# Chitinase 6 is required for procuticle thickening and organ shape in Drosophila wing

Wei Dong, Justin Flaven-pouchon, Ying-hao Gao, Chen-yang Song, Abeer El Wakil, Jian-zhen Zhang, Bernard Moussian

► To cite this version:

Wei Dong, Justin Flaven-pouchon, Ying-hao Gao, Chen-yang Song, Abeer El Wakil, et al.. Chitinase 6 is required for procuticle thickening and organ shape in Drosophila wing. Insect Science, 2022, 30 (2), pp.268-278. 10.1111/1744-7917.13115 . hal-04292418

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Insect Science (2023) 30, 268–278, DOI 10.1111/1744-7917.13115

ORIGINAL ARTICLE

# Chitinase 6 is required for procuticle thickening and organ shape in *Drosophila* wing

Wei Dong<fnref n="1" /> ID, Justin Flaven-Pouchon², Ying-Hao Gao<fnref n="1" />,³, Chen-Yang Song<fnref n="1" />,³, Abeer El Wakil⁴, Jian-Zhen Zhang<fnref n="1" /> ID and Bernard Moussian<fnref n="1" />,²,⁵

**Abstract** The polysaccharide chitin is a major scaffolding molecule in the insect cuticle. In order to be functional, both chitin amounts and chitin organization have been shown to be important parameters. Despite great advances in the past decade, the molecular mechanisms of chitin synthesis and organization are not fully understood. Here, we have characterized the function of the Chitinase 6 (Cht6) in the formation of the wing, which is a simple flat cuticle organ, in the fruit fly *Drosophila melanogaster*. Reduction of Cht6 function by RNA interference during wing development does not affect chitin organization, but entails a thinner cuticle suggesting reduced chitin amounts. This phenotype is opposed to the one reported recently to be caused by reduction of *Cht10* expression. Probably as a consequence, cuticle permeability to xenobiotics is enhanced in Cht6-less wings. We also observed massive deformation of these wings. In addition, the shape of the abdomen is markedly changed upon abdominal suppression of Cht6. Finally, we found that suppression of *Cht6* transcript levels influences the expression of genes coding for enzymes of the chitin biosynthesis pathway. This finding indicates that wing epidermal cells respond to activity changes of Cht6 probably trying to adjust chitin amounts. Together, in a working model, we propose that Cht6-introduced modifications of chitin are needed for chitin synthesis to proceed correctly. Cuticle thickness, according to our hypothesis, is in turn required for correct organ or body part shape. The molecular mechanisms of this processes shall be characterized in the future.

**Key words** abdomen; chitin; Cht6; *Drosophila melanogaster*; procuticle; wing

**Introduction**

The insect body cuticle serves as a barrier against a potentially hostile environment. It is a layered apical extracellular-matrix (ECM) secreted by epidermal cells (Galko & Krasnow, 2004; Shaik *et al.*, 2011;

Correspondence: Wei Dong, Jian-Zhen Zhang and Bernard Moussian, Institute of Applied Biology, Shanxi University, Taiyuan 030006, China. Email: dongwei@sxu.edu.cn; zjz@sxu.edu.cn; bernard.moussian@unice.fr

Curtis *et al.*, 2013; Jaspers *et al.*, 2014). The outer layer is the lipid-rich envelope supported by the proteinaceous epicuticle. Underneath the epicuticle, the inner procuticle harbors various proteins that interact with chitin, a linear polysaccharide chain of β-1,4-linked N-acetylglucosamines (GlcNAc) (Moussian, 2010). In insects, chitin microfibrils are arranged in parallel to each other forming flat sheets named laminae (Moussian *et al.*, 2015). These laminae may be stacked helicoidally or with a preferred orientation of the microfibrils in the laminae. The physical and chemical properties of the cuticle are proposed to depend on these

<footnote n="1">¹Institute of Applied Biology, Shanxi University, Taiyuan, China; ²Interfaculty Institute of Cell Biology, University of Tübingen, Tübingen, Germany; ³College of Life Science, Shanxi University, Taiyuan, China; ⁴Faculty of Education, Department of Biological and Geological Sciences, Alexandria University, Alexandria, Egypt and ⁵INRAE, CNRS, Université Côte d'Azur, Institut Sophia Agrobiotech, Sophia Antipolis, France</footnote>
quasi-crystalline chitin arrangements (Vincent & Wegst, 2004).

In the *D. melanogaster* embryo, the chitinous procuticle is produced when the envelope is already continuously covering the body surface (Moussian *et al.*, 2006). Similarly, the wing procuticle is synthesized later than the envelope. Chitin is observed firstly at 42 h after the white prepupae (awp) stage, subsequently, the procuticle layer thickens after 62 h awp (Adler *et al.*, 2013; Sobala *et al.*, 2015; Sobala & Adler, 2016). The fly wing provides several advantages for cuticle studies. It is a flat and thin tissue allowing easy observation of possible phenotypes including severe ones as it is not required for survival under the laboratory conditions. Compared to the body, the adult wings can be dissected cleanly from other tissues such as muscles and the fat body reducing contamination. Sobala and Adler (2016) described the gene expression program in depth during wing cuticle formation. Numerous genes including some *chitinases* (*Cht*) are expressed at high levels in pupal wings. Insect *Chts* belong to family 18 glycosylhydrolases, which catalyze the hydrolysis of β-1,4-linkages in chitin (Kramer & Muthukrishnan, 1997). Chitinases are well characterized in the red flour beetle *Tribolium castaneum* and the fruit fly *Drosophila melanogaster* (Zhu *et al.*, 2008; Arakane & Muthukrishnan, 2009; Pesch *et al.*, 2016). *TcCht5* has been reported that to be dispensable for pupal-adult molting in *T. castaneum* using RNA interference (RNAi). While *TcCht10* is involved in many developmental processes including larval hatching, larval molting and adult metamorphosis. A recent research revealed that *TcCht7* played a vital role in chitin organization in *T. castaneum* (Noh *et al.*, 2018). Unlike above-mentioned chitinases, *Cht2* has been demonstrated to promote procuticle thickening and pore-canal formation (Pesch *et al.*, 2017).

During wing cuticle formation in *D. melanogaster*, *Cht6*, *Ch10*, and *krotzkopf verkehrt* (*kkv*, coding for the chitin synthase 1) were highly expressed between 42 and 62 h awp, when the procuticle is thickening (Sobala & Adler, 2016; Dong *et al.*, 2020). We hypothesised that these two chitinases cooperate with the chitin synthase in chitin deposition and organization in fly wings. Genetic analyses of *Cht10* functions in the *Drosophila* wing provided evidence that *Cht10* is needed to ensure chitin amounts to ensure chitin lamellar organization (Dong *et al.*, 2020). In order to understand another candidate enzyme *Cht6*, we have used RNAi to investigate the functions of *Cht6* in wing cuticle of *D. melanogaster*. We found that depletion of *Cht6* affects the procuticle thickening in the wing vein of *Drosophila*.

## Materials and methods

### Fly stocks

The ci-Gal4, en-Gal4, UAS-GFP, UAS-brk fly lines were obtained from Prof. Jie Shen (China Agricultural University), the *UAS-Cht6-RNAi* (BS54823) line was purchased from Bloomington *Drosophila* Stock Center (BDSC), the *UAS-Cht6-RNAi* (VDRC102293) line was purchased from Vienna *Drosophila* RNAi Center (VDRC) (Table S1). According to the VDRC site, this hairpin RNA does not have any off-target in the *D. melanogaster* genome; moreover, its efficiency was determined being 99% by Pesch *et al.* (2016). All fly lines were maintained on standard cornmeal medium at 25 °C. For RNAi analysis, ci-Gal4 and en-Gal4 flies were crossed with *UAS-Cht6-RNAi* flies. For overexpression analysis, en-Gal4 flies were crossed with UAS-brk flies. The crossed flies were raised at 18 °C and their offspring was transferred to 30 °C to allow target gene expression.

### Reverse transcription quantitative PCR (RT-qPCR)

Wing buds were carefully dissected every day during pupal stage and total RNA was isolated using RNAiso Plus (TaKaRa, Japan). 1 µg of total RNA was used to synthesize the first strand of cDNA using M-MLV Reverse Transcriptase (TaKaRa, Japan). The obtained cDNA samples were diluted 10 times for qPCR analysis. qPCR reactions solution contained 3 µL template, 7.5 µL SYBR® Premix EX Taq™ (TaKaRa, Japan), 0.6 µL specific primers, and 3.3 µL RNase free dd H₂O. The 2⁻⁰Δct method was used to quantify relative mRNA levels and *rp49* was used as the reference. Four biological replicates were examined. The primer information is listed in Table 1.

### Imaging

The 3d-old adults were anesthetized with ethanol vapor. Wings were removed from flies using tweezers and washed several times with 50% ethanol. The washed wings were dried and photographed using Multifocus Imaging System of a microscope (MV PLAPO 1X, Olympus, Japan) equipped with a CCD camera (DFC450 C, Leica, Germany) under white light. For wing interference pattern analysis, isolated wings were dried and horizontally fixed on a black background. Photos were taken with a CCD camera using white light. Fiji software (Schindelin *et al.*, 2012) was used, for image preparation and wing size measurements. For abdomen analysis,
<table><caption>**Table 1** Primers for PCR amplification.</caption><thead><tr><th>Gene</th><th>Primer</th><th>Primer sequences</th><th>Product size (bp)</th></tr></thead><tbody><tr><td rowspan="2">rp49</td><td>Forward</td><td>GACAGTATCTGATGCCCAACA</td><td rowspan="2">170</td></tr><tr><td>Reverse</td><td>CTTCTTGGAGGAGACGCCGT</td></tr><tr><td rowspan="2">Cht6 (CG43374)</td><td>Forward</td><td>AAGAAGATACTGACGAGCTGTC</td><td rowspan="2">146</td></tr><tr><td>Reverse</td><td>CGATCCATCCTTGTTACGC</td></tr><tr><td rowspan="2">Tre (CG9364)</td><td>Forward</td><td>GCTCCCAGCCGCCACTTCTA</td><td rowspan="2">223</td></tr><tr><td>Reverse</td><td>TCCTCGCCGGTCTCCACATC</td></tr><tr><td rowspan="2">Gfat1 (CG12449)</td><td>Forward</td><td>TCGCCTAAAGAAGAGCCT</td><td rowspan="2">252</td></tr><tr><td>Reverse</td><td>GTGCCACATCCAATCAAC</td></tr><tr><td rowspan="2">Gfat2 (CG1345)</td><td>Forward</td><td>GGACGCATCGGCTGTTAT</td><td rowspan="2">118</td></tr><tr><td>Reverse</td><td>ATCGGATGATTTGTTTAGGC</td></tr><tr><td rowspan="2">nst (CG10627)</td><td>Forward</td><td>CACCTGTAGAGCGGAATG</td><td rowspan="2">144</td></tr><tr><td>Reverse</td><td>CAGAATAAATGCGTTTGAGA</td></tr><tr><td rowspan="2">mmy (CG9535)</td><td>Forward</td><td>TCCGTTTGCCCATGTAAT</td><td rowspan="2">183</td></tr><tr><td>Reverse</td><td>CTGAAGTTGCTGCGACCC</td></tr></tbody></table>

3-d-old adults were anesthetized at –20 °C, wings and legs were removed to ensure all individuals were placed in the same axes. Pictures were taken under a Leica binocular with a CCD camera. Abdomen perimeter and circularity were measured using Image J (freehand polygons) and the angle between the ventral cuticle and 5–6 segmental intersection was measured.

### Eosin Y staining

The 3d-old adults were collected and anesthetized with CO₂. Flies were transferred into Eosin Y dye solution (0.5% (W/V) and 0.1% Triton X-100) and incubated at 55 °C (Wang *et al.*, 2016). After 35 min of staining, stained flies were washed several times with distilled water and the wings were dissected and mounted with glycerin. Images were captured by using a MV PLAPO 1X Microscope.

### Wing interference patterns (WIPs)

Wings were dissected from 3d-old adults and dried at room temperature. The dry wings were horizontally fixed on a black background with glass cover (Shevtsova *et al.*, 2011). Images were taken with a CCD camera (DFC450C, Leica, Germany) using the imaging software Multifocus Imaging System using white light.

### Immunostaining of imaginal disc

Wing discs were dissected from 3rd-instar larvae and fixed in 4% formaldehyde in PBT (PBS, BSA, and Triton X-100) for 40 min. The fixed tissues were rinsed

four times in PBT and then washed for 1 h in PBT. Wing imaginal discs were incubated overnight with the primary antibody at 4 °C. Before adding the second antibody, the dissected tissue was rinsed four times in PBT and then washed for 1 h in PBT. Discs were incubated with the secondary antibody at room temperature for 1 h. Rabbit anti-Caspase-3 was purchased from Cell Signaling (1 : 100). The secondary goat anti-rabbit DyLight 549 antibody was purchased from Agrisera (1 : 200). Images were collected using a fluorescence microscope EVOS fl.

### Ultrastructure analysis

Transmission electron microscopy (TEM) was used to observe the ultrastructure of the wing cuticle (Liu *et al.*, 2009). The wings were removed from 3-d-old flies and fixed with 3% glutaraldehyde in a phosphate buffer at 4 °C for 48 h. The wings were rinsed 3 times using phosphate buffer followed by postfixation in 1% osmium tetroxide for several hours at 4 °C. The wings were washed twice, each for 10 min, and dehydrated in a series of ascending concentration of acetone (50%, 70%, 80%, 90%, and 100%). The sample was embedded in Epon 812 for 2 h and trimmed to prepare ultrathin sections. A JEM-1200EX transmission electron microscope (TEM, JEOL, Japan) was used to observe the ultrastructure of the wing cuticle (TEM, JEOL, Japan).

### Statistical analysis

Data were analyzed by one-way (ANOVA) test and Tukey's test a *post hoc* using the SPSS software (SPSS Inc. Chicago, USA) after checking normality and
<figure><img src="image_3.png" /><figcaption>**Fig. 1** Expression of *Cht6* in the whole body and in pupal wings. (A) Expression of *Cht6* in the entire body throughout developmental stages. (B) Expression of *Cht6* in pupal and adult wings at different days as detected by RT-qPCR (pupal day 1–5, P1–P5, adult day 1, 2, A1–2). Data are shown as means ± SD of five independent biological replications. Data differences were analyzed using one-way analysis of variance (ANOVA). Different letters above bars indicate significant difference (*P* < 0.05).</figcaption></figure>

homoscedasticity using Shapiro test and Bartlet test respectively. When only two groups were analyzed, Student's tests were performed following Shapiro and Fischer tests. The significance threshold was set at *P* < 0.05.

## Results

*The expression profile of Cht6 in pupal primordia and adult wings*

To obtain the expression profile of *Cht6* in pupal wings, RT-qPCR was used to analyze the *Cht6* expression at different developmental stages. In these experiments, *Cht6* was expressed throughout all stages of pupal wing development. At the early pupal stage, the *Cht6* expression increased gradually and reached a peak at P3 (3 d after pupation). A drastic reduction of *Cht6* expression was detected at P4 and P5 (Fig. 1). Interestingly, *Cht6* was also expressed at the adult stage. The *Cht6* expression coincides with the expression of *Cht10* and *kkv*, although the expression peaks were distinct.

*Suppression of Cht6 leads to wing size reduction*

In order to explore the role of Cht6 during wing developmental, *Cht6* expression was suppressed by RNAi in a tissue-specific manner. The hairpin RNA against *Cht6* was expressed in the anterior or posterior wing using ci-Gal4 and en-Gal4, respectively. Compared to control wings, both suppression of *Cht6* in the anterior and posterior wing halves caused wing size reduction (Fig. 2). The wing size in ci>*Cht6*-RNAi flies decreased

by 36.71% in males and by 25.64% in females, while in en>*Cht6*-RNAi flies, the wing size decreased by 63.16% in males and by 55.25% in females (Fig. 2B). In the control flies, the ratio of anterior to posterior wing areas is 67.5%. Suppression of *Cht6* expression in the anterior half of the wing caused a slight reduction in the anterior/posterior ratio, whereas suppression of *Cht6* expression in the posterior half of the wing caused increase of the anterior/posterior ratio (Fig. 2C). These results suggest that reduced expression of *Cht6* affects wing size. However, suppression of *Cht6* in the posterior half of the wing had a more severe effect on wings as the posterior area of the wings was curled (Fig. 2A). To confirm the *Cht6* knockdown phenotype, another *UAS-Cht6-RNAi* line (VDRC102293) was used. Most en>*Cht6*-RNAi (V102293) animals died when raised at 30 °C. Considering that temperature might affect interference efficiency, we suppressed *Cht6* (V102293) transcript levels at 28 °C (Zhang *et al.*, 2020). There is a slight, but insignificant decrease in wing size of en>*Cht6*-RNAi (V102293) flies at 28 °C. As this phenotype was rather weak, we used the wing-specific Gal4 driver nubbin (nub)-Gal4 to suppress *Cht6* expression in the entire wing (Fig. S1). The wings of nub>*Cht6*-RNAi(V102293) flies were significantly smaller than control wings. Thus, these experiments underline that *Cht6* is needed for correct organ shape (Fig. S1).

The strong size reduction in the whole wing may be associated with increased cell death. We, therefore, detected caspase-3 in wing discs in control and in en>*Cht6*-RNAi (BS54823) larvae as a marker for apoptosis. Some caspase-3 signal was detected in wing discs of wild-type control larvae, while in the posterior
<figure><img src="image_4.png" /><figcaption>**Fig. 2** Knockdown of *Cht6* results in wing size reduction. (A) Wings of control flies are oval. The wing shape of ci>*Cht6-RNAi* appears to be normal, while wings of en>*Cht6*-RNAi flies shows a slightly crinkled phenotype. Scale bar: 0.5 mm. (B) Comparison of wing size in *Cht6*-knockdown and control flies. Ten wings size of each genotype were measured. The wing size of ci>* Cht6-RNAi* flies is reduced by 36.71% in males and by 25.64% in females, while the wing size of en>*Cht6*-RNAi flies is reduced by 63.16% in males and by 55.25% in females. (C) The anterior/posterior wing ratio in *Cht6*-knockdown and control flies. In ci>*Cht6*-RNAi flies, we observe a slight reduction of the anterior/ posterior ratio, while in en>*Cht6*-RNAi flies, the anterior/posterior ratio increases by 58.4%. (D) Caspase-3 (red) marks a mild apoptosis in wing disc of wild-type larvae and increased apoptosis in posterior compartment of wing disc of en>* Cht6-RNAi* larvae. en>*brk* larvae were used as positive controls as excess *brk* expression has been shown to trigger apoptosis. DAPI (blue) stains the nuclei. Scale bar = 100 µm.</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>**Fig. 3** Knockdown of *Cht6* results in abdomen size reduction. (A) en>**Cht6**-RNAi flies (right) showed a smaller chubby abdomen compared to control (left). P+C (right panel) and A (left panel) show the selections used to quantify Perimeter, Circularity and urogenital Angle shown in B. (B) Quantification of abdomen Perimeters, Circularity and urogenital Angle in *Cht6*-knockdown flies (en>**Cht6**-RNAi in green) and control flies (en> + in orange). Perimeter (shown in en>**Cht6**-RNAi female in A by a black line) is expressed in mm. Circularity close to one indicates a perfect circle was computed from the same black line. Urogenital zone angle informs about last segment curvature. Data were analyzed using Student's tests. \*P < 0.05, \*\*P < 0.01, \*\*\*P < 0.001. *n* = 10 flies for each group.</figcaption></figure>

half of wing discs of positive control flies overexpressing the apoptosis-related gene *brinker* (*brk*) (Martin *et al.*, 2004), the caspase-3 signal was visibly enhanced. Compared to the controls, the caspase-3 signal was slightly increased in the posterior compartment of wing discs of en> *Cht6-RNAi* larvae (Fig. 2D). These results indicate that suppression of *Cht6* may induce apoptosis.

Interestingly, *engrailed* (*en*)>Cht6-RNAi flies showed abnormal abdominal shape (Fig 3). Of note, *en* has been shown to be expressed in the posterior half of wings and in the posterior part of each abdominal segment (Struhl *et al.*, 1997). We therefore sought to quantify these shape defects. Similarly, to our observations in wings, both *en*>Cht6-RNAi females and males exhibited a smaller abdomen compared to their wild-type respective controls (Fig. 3A). As revealed by the measurement of abdomen circularity and the urogenital zone angle, the abdomen curvature was higher in both *en*>Cht6-RNAi males and females (Fig. 3B). Thus, Cht6 is necessary for the correct shape of the abdomen.

*Suppression of Cht6 enhances wing cuticle permeability and disrupts wing interference patterns (WIPs)*

To investigate whether Cht6 is involved in wing cuticle permeability, an Eosin Y dye penetration assay was performed (Wang *et al.*, 2016). In control flies, two areas in the posterior half of the wing blade were penetrated by Eosin Y at 55 °C (Fig. 4A). By contrast, the anterior area of wing blades of 50% of ci>Cht6-RNAi flies were stained with Eosin Y (Fig. 4B). Likewise, suppression of *Cht6* expression in the posterior half of the wing of en>Cht6-RNAi flies caused Eosin Y penetration in the posterior area of all wings (Fig. 4C). These results indicate that reduced expression of *Cht6* affects wing cuticle permeability.

In order to characterize the influence of Cht6 on wing cuticle properties, the wing interference patterns, a stable structural color pattern, was investigated in control and *Cht6* knocked-down flies (Fig. 4D). On a black background, whole wings of control flies exhibited a stereotypic color pattern, whereas wings of ci>Cht6-RNAi and
<figure><img src="image_6.png" /><figcaption>**Fig. 4** Depletion of *Cht6* affects wing permeability and interference color. (A) In control flies, the two areas in the posterior half of the wing are penetrated by Eosin Y at 55 °C. (B) In 50% of ci>*Cht6-RNAi* flies, the anterior region of the wings is penetrated by Eosin Y. (C) In all en > *Cht6-RNAi* flies, the posterior region of wings is penetrated by Eosin Y. Depletion of *Cht6* causes excessive Eosin Y penetration in the posterior region of wings. (D) The control wings exhibited a vivid structural color pattern under black background. (E, F) Depletion of Cht6 disrupts this pattern in the anterior or the posterior region of wings by using the ci-Gal4 or en-Gal4 drivers in RNAi experiments, respectively. Scale bar: 0.5 mm</figcaption></figure>

en>*Cht6-RNAi* flies showed a pale color in the anterior or posterior half of the wing, respectively (Fig. 4E, F). In summary, reduced expression of *Cht6* affects wing interference patterns, implying that wing surface microstructures are disrupted in *Cht6* knockdown flies.

*Cht6 depletion affects wing cuticle thickening*

In order to investigate the effects of *Cht6* suppression on cuticle ultrastructure, we performed ultrastructural analyses with wings of ci>*Cht6-RNAi* and en>*Cht6-RNAi* lines. The vein cuticle of wild-type control flies consisted of a thick laminar procuticle and the above thin epicuticle. The procuticle of anterior wing halves was thinner in ci>*Cht6-RNAi* flies than in control flies. Consistently, the procuticle of posterior wing was thinner in en>*Cht6-RNAi* flies than in control flies (Fig. 5A). The thickness was reduced by 33.87% in ci > *Cht6-RNAi* and 49.5% in en>*Cht6-RNAi* flies compared with the control (Fig. 5B). We further observed and analyzed the chitin-laminae structure. There were fewer laminae in the wings of Cht6 knockdown flies than in the wings of control flies, indicating that the cuticle thickness reduction caused by Cht6 depletion is, at least partly, due to reduced laminae. However, the laminar organization of the procuticle was unaffected in flies with suppressed *Cht6* expression in the wing (ci>*Cht6-RNAi*

and en>*Cht6-RNAi*). These results indicate that *Cht6* is needed for wing cuticle thickening.

*Cht6 depletion affects expression of chitin biosynthesis genes*

Chitin is one of main components of the procuticle. The changes in chitin biosynthesis or assembly processes may disturb the chitin-matrix structure causing procuticle defects. To further understand the *Cht6* depletion phenotypes, we analyzed the expression of genes coding for enzymes of the chitin biosynthesis pathway in *Cht6* suppressed flies. After *Cht6* depletion, the expression of the gene coding for trehalase is reduced, while the expression of the gene coding for Glutamine fructose-6-phosphate aminotransferase 1 was increased. The gene coding for the last two enzymes of the pathway of chitin synthesis *nesthocker* (*nst*), *mummy* (*mmy*) (Schimmelpfeng *et al.*, 2006; Tonning *et al.*, 2006), is unchanged in *Cht6* suppressed flies (Fig. 6A). Taken together, Cht6 activity seems to be sensed by a genetic program regulating the expression of genes of the chitin biosynthesis or assembly pathway and cuticle thickening (Fig. 6B).

**Discussion**

In insects, 18 glycosyl-hydrolases constitute a conserved multigene family comprising a dozen of chitinases (Chts)
<figure><img src="image_7.png" /><figcaption>**Fig. 5** Ultrastructural analysis of the wing cuticle in control and *Cht6* suppressed flies. (A) The ultrastructure of the vein cuticle in adult wings. The anterior (L2 vein) or posterior (L4 vein) cuticle of control wings has a laminate procuticle and the above epicuticle (the outermost envelope is not discernable in these sections). The black dotted lines delimit laminae. The procuticle of anterior or posterior wing halves is thinner in ci>*Cht6-RNAi* flies or in en>*Cht6-RNAi* flies than in control flies. (B) The thickness is reduced by 33.87% in ci>*Cht6-RNAi* and 49.5% in en>*Cht6-RNAi* flies. Scale bar: 500 nm</figcaption></figure>

and several Imaginal-disc-growth-factors (Idgfs) that seem to have lost their hydrolase activity (Zhu *et al.*, 2008; Pesch *et al.*, 2016). Usually, chitinases contribute to chitin degradation and cuticle molting at stage transitions. Recent studies reported that some chitinases were involved in cuticle formation and chitin organization (Pesch *et al.*, 2017; Noh *et al.*, 2018; Behr & Riedel, 2020; Dong *et al.*, 2020). Pesch *et al.* (2016) reported phenotypes of chitinase depletion based on RNAi in *D. melanogaster*. Individual suppression of *Cht2*, *5*, *6*, and 7 transcript levels in the epidermis caused pupal lethality. It has been shown that tracheal-specific knockdown of *Cht2*, *Cht5*, or *Cht6* genes caused abnormal tracheal tubes in embryos (Behr & Riedel, 2020). Interestingly, *Cht5* and *Cht6* knockdown flies showed molting defects and problems with wing extension (Pesch *et al.*, 2016).

*Cht6 is needed for procuticle formation in the wing*

*Cht6* as a member of chitinase group VI is predicted to possess one GH18 domain and two chitin-binding domains. Putative *Cht6* orthologues are conserved in

several insect species (Zhang *et al.*, 2011). For example, the putative *Cht6* orthologue in the migratory locust *Lo-custa migratoria* and the noninsect carmine spider mite *Tetranychus cinnabarinus* has a single GH18 domain and two chitin-binding domains like the *D. melanogaster* counterpart (Fig. S2). The presence of relatively conserved residues in the GH18 domain implies that *Cht6* exerts its function as a chitinase. In this study, we found that the *Cht6* gene is highly expressed during early pupal stage peaking at day 3 and its expression pattern coincides with the expression of *kkv* (chitin synthase coding gene). This is consistent with recent large-scale expression profiling data on genes expressed in the wing primordia (Sobala & Adler, 2016). This coincidence suggests that *Cht6* acts on newly formed chitin fibers in the wing. Besides *kkv* and *Cht6*, the chitinase coding genes *Cht2* and *Cht10* as well as *knickkopf* (*knk*) coding for a chitin-organizing protein (Li *et al.*, 2017; Dong *et al.*, 2020) are expressed in the early pupa. Our previous study showed that *Cht10* is involved in wing cuticle formation by controlling chitin content in *D. melanogaster* (Dong *et al.*, 2020). We had also shown that *Knk* is needed for chitin organization in the wing procuticle. Finally, *Cht2* that was reported to be essential for chitin organization in the *D. melanogaster*
<figure><img src="image_8.png" /><figcaption>**Fig. 6** Relative expression levels of key genes of the chitin synthesis pathway in *Cht6* suppressed flies. (A) Effect of *Cht6* knockdown on expression of genes coding for chitin synthesis detected by RT-qPCR, including trehalase (Tre), Glutamine:fructose-6-phosphate aminotransferase 1 (Gfat1), Glutamine:fructose-6-phosphate aminotransferase 2 (Gfat2), Nesthocker (Nst) and Mummy (Mmy). The *Cht6* silencing efficiency was 53.15%. The *Tre* transcript expression is significantly reduced and the *Gfat1* transcript expression is significantly increased in *Cht6* knockdown flies, while the expression of other chitin biosynthesis genes is unchanged. *rp49* was used as the reference control. Data are shown as means ± SD of four independent biological replications, asterisks indicate significant difference (*P* < 0.05). (B) A proposed model for the role of *Cht6* in cuticle development in *Drosophila*. Reduction of *Cht6* expression causes a reduced, thin procuticle. With reduced *Cht6* expression, the normal course of the chitin synthesis pathway is modified as the levels of two transcripts coding for enzymes of this pathway are changed. In particular, *Tre* coding transcripts are suppressed, while those of *Gfat1*-coding transcripts are enhanced. In the wild-type situation, we hypothesize that procuticle thickness depends on *Cht6* activity, *Cht6* indirectly enhances *Tre* expression and suppresses *Gfat1* expression (gray arrow and gray inhibition sign) in order to ensure correct procuticle thickness. This, in turn, is needed for correct organ shape.</figcaption></figure>

larva might also be needed for this process in the adult wing. Taken together, based on the *Cht6* expression profile, we speculate that *Cht6* might be implicated in wing cuticle formation together with *Kkv*, *Knk*, *Cht2*, and *Cht10*.

What might be the cellular function of Cht6? Although both *Cht6* and *Cht10* are expressed during early pupal wing development, they play opposite roles in wing cuticle formation. Unlike the *Cht6* knockdown phenotype, knockdown of *Cht10* affects chitin organization in the wing vein. Moreover, chitin content is slightly increased in *Cht10* suppressed flies, whereas the cuticle is thinner in *Cht6* knockdown flies suggesting reduction of chitin amounts. Besides *Cht6* and *Cht10*, a membrane-bound chitinase *TcCht7* with two catalytic GH18 domains has been suggested to be essential for correct chitin organization in *T. castaneum* (Noh *et al.*, 2018). *Cht2*, as described above, with a single GH18 domain has been proposed to participate in cuticle thickening in larvae of *D. melanogaster* (Pesch *et al.*, 2017). Taken together, several chitinases act in concert during chitin production and organization. *Cht6* is needed for normal chitin amounts but not for chitin organization in this process.

*Cht6 is involved in cuticle organ shape*

The wing size is reduced after *Cht6* depletion. Likewise, the shape of the abdomen is altered when *Cht6* expression is reduced. Thus, *Cht6* activity is important for normal organ and body shape. How does *Cht6* activity participate at these global processes? In a simple scenario, procuticle thickness is important in organ and tissue formation. This is in line with the finding that the cuticle-cell interaction requiring the cuticle protein Dumpy is decisive for wing expansion in *D. melanogaster* (Etournay *et al.*, 2015). Thus, a thinner procuticle in the wing and abdomen with reduced *Cht6* expression may be responsible for the observed shape changes. Reduction of the wing size may also be due to a slightly enhanced apoptosis in *Cht6*-depleted wing discs. This is consistent with the view that apoptosis is needed for wing shape determination in *D. melanogaster* (Umemori *et al.*, 2009).

*Cht6 activity is sensed by the chitin synthesis pathway*

Chitin biosynthesis is a fundamental process for cuticle formation in insects involving a series of
enzymes (Liu *et al.*, 2019). The reaction starts with the disaccharide trehalase and ends with the synthesis of UDP-N-acetylglucosamine (UDP-GlcNAc), which is the chitin monomer. This pathway requires the enzymes trehalase, hexokinase, glutamine-fructose-6-phosphate aminotransferase, glucosamine-6-phosphate N-acetyltransferase, phosphoglucosamine mutase, and UDP-N-acetylglucosamine pyrophosphorylase (UAP) (Jaworski *et al.*, 1963; Cohen, 1987; Merzendorfer & Zimoch, 2003). Interestingly, levels of transcripts coding for trehalase (Tre) and glutamine:fructose-6-phosphate aminotransferase 1 (Gfat1) were changed in response to Cht6 depletion. Based on these data, we propose that the chitin biosynthesis pathway is sensitive to the activity of Cht6 during cuticle formation. In other words, cuticular defects caused by reduction of Cht6 activity have an impact on the chitin synthesis pathway through the modulation of Gfat1 and Tre transcript levels. For a detailed comprehension of the Cht6 function, the localization and site of activity of Cht6 remains to be investigated during chitin biosynthesis and cuticle formation.

## Acknowledgments

We thank the Bloomington *Drosophila* Stock Center and Core Facility of *Drosophila* Resource and Technology and the Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences for fly stocks. This work was supported by the National Natural Science Foundation of China (Grant No. 32170505; 32170526), the NSFC-DFG (Grant No. 31761133021), Natural Science Foundation of Shanxi Province (Grant No. 20210302123473), Research Project Supported by Shanxi Scholarship Council of China (HGKY2019011), the Fund for Shanxi “1331 Project.” BM’s contribution was supported by the German Research Foundation (DFG grant MO1714/10-1).

Open Access funding enabled and organized by Projekt DEAL.

## Disclosure

All authors have seen and agree with the contents of the manuscript and there is no conflict of interest, including specific financial interest and relationships and affiliations relevant to the subject of manuscript.

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Manuscript received January 19, 2022

Final version received August 9, 2022

Accepted August 18, 2022

## Supporting Information

Additional supporting information may be found online in the Supporting Information section at the end of the article.

<caption>**Table S1** Fly stocks information.</caption>

**Fig. S1** Downregulation of *Cht6* using a different RNAi line leads to reduced wing size.

<caption>**Fig. S2** Domain composition of insect Cht6 proteins.</caption>