf1, Idgf2 and Idgf3. These genes are not one-to-one orthologs of the Drosophila Idgf1, Idgf2 and Idgf3 genes, as phylogenetic analysis suggests that Idgf genes have duplicated independently in these two insect lineages (Figure 3). All three Idgf genes in D. citri are found in a 1.25-megabase pair (Mbp) region of chromosome 2, with Idgf1 and Idgf2 adjacent to one another on the same strand. Idgf1 and Idgf3 form their own clade in our phylogenetic tree, while Idgf2 is an outgroup to the other group V chitinases, suggesting it has diverged more extensively than the other two paralogs (Figure 3).

As seen in group V chitinases of other insects, all three D. citri Idgf proteins have only one catalytic domain and they do not contain a CBD (Figure 2). The catalytic domain of Idgf proteins is inactive because of a mutation that produces an aspartic acid to alanine substitution in conserved motif II [2, 26]. This mutation is present in all three D. citri Idgf genes, confirming their identity.

Group VI Chitinases

In insects, the group VI chitinases are usually named Chitinase 6 (Cht6) (Table 4) [2]. In holometabolous insects, group VI chitinases have a similar domain structure to group I chitanases with an N-terminal catalytic domain and one CBD, but additionally have a long serine/threonine (S/T)-rich region at the C-terminus [2]. The hemipterans N. lugens and A. pisum each have a single group VI chitinase. These proteins differ from their holometabolous orthologs in that they have a second CBD near the C-terminus [4, 23]. In D. citri, we identified one Cht6 gene that also encodes a protein with a second CBD (Figure 2). The D. citri Cht6 protein also contains a long stretch of amino acids between the CBDs, which contains approximately 25% S/T residues, supporting its classification as a group VI chitinase. We identified two isoforms of Cht6 in D. citri, which differ only in the length of the S/T-rich region between the CBDs. Similar isoforms have been reported for S. furcifera Cht6 [7].

In contrast to the other chitinase groups, the group VI orthologs do not all cluster together in our phylogenetic tree (Figure 3). The hemipteran group VI proteins form one cluster, while the T. castaneum and D. melanogaster Cht6 orthologs are in a separate cluster with N. lugens Cht10, which has been classified in group II [23]. BtCht2, which was formerly classified as group VII [6], also clusters with the group VI genes, albeit with low bootstrap values (Figure 3). Moreover, D. melanogaster Cht8, which is considered a group IV member, is the closest outgroup to the hemipteran group VI proteins.

Group VII chitinases

Group VII chitinases are typically named Chitinase 2 (Cht2) in insects [2]. Within hemipterans, the planthoppers N. lugens and S. furcifera have a group VII chitinase gene [7, 23], but A. pisum does not (Table 3) [4]. B. tabaci was reported to have a group VII gene, which was consequently named BtCht2 [6]. However, the placement of BtCht2 in group VII was only weakly supported by phylogenetic analysis and, in our phylogenetic tree (Figure 3), it clusters with the group VI genes as discussed above. Although the proper classification of BtCht2 is unclear, our interpretation is that B. tabaci lacks a group VII gene (Table 4). Likewise, we found no group VII chitinase gene in the genome of D. citri. Since the three hemipterans lacking group VII genes are all sternorrhyncans, these results suggest that the group VII chitinase may have been lost after the divergence of the Sternorrhynca from other hemipterans.

Group VIII chitinases

Group VIII chitinases are typically called Chitinase 11 (Cht11) in insects (Table 4) [2]. To our knowledge, all insects examined to date have only one group VIII chitinase gene. We too identified only one group VIII chitinase in the D. citri genome. Like several other group VIII chitinases, D. citri Cht11 has an N-terminal transmembrane domain and a catalytic domain, but no CBD [2, 4].

Group IX chitinases

Group IX chitinases appear to be an ancient group, since orthologs are found in organisms as distantly related to arthropods as sea urchins and nematodes [5]. However, no group IX chitinases have been found in hemipteran genomes thus far [4, 6, 7, 23]. As expected, we were also unable to identify a group IX gene in D. citri (Tables 3 and 4).

Group X chitinases

Group X chitinases, most of which are named Cht3 (Table 4), were first recognized as a separate group by Tetreau et al. [5]. Several members of this new group had previously been assigned to group IV, although their membership in that group was always uncertain. Group X genes are found only in arthropods and seem to have been lost in the dipteran lineage [5]. The proteins encoded by group X genes have a unique, highly conserved structure consisting of a single catalytic domain followed by two closely spaced CBDs, a long intervening region with many potential glycosylation sites, and a third CBD near the C-terminus [5–7, 23]. We identified and annotated one Cht3 gene in D. citri. The encoded protein clusters with group X members in our phylogenetic analysis (Figure 3) and shares the same domain structure (Figure 2).

ENGases

The endo-beta-N-acetylglucosaminidase (ENGase) proteins are part of the GH18 chitinase-like superfamily, and have therefore been included in recent phylogenetic analyses of chitinases [4, 23]. Like the group V chitinases, these proteins lack chitinase activity because of a change in the catalytic domain. ENGase orthologs have been found in various insects, including in hemipterans [4, 6, 7, 23]. In the D. citri genome, we identified one ENGase ortholog (Tables 2, 3 and 4).

Chitinase PE

D. citri has one chitinase gene that could not be classified based on the currently defined groups. In our tree, it clusters with A. pisum Cht7, which also has not been definitively classified [4], and B. tabaci Cht8 and Cht9, which had been tentatively included in group IV [6].

The A. pisum and D. citri proteins have an unusual structure: an N-terminal signal peptide, a long N-terminal region where the only recognizable sequence is a PAN/Apple domain, and a DNA/RNA non-specific endonuclease domain in the central portion of the protein, followed by the chitinase catalytic domain and multiple CBDs. We have named the D. citri gene Chitinase PE (ChtPE) to denote the presence of the PAN domain and endonuclease domain.

Previously, it was noted that the three CBDs in A. pisum Cht7 are ChtBD1-type domains (typically found in plants and fungi) rather than the ChtBD2 type found in other insect chitinases [4]. We analyzed the domain structure of D. citri ChtPE and B. tabaci Cht8 and Cht9 and found that these proteins also have ChtBD1 domains, although the D. citri protein has only two.

BLAST analysis suggests that these novel chitinases have a very unusual phylogenetic distribution. Within the Hemiptera, they are present in several, but not all, of the sequenced genomes from sternorrhyncans (aphids, psyllids and whiteflies). Orthologous genes encoding all the domains found in ChtPE are also found in a few other phylogenetically dispersed insects, as well as in several spider mites, springtails and rotifers.

The presence of plant/fungi-like CBDs and the limited phylogenetic distribution of the gene suggest that ChtPE may have arisen by horizontal gene transfer (HGT), although the source of the gene is not clear. There have been previous reports of HGT involving chitinases. Many lepidopterans have a Cht-h gene that seems to have been horizontally transferred from bacteria [5]. A separate instance of HGT of a bacterial chitinase has been reported in spider mites [27]. However, BLAST analysis, domain content and phylogenetic analysis show that these proteins are clearly distinct from ChtPE (Figure 3).

It is unclear how the phylogenetic distribution of ChtPE-like genes arose, since this would seem to require either horizontal transfer into multiple lineages, or an ancient horizontal transfer followed by loss in most lineages. Neither scenario is particularly parsimonious. The presence of ChtPE-like genes in several sternorrhynchans but very few other hemipterans suggests there may have been a horizontal transfer event early in the sternorrhyncan lineage. However, it is unclear whether the B. tabaci genes BtCht8 and BtCht9 are orthologous to ChtPE. BtCht8 and BtCht9 are unusual in that they are single exon genes [6], while the related A. pisum and D. citri genes have multiple exons. Moreover, the encoded B. tabaci proteins have the chitinase catalytic domain and the ChtBD1 domains, but lack the PAN/Apple and endonuclease domains. Regardless of the number of HGT events, A. pisum Cht7, BtCht8 and BtCht9 belong with the HGT chitinases (Table 4) rather than in group IV where the B. tabaci proteins were previously placed [6].

Expression of chitinase genes in D. citri

We assessed expression of the chitinase genes in D. citri using the Citrus Greening Expression Network [17] found on the Citrus Greening website [11] (Figure 4, Table 5). This tool allows comparison of gene expression levels in various publicly available D. citri RNA-seq datasets that vary by life stage, tissue, food source, and CLas exposure. In D. citri, Cht5, Cht10-1, and Cht11 are expressed at highest levels in eggs with somewhat lower levels in nymphs, while Cht3, Cht6, and Cht7 are most highly expressed in nymphs. The unusual group II gene Cht10-2 is expressed at low-to-moderate levels in all stages and in most tissues. IDGF2 expression is mostly restricted to eggs, while IDGF1 and IDGF3 are expressed at all stages, but highest in adults. ENGase shows low levels of expression in all samples, with the highest expression in eggs and female abdomens. A few of the chitinases (Cht5, Cht11, IDGF1 and IDGF3) show moderate expression in the gut. ChtPE is expressed in all stages and tissues, with the highest expression in head, thorax and midgut. These expression trends are consistent with reports from other hemipterans, particularly for the stage showing the highest expression for each gene [4, 6, 7, 23].

Figure 4.

Expression of chitinase genes in Diaphorina citri. The heatmap was created from publicly available RNA-Seq expression data ([28–32] and NCBI Bioprojects PRJNA609978 and PRJNA448935) obtained from CGEN [17]. Expression is scaled by gene. Hierarchical clustering has been applied to both genes and RNA-seq samples such that those with similar expression are grouped together. Expression data used to create the heat map are provided in Table 4.

Table 5.

Expression counts of Diaphorina citri chitinase genes.

Gene ID	Cht5	Cht10-1	Cht10-2	Cht7	ChtPE	Idgf1	Idgf2	Idgf3	Cht6	Cht11	Cht3	ENGase
Egg: C. macrophylla CLas− Whole body	80.44	17.67	5.15	181.38	6.15	185.72	110.96	461.11	22.37	108.89	17.17	15.86
Nymph: C. medica CLas+ Low infection Whole body	17	5.07	22.3	598.95	30.46	580.75	1.47	615.06	53.69	31.59	125.87	7.96
Nymph: C. sinensis CLas+ High infection Whole body	26.56	8.01	15.54	505.99	16.6	594.6	2.09	611.67	42.55	25.11	98.39	6.42
Nymph: C. sinensis CLas− Whole body	14.85	2.45	17.26	305.92	27.69	615.89	0.58	423.62	34.1	38.01	68.42	8.25
Nymph: C. macrophylla CLas− Whole body	12.88	4.14	53.68	471.04	33.3	356.48	1.19	350.74	19.36	51.44	135.21	4.17
Nymph: Citrus spp. CLas− Whole body	1.27	1.53	70.52	87.77	124.54	604.06	0.6	434.54	4.26	43.16	44.6	1.75
Nymph: Citrus spp. CLas+ Whole body	6.1	5.77	27.63	83.58	11	410.24	0.94	326.6	5	44.81	35.35	10.03
Adult: C. medica CLas− Gut	0.05	0.05	0.69	1.56	32.01	38.62	0	37.36	0	57.91	0.02	6.69
Adult: C. medica CLas+ Gut	0.07	0.03	1.25	0.57	39.99	30.13	0.03	37.35	0.02	72.09	0.03	6.49
Adult: C. medica CLas+ High infection Whole body	2.97	0.22	24.1	98.87	39.78	790.73	0.91	1123.64	6.69	38.29	20.34	8.69
Adult: C. medica CLas+ Low infection Whole body	2.98	0.28	33.97	104.33	30.33	735.91	1.16	846	6.79	40.08	19.07	11.88
Adult: C. medica CLas− Whole body	6.05	0.59	8.82	179.81	41.13	725.31	0.91	896.67	7.03	34.41	54.43	4.84
Adult: C. macrophylla CLas− Whole body	0.9	0.06	22.35	7.41	29.62	788.05	1.49	1098.32	1.34	49.79	2.32	13.07
Adult: Citrus spp. CLas− Whole body	0	0.09	11.7	2.99	88.19	533.99	1.42	368.82	0	42.11	0.86	6.88
Adult: Citrus spp. CLas+ Whole body	0	0.13	26.22	2.55	73.27	575.93	1.51	1055.86	0	44.96	0.51	9.02
Adult: Citrus spp. CLas− midgut	1.21	0.03	1.47	0.44	86.49	71.33	0	89.28	0.03	70.69	0.35	8
Adult: Citrus spp. CLas+ midgut	0.53	0.03	2.27	4.83	140.65	186	0.08	116.46	0.1	40.18	1.46	10.08
Adult: C. reticulata CLas− Female abdomen	0.46	0.16	7.75	1.32	81.55	946.67	0.48	883.26	0.13	29.65	0.35	21.83
Adult: C. reticulata CLas− Female antennae	0	0	32.8	18.89	85.16	1723.19	0	1750.55	0.22	23.29	0.64	4.25
Adult: C. reticulata CLas− Female head	0.21	0	32.76	10.97	181.03	1662.39	0.18	1915.3	0.25	19.52	0.43	3.33
Adult: C. reticulata CLas− Female leg	0.02	0	8.5	2.73	69.41	1315.12	0	2022.88	0.18	20.17	0.27	12.84
Adult: C. reticulata CLas− Female terminal abdomen	0.64	0	89.66	6.12	47.48	609.54	0.16	1068.81	0	13.73	1.61	17.82
Adult: C. reticulata CLas− Female thorax	0	0	15.82	0.96	131.37	1221.78	0	1482.48	0.58	19.88	0.91	6.94
Adult: C. reticulata CLas− Male abdomen	0.48	0.09	5.75	2.3	75.05	1139.68	0.26	1490.21	0.1	37.33	1.02	6.32
Adult: C. reticulata CLas− Male antennae	0.33	0	14.25	37.15	55.26	1689.26	0.16	1918.4	0.52	30.12	1.69	2.78
Adult: C. reticulata CLas− Male head	0	0	31.07	9.79	136.84	1761.42	0	1936.55	0.31	28.17	0.76	3.18
Adult: C. reticulata CLas− Male leg	0	0	14.41	1.03	77.63	1994.31	0.5	2939.9	0.16	27.21	0.73	5.36
Adult: C. reticulata CLas− Male terminal abdomen	1.83	0.02	13.6	8.5	24.59	823.39	0.37	1357.2	0.22	22.44	1.47	5.79
Adult: C. reticulata CLas− Male thorax	0	0	12.45	0.37	203.18	1386.57	0.03	1798.49	0.31	22.61	0.82	6.29
Adult: C. reticulata CLas− Female antennae [28]	0.53	0	25	8.71	151.75	1835.97	0.49	2699.83	0.57	29.29	0.65	8.61
Adult: C. reticulata CLas− Female terminal abdomen [28]	0.77	0	12.95	1.87	61.81	980.79	0.37	1114.55	0.07	23.81	0.6	16.12
Adult: C. reticulata CLas− Male antennae [28]	0.44	0	23.52	20.19	140.14	2104.17	0.76	3582.55	1.36	37.96	1.61	5.23
Adult: C. reticulata CLas− Male terminal abdomen [28]	1.26	0.06	12.11	1.64	63.5	1132.34	0.98	1963.58	0	45.04	1.77	7.95

Expression values in transcripts per million (TPM) obtained from the Citrus Greening Expression Network [17] for annotated Diaphorina citri chitinase genes. Sample metadata including developmental stage, tissue, food source, and CLas exposure status are recorded in the first column. Cht: Chitinase; IDGF: Imaginal disc growth factor; ENGase: endo-B-N-acetylglucosaminidase.

That chitinase genes in hemipterans are generally conserved suggests that the genes may also have conserved functions. Based on expression data and RNAi studies in other insects, including several hemipterans [2, 6, 7, 23], the D. citri Cht5, Cht7 and Cht10 orthologs are the most likely to be required for molting during development. Thus, these genes should be prioritized as potential targets for RNAi-based pest control. Knockdown of the other chitinase genes will probably have only subtle effects, possibly because of redundancy, and understanding the function of these genes will require more extensive analysis. While this manuscript was under review, Wu et al. [33] published an independent characterization of D. citri chitinase genes with very similar results. They performed RNAi with each of the genes and, as we predicted, found that only DcCht5, DcCht7, DcCht10-1 and DcCht10-2 affected molting.

Conclusions

We have annotated 12 genes of the chitinase family from the citrus greening vector D. citri. We used BLAST, domain content and phylogenetic analysis to assign the predicted chitinase proteins into groups according to the current classification system [5]. D. citri has members of all chitinase groups except groups IV, VII, and IX (Table 4). We also determined that D. citri and several other sternorrhyncan hemipterans have a novel chitinase gene that appears to be the result of horizontal gene transfer.

Re-use potential

Our curation of chitinase gene models and classification of chitinase proteins will be helpful to scientists wishing to carry out additional research on these genes. Chitinases are considered good targets for gene-based pest control methods, but research in other insects has shown that not all chitinases are essential. Our analysis will help researchers choose the best genes to target and will provide accurately annotated genes as a foundation for their work.

Funding Statement

This research was funded by USDA-NIFA grant 2015-70016-23028, HSI 1300394, 2020-70029-33199 and an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103418.

Data availability

The gene models are part of an updated official gene set (OGS) for D. citri submitted to NCBI under Bioproject PRJNA29447. Sequences of the annotated genes described here are available in the GigaScience GigaDB repository [34]. They are also included in an updated official gene set (OGS) linked to the same NCBI Bioproject. Genome assembly, transcriptome and official gene set sequences are currently available for BLAST and expression analysis on the Citrus Greening Solutions website [11].

Editor’s note

This article is one of a series of Data Releases crediting the outputs of a student-focused and community-driven manual annotation project curating gene models and, if required, correcting assembly anomalies, for the Diaphorina citri genome project [35].

List of abbreviations

CBD: Chitin binding domain; CGEN: Citrus Greening Expression Network; Cht: Chitinase; CLas: Candidatus Liberibacter asiaticus; ENGase: endo-beta-N-acetylglucosaminidase; HGT: horizontal gene transfer; Idgf: Imaginal disc growth factor; MCOT: Maker, Cufflinks, Oases and Trinity; NCBI: National Center for Biotechnology Information; OGS: official gene set; RNA-seq: RNA sequencing; S/T: serine/threonine; TPM: transcripts per million.

Ethical approval

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Funding

This research was funded by USDA-NIFA grant 2015-70016-23028, HSI 1300394, 2020-70029-33199 and an Institutional Development Award (IDeA) from the National Institute of General Medical Sciences of the National Institutes of Health under grant number P20GM103418.

Authors’ contributions

WBH, SJB, TD and LAM conceptualized the study; TD, SS, TDS and SJB supervised the study; SJB, TD, SS, and LAM contributed to project administration; SM, TDS, and BT conducted the investigation; PH, MF-G, and SS contributed to software development; SS, TDS, PH, and MF-G developed methodology; SJB, TD, WBH, and LAM acquired funding; TDS and SM prepared and wrote the original draft; SS, WBH and SJB reviewed and edited the draft.

Acknowledgement

We thank Dr. Josh Benoit for assistance with visualization of expression data.