Introduction
Juvenile hormone (JH) is secreted by the insect corpora allata (Hang et al., Reference Hang, Li, Zhong and Cao2004), which maintains larval characteristics and the prothoracic gland, promotes ovarian maturation, and regulates insect development, metamorphosis, and reproduction (Shelby et al., Reference Shelby, Madewell and Moczek2007; Riddiford et al., Reference Riddiford, Truman, Mirth and Shen2010; Kayukawa et al., Reference Kayukawa, Minakuchi, Namiki, Togawa, Yoshiyama and Kamimura2012; Hiruma and Kaneko, Reference Hiruma and Kaneko2013; Hu et al., Reference Hu, Tian, Yang, Tang, Qiu and He2020). JH regulates the downstream response gene krüppel homolog1 (Kr-hl) through the JH receptor methoprene-tolerant (Met) gene, thus leading to corresponding physiological responses (Jin and Lin, Reference Jin and Lin2014). The regulatory mode of JH-Met-Kr-h1 is complex, and research on Kr-h1 function gene has primarily focused on its role in regulating insect growth and development, neuronal cell formation, foraging behaviour, and sexual maturation (Jin and Lin, Reference Jin and Lin2014; Gassias et al., Reference Gassias, Maria, Couzi, Demondion, Durand, Bozzolan, Aguilar and Debernard2021).
Coccinella septempunctata L. (Coleoptera: Coccinellidae) is an important predatory insect that can effectively control aphids, white flies, jassids, and small lepidoptera larvae in the field (Zhou et al., Reference Zhou, Cheng, Jin, Li and Li2017; Chatha and Naz, Reference Chatha and Naz2020; Hakeem et al., Reference Hakeem, Qayoom, Muhammad, Mithal, Ali, Ali and Yasmeen2021; Bajracharya et al., Reference Bajracharya, Budha and Baral2023). Due to climate change and the application of pesticides, field population of C. septempunctata have decreased (Sun and Wan, Reference Sun and Wan1999; Zhang et al., Reference Zhang, Chen and Li2014), and it is now necessary to artificially rear C. septempunctata. The cost of feeding of C. septempunctata with an aphid diet is high, so it is necessary to develop artificial food for ladybird; unfortunately, artificial diets are generally inferior to aphid diets with regard to weight gain, pupation rates, and eclosion rates as compared with aphid diets (Sarwar and Saqib, Reference Sarwar and Saqib2010; Yazdani and Zarabi, Reference Yazdani and Zarabi2011; Cheng et al., Reference Cheng, Yu, Zhou and Li2022, Reference Cheng, Zhou, Ran and Li2023). A diet of artificial food impacts the normal secretory activities of C. septempunctata neurosecretory cells and the pharyngeal lateral body. Low levels of endogenous hormones inhibit the synthesis and release of vitellogenin protein, thus affecting ovary development and oocyte maturation, resulting in more female individuals entering reproductive diapause (Fu and Chen, Reference Fu and Chen1984). The impact on gonadal development and semen proteins has not been reported yet.
In the early stages of optimising the ladybird artificial diet, we observed that the addition of JH to the diet resulted in adult egg production and hatching rates that were respectively four- and three-fold higher than diets lacking JH. However, amendment of the diet with JH significantly prolonged the duration of larval development and reduced the pupation rate (Cheng et al., Reference Cheng, Yu, Zhou and Li2022, Reference Cheng, Zhou, Ran and Li2023). The underlying mechanism by which JH regulates the expression of Met and Kr-hl genes in C. septempunctata remains unclear.
Our group recently cloned and monitored spatiotemporal expression of the C. septempunctata Met (CsMet) gene; this was expressed in different developmental stages and was most highly expressed in 3rd instar larvae (unpublished). CsMet expression levels gradually increased in 20- to 30-d-old female adults. With the exception of 20-d-old female adults, the expression level of CsMet was higher in females as compared to male adults. CsMet expression levels in male and female adult adipose tissue were significantly higher than expression in the head, thorax, and reproductive systems.
In this study, we evaluate whether the expression of Kr-hl in C. septempunctata is similar to CsMet. The transcriptome database of C. septempunctata (Cheng et al., Reference Cheng, Zhi, Li, Wang, Zhou and Jin2020) was used to clone the cDNA sequence of Kr-h1, and its expression was characterised in different developmental stages and tissues to evaluate potential improvement of the artificial ladybird diet.
Materials and methods
Insects
C. septempunctata were collected from Jinzhu Town, Huaxi District, Guiyang City, Guizhou Province. Ladybugs were reared indoors on Aphis craccivora Koch (Hemiptera: Aphididae) for over 20 generations at the Institute of Plant Protection, Guizhou Academy of Agricultural Sciences. Experiments were performed in environmental chambers at 25 ± 1°C, 70 ± 5% RH with a 16: 8 h light : dark photoperiod.
RNA extraction and cDNA synthesis
Ten-day-old female adults were grinded to a powder in liquid nitrogen and then transferred to 1.5 mL RNase-free microcentrifuge tubes. Trizol (1 ml) was added, and the mixture was incubated for 5 min at room temperature; trichloromethane (200 μl) was then added, gently mixed, incubated at room temperature for 3 min, and then centrifuged at 12,000 × g for 15 min at 4°C. A 600 μl volume of the supernatant was transferred into a new microcentrifuge tube, 500 μl of 100% isopropanol was added, and the mixture was incubated at room temperature for 10 min. The suspension was then centrifuged at 12,000 × g at 4°C for 10 min; the supernatant was then removed and 75% ethanol was added, gently inverted eight times, and centrifuged at 7500 × g for 5 min at 4°C. Ethanol was then removed and RNA pellets were allowed to air dry for 5–10 min. RNA (1 μg) was used as a template, and the first strand of cDNA was synthesised using the RevertAid First Strand cDNA Synthesis Kit (Fermentas Co.) and stored at −80°C for future use.
Cloning and sequencing of Kr-h1
Based on the transcriptome database of C. septempunctata constructed in our laboratory, the ORF encoding Kr-h1 was selected for cloning. cDNA from 10-d-old female adults was used as a template; primers were designed using ClustalX and Primer Premier 5.0 and synthesised by Sangon Biotech Co. (Shanghai) (Table 1). Using the 3’ and 5’ first-strand cDNA as templates, full-length amplification of CsKr-h1 was performed using the SMARTer RACE cDNA Amplification Kit (Clontech Co.) and the 5’ RACE System for Rapid Amplification of cDNA Ends (Invitrogen Co.). PCR products were purified and recovered by 1% agarose gel electrophoresis, and then connected to pmd-18t carrier, transformed DH5a competent cells, and sequenced by Sangon Biotech Co. The ORF Finder program (https://www.ncbi.nlm. nih.gov/orffinder/) was used to identify the coding region for Kr-h1, and the ExPASy program (https://web.expasy.org/compute_pi/) was used to identify the isoelectric point and molecular weight of the Kr-h1-encoded protein. SMART 8 software (https://prosite.expasy.org/) was used to identify the structural domain of the encoded protein.
Table 1. Primers used in this study
BLAST and phylogenetic analyses of Kr-hl
The NCBI BLAST program (https://blast.ncbi.nlm.nih.gov/Blast) was used to analyse similarity between C. septempunctata Kr-hl (CsKr-hl) and orthologs in other insect species. To further investigate evolutionary relationships, deduced amino acids of insect Kr-hl sequences were downloaded from NCBI, and a phylogenetic tree was constructed using the neighbour-joining method by MEGA 6 software.
Spatio-temporal expression of CsKr-hl
Samples of different developmental stages (e.g., 2-d-old eggs, 1st −4th instar larvae, pupae, and 1-, 5-, 10-, 15-, 20-, 25- and 30-d-old female and male adults) were collected along with heads, chest, adipose, and reproductive systems of 10-d-old female and male adults. A single replicate consisted of the following: 60 eggs; 30, 1st instar larvae; 15, 2nd instar larvae; 10, 3rd instar larvae; four 4th instar larvae; four pupae; and four adults. The head, chest, adipose, and reproductive system were dissected from 6 10-d-old female and male adults, respectively, and considered as one replicate (Table 2). Each sample was replicated three times. Collected samples were immediately frozen in liquid nitrogen and stored at −80℃ for future use.
Table 2. Sample size for the spatio-temporal expression of CsKr-hl
Total RNA was extracted from each sample according to the instructions included with the Eastep® Super Total RNA Isolation Kit (Promega Co.). The IScript cDNA Synthesis Kit (Bio-Rad Co.) was used to reverse transcribe and synthesise cDNA, and samples were stored at −20℃ for future use. The Kr-hl-specific primers, Kr-hl-QF/ Kr-hl-QR, and Actin-F/Actin-R (internal standard, Liu et al., Reference Liu, Wang, Wang, Gao, Zhang, Li, Zang and Zhang2019) were used to measure expression in different developmental stages and tissues (Table 1). qPCR was conducted in a 20 μl volume containing the following: cDNA template, 2 μl; upstream and downstream primers, 2 μl each; Sso Advanced Universal SYBR Green Supermix, 10 μl; and ddH2O, 4 μl. The qPCR reaction conditions included pre-denaturation at 95℃ for 2 min; 95℃ denaturation for 5 s, and 60℃ annealing for 30 s for a total of 39 cycles. The relative expression level of Kr-hl was calculated using the 2−ΔΔCt method (Pfaffl, Reference Pfaffl2001).
Statistical analysis
One-way ANOVA was performed on the experimental data, and the multiple comparison LSD method was used to determine significance with DPS 17.0 software (Tang and Zhang, Reference Tang and Zhang2013).
Results
Cloning and sequence analysis of C. septempunctata Kr-hl
Based on our transcriptome data of C. septempunctata, cDNA from female adults was used as a template to clone CsKr-hl (GenBank accession no. OR183710). Sequence analysis showed that CsKr-hl cDNA was 2694 bp and encoded a 1338 bp ORF consisting of 445 predicted amino acids and 5’ and 3’ noncoding regions of 126 and 1230 bp, respectively (fig. 1). The molecular weight of the predicted CsKr-hl protein was 51.01kD, and its isoelectric point was 8.50. There were eight conserved domains, namely ZINC_FINGER_C2H2_2, Zinc finger C2H2 type domain profile, ZINC_FINGER_C2H2_1, and Zinc finger C2H2 type domain signature repeat; these were located at amino acid residues 211–238, 239–266, 267–294, 295–322, and 213–233, 241–261, 269–289, 297–317, respectively.
Figure 1. Nucelotide and deduced amino acid sequence of CsKr-hl.
BLAST comparison and phylogenetic analysis of CsKr-hl
BLAST analysis of CsKr-hl in NCBI revealed 83.29% similarity with HmKr-hl (Harmonia axyridis), and then 63.09%, 65.07%, and 63.12% similarity with AtKr-hl (Aethina tumida), DvKr-hl (Diabrotica virgifera), and AgKr-hl (Anthonomus grandis), respectively (fig. 2). Phylogenetic analysis indicated that CsKr-hl and AtKr-hl clustered together in one branch (fig. 3).
Figure 2. Multiple sequence alignments of CsKr-hl from Coccinella septempunctata and orthologs in other insect species. Abbreviations: Cskr-hl, Coccinella septempunctata; HaKr-hl, Harmonia axyridis; AtKr-hl, Aethina tumida; DvKr-h, Diabrotica vir gifera; and AgKr-hl, Anthonomus grandis.
Figure 3. Phylogenetic tree of CsKr-hl from Coccinella septempunctata and orthologs in other insect species.
Spatiotemporal expression analysis of CsKr-hl
There were significant differences in CsKr-hl expression levels in the egg to pupal stages at different developmental stages (F = 12.06, df = 5,10, P = 0.0006) (fig. 4). Expression gradually increased in 1st to 3rd instar larvae, decreased in 4th instar larvae, and increased significantly in the pupal stage. The relative expression level of CsKr-hl in 1st instar larvae was set to 1. From egg to pupal stage, the expression level of 4th instar larvae was the lowest at 0.58 times the number of 1st instar larvae, while the expression level of pupa was the highest at 7.48 times the number of 1st instar larvae.
Figure 4. Relative expression levels of CsKr-hl in different developmental stages of Coccinella septempunctata. Columns labelled with different letters indicate significance at P < 0.05 using the LSD test.
There were obvious differences in CsKr-hl expression levels when comparing female and male adults at different developmental stages (fig. 5). Expression levels in 1 to 15-d-old female and male adults were relatively low, and only CsKr-hl expression in1-d-old female adults was higher than male adults. The relative expression level of CsKr-hl in 1-d-old male adults was set to 1. The expression levels of CsKr-hl in 1, 5, 10, 15, and 20-d-old female adults were higher at 3.26, 1.28, 1.05, 1.35, and 2.00 times the number of 1-d-old male adults, respectively. The expression levels of CsKr-hl in 1, 5, 10, and 15-d-old male adults were 1, 1.36, 0.91, and 1.31 times the number of 1-d-old male adults, respectively. Expression levels gradually increased in 20-d-old adults and was significantly higher in males vs females at ages 20 d (F = 35.79, df = 1,2, P = 0.0268) and 25 d (F = 54.38, df = 1,2, P = 0.0179). The expression level of CsKr-hl in 30-d-old female adults was significantly higher than in males (F = 37.36, df = 1,2, P = 0.0257) (fig. 6).
Figure 5. Relative expression levels of CsKr-hl in different ages of Coccinella septempunctata adults. Data are means ± SD. Columns labelled with different letters indicate significance at P < 0.05 using the LSD test.
Figure 6. Relative expression levels of CsKr-h1 in different tissues of Coccinella septempunctata adults. Panels: (a) female adults; (b) male adults; and (c) female and male adults. Data are mean ± SD. Columns labelled with different letters indicate significance at P < 0.05 using the LSD test.
The relative expression level of CsKr-hl in thorax was set to 1. The expression levels of CsKr-hl gene in female adults in head, adipose, and ovary were 25.96, 6.69, and 6.57 times the number of thorax, respectively. The expression levels of CsKr-hl gene in male adults in head, adipose, and ovary were 19.79, 2.86, and 2.75 times the number of thorax, respectively. The expression level of CsKr-hl in females and males was highest in the head. The relative expression level of Cskr-hl in the head (F = 22.33, df = 1,2, P = 0.0420), adipose (F = 2226.19, df = 1,2, P = 0.0004), and reproductive system (F = 75.52, df = 1,2, P = 0.0130) of females was significantly higher than males; however, there was no significant difference in Cskr-hl expression in the thorax of the two sexes (F = 0.0369, df = 1,2, P = 0.8655).
Discussion
JH is secreted by the corpora allata and forms a complex with carrier protein and ultimately reaches the nucleus through blood circulation. JH then binds specifically to Met or related complexes to convert hormonal signals and initiate Kr-h1 transcription, which affects growth and development and causes morphological changes in insects (Jindra et al., Reference Jindra, Palli and Riddiford2013). Kr-h1 protein is a transcription factor containing C2H2 zinc fingers (Jin and Lin, Reference Jin and Lin2014). CsKr-h1 has eight zinc fingers, and phylogenetic analysis indicated that CsKr-h1 is closely related to Kr-h1 in Aethina tumida, which is also a member of the Coleoptera, which indicates a high level of evolutionary conservatism among members of the Coleoptera.
In this study, qPCR revealed that CsKr-h1 is expressed in various developmental stages of C. septempunctata. In the larval stage, the expression of CsKr-h1 was consistent with CsMet, and expression levels in 3rd instar larvae were highest; this may be caused by the rapid growth rate of 3rd instar larvae and the need for larger quantities of JH. CsKr-h1 expression rapidly decreased as larvae entered the 4th instar stage. Furthermore, Kr-h1 is regarded as an early-stage inducible gene that is responsible for the repression of metamorphosis (Kayukawa et al., Reference Kayukawa, Nagamine, Ito, Nishita, Nishita, Ishikawa and Shinoda2016). The 4th instar larvae of ladybirds metamorphose into pupal stage, and CsKr-h1 may be reduced by regulation. When C. septempunctata entered the pupal stage, CsKr-h1 expression levels were significantly upregulated, which prepares the insect for reproduction in the adult stage. Addition of JH to the artificial diet of the C. septempunctata prolonged the larval lifespan and decreased pupation rates (Cheng et al., Reference Cheng, Yu, Zhou and Li2022, Reference Cheng, Zhou, Ran and Li2023). Collectively, our results indicate that JH regulates insect metamorphosis via CsKr-h1; however, further investigation is needed to determine whether changes in JH levels impact the transcription of CsKr-h1.
The expression of CsKr-h1 in female adults of C. septempunctata was lower from 1 to 15 days than 20 to 30 days after eclosion. During the peak period of egg deposition (20–30 days after eclosion), the expression levels of CsKr-h1 gradually increased; this suggests that CsKr-h1 is involved in the regulation of reproduction. Furthermore, CsKr-h1 expression levels in males were significantly higher at 20–30 days post-emergence as compared to 1–15 days. Gassias et al. (Reference Gassias, Maria, Couzi, Demondion, Durand, Bozzolan, Aguilar and Debernard2021) highlighted the involvement of the JH-Met-Kr-h1 signalling pathway in the development and secretory activity of male accessory glands in Agrotis ipsilon. We speculate that Kr-h1 in C. septempunctata was also involved in the development and secretory activity of male accessory glands, and its peak secretion was consistent with the peak period of egg deposition by female adults, with both occurring 20–30 days after eclosion. Injection of JH-II into newly emerged A. ipsilon adult males induced the transcription of Met1, Met2, and Kr-h1 associated to an increase in the length and protein content of the male accessory glands (Gassias et al., Reference Gassias, Maria, Couzi, Demondion, Durand, Bozzolan, Aguilar and Debernard2021). In future studies, we will utilise gene silencing to verify whether the CsKr-h1 is involved in the regulation of male accessory gland development and secretion activity. Analysis of tissue expression patterns showed that CsKr-h1 was most highly expressed in adult ladybug heads, whereas Kr-h1 was higher expressed in larval heads of Dendroctonus armandi (Sun et al., Reference Sun, Fu, Liu, Wang and Chen2022). In Drosophila photodetector and Agrotis ipsilon, Kr-h1 promotes neuronal cell formation in head (Duportets et al., Reference Duportets, Bozzolan, Abrieux, Maria, Gadenne and Debernard2012; Fichelson et al., Reference Fichelson, Brigui and Pichaud2012), which is also a possible function of CsKr-h1 in C. septempunctata. CsKr-h1 was highly expressed not only in the heads but also in the adipose tissue and reproductive system, while Kr-h1 of H. axisis was highly expressed in wings, legs, and adipose tissue (Han et al., Reference Han, Feng, Han, Chen, Wang and He2022).
In summary, we cloned the JH response gene CsKr-h1 from C. septempunctata and analysed its transcription, thus providing a foundation for clarifying the role of JH in regulating reproductive development in ladybird beetles. In the future, JH will be added to artificial diets and CsKr-h1 transcription will be assessed. Gene silencing will be used to analyse development of the reproductive system, egg production, and hatchability of C. septempunctata, and the regulatory effects of CsKr-h1 on the growth and reproduction of ladybird beetles will be explored.
Acknowledgements
This project was funded by the National Natural Science Foundation of China (grant no. 31960562).
