Molecular Cloning of the Vitellogenin Gene and the Effects of Vitellogenin Protein Expression on the Physiology of Harmonia axyridis (Coleoptera: Coccinellidae)

Vitellogenin (Vg), the main egg storage protein precursor, plays an integral role in many oviparous animals, including Harmonia axyridis, an important agent for the biological control of many insect pests. In this study, the full-length Vg gene of was cloned. The open reading frame (ORF) of H. axyridis Vg cDNA is 5,403 bp in length and encodes 1,800 amino acids, with a predicted molecular mass of 211.88 KDa (accession number in NCBI: KX442718). Recombinant protein (18 kDa) expressed by the cloned Vg gene was characterized, and the effects of the expression of this protein on the physiology of H. axyridis were investigated. We found that Vg fragment significantly increased the egg production of H. axyridis. Furthermore, we also found that the activities of trypsin and lipase in H. axyridis were significantly higher in the groups treated with Vg fragment compared with those of the controls. The data from this study also reveals that Vg expression has significant effects on the physiology of H. axyridis and leads to increased egg production in these insects. These results may have future implications for increasing the reproduction rates of beneficial insects.

The aims of this study were to clone the full-length Vg gene of H. axyridis and investigate its effects on H. axyridis reproduction (egg production and hatching) and biochemistry (general lipase and trypsin activities). Through a better understanding of the effects of Vg on the physiology of H. axyridis, we may develop improved techniques to increase the population of this important insect 22 .

Results
Molecular cloning and analysis of cDNA sequencing. The full-length H. axyridis Vg cDNA was cloned using RT-PCR. This gene contains a 5,403 bp ORF that encodes for 1,800 amino acids (accession number in NCBI: KX442718). According to the online SignalP 4.0 software used for sequence analysis, the Vg protein contains a signal peptide before amino acid 17 with a signal peptide cleavage site between amino acids 17 and 18. The theoretical pI is 4.71, and the molecular weight is 211.88 KDa.
A Vg-N domain in the Vg protein (amino acids sites: 38-753) was found using NCBI BLAST to analyze the amino acid sequence in the middle region of the Vg protein. A DUF1934 domain (amino acids sites: 793-1077) and a Willerbrand factor type D (VWD) domain were found in the C-terminus (amino acids sites: 1465-1651), and these are specific to the Vg structure 23 .

Comparison of the H. axyridis Vg gene with the Vg genes of other insects. H. axyridis Vg was
compared with the Vg of other insects using BLASTX. Through BLAST alignment and phylogenetic tree analysis, we found that the H. axyridis Vg gene and its amino acid sequence shares varying levels of homology with the following insects: Tribolium castaneum (38% homology), Rhynchophorus ferrugineus (34% homology), Bombus hypocrite (28% homology) (Fig. 1). H. axyridis Vg is the most homologous with the Vg of T. castaneum.
Expression and purification of Vg fragment. The recombinant plasmid pET-28-VWD was transferred into E. coli, and its expression was induced with 0.5 M IPTG. The resultant recombinant protein was purified and examined on SDS-PAGE (Fig. 2). The level of protein expression increased with a longer induction time.

The effects of the Vg fragment on H. axyridis reproduction. The effects of the 18 KDa recombinant
Vg fragment on the development and reproduction of H. axyridis were evaluated. There were no significant effects on pre-oviposition time (the period from the emergency to the first egg-laying) (F = 0.876; df = 3, 15; P = 0.481) (Fig. 3) of H. axyridis. However, our results indicate that the total number of eggs in the treatment groups were significantly higher (F = 9.274; df = 3, 15; P < 0.05) than those in the control groups (Fig. 4). During the one month period, there were a total of 119 and 121 eggs produced from the groups treated with 60 μg/mL and 30 μg/ mL of Vg fragment, respectively, and 69 and 70 eggs from the control groups treated with 60 μg/mL and 30 μg/ mL of BSA, respectively. Similarly, the mean number eggs of 26.75 and 28 per female for treated groups were significant higher than that of the blank control (F = 16.301; df = 2, 11; P = 0.044) (Fig. 5). The groups treated with 60 μg/mL and 30 μg/mL of Vg fragment were 2.14-and 2.24-fold higher than that of the control (blank control and no adding Vg fragment). In addition, our data showed that the mean egg hatching rate (78%) in the treatment group (adding 30 μg/mL of Vg fragment) was higher than that (51%) of the control.   Vg mRNA expression in H. axyridis. qRT-PCR was used to probe Vg gene expression during the different stages of the H. axyridis life cycle. The relative mRNA expression levels of the Vg gene showed markedly significant differences between the treatment groups and the control groups collected on the days 9 (F = 156.066; df = 3, 11; P < 0.001), 18 (F = 131.693; df = 3, 11; P < 0.001), 26 (F = 257.967; df = 3, 11; P < 0.001) and 32 (F = 376.706; df = 3, 11; P < 0.001).The Vg mRNA expression levels in insects treated with 60 μg/mL Vg protein were 27-, 51-, 6-, and 2.5-fold higher than those of control groups on days 9, 18, 26, and 32, respectively. Similarly, the Vg mRNA expression levels in insects treated with 30 μg/mL Vg protein were 51-, 160-, 6-, and 1.3-fold higher than those of control groups on days 9, 18, 26, and 32, respectively (Fig. 6).

Discussion
In recent years, the Vg gene of many insects has been cloned at different stages of growth and ovarian development 24 . Several studies have reported that the C-terminal and the VWD domain of Vg are related to the vitelline coat, which participates in fertilization as the binding partner of sperm proteases 25 . In this study, we cloned the complete cDNA of the H. axyridis Vg gene. Our primary structural analysis shows that the Vg-N, VWD, and DUF1943 domains are highly conserved in the Vg genes of oviparous animals 26,27 . Through our analysis using the NCBI Conserved Domain Search, we found three functional domains in the H. axyridis Vg gene. The VWD  Adults sustained on 60 μg/mL Vg, 30 μg/mL Vg, 60 μg/mL BSA and 30 μg/mL BSA (mean ± SE). Different letters above the bars indicate highly significant differences (least significant difference test, P < 0.0001).
is a conserved domain and existed in the all known Vg protein sequences in insects. Liang et al. (2015) is also expressed VWD domain with Vg gene of Geocoris pallidipennis 28 . The Vg gene plays an important role in embryonic development and serves as the main source of nutrients for oocyte growth and development 29 . Zeng et al. (1997) found that the expression of juvenile hormone was related to Vg production in insects 30 . This hormone has been reported to play an important role in the regulation of insect reproduction 31,32 . The regulation of Vg expression by hormones and social behavior is also found in honeybees 33,34 . However, the data from this study showed that the Vg gene expression can be affected by adding the VWD in the artificial diet to feed insects.
Niijima 35 found that an artificial chemical diet sustains adult H. axyridis animals, however the diet did not assist in egg production. The major nutritional composition of artificial diet used in this study was analyzed and the soluble protein, sugar and lipid were about 10.9%, 1.95%, 1.72% respectively. There were no significant differences in total protein contents of artificial diets with different treatments (Supplementary information Fig. 1). Our results indicate that H. axyridis adults sustained on this artificial diet with the Vg fragment produced significantly more eggs than adults fed artificial diets with BSA. Furthermore, our addional experiment also show H. axyridis adults sustained on an artificial diet with the recombinant Vg fragment produced significantly more eggs than the control with no aditional protein. In other words, the recombinant Vg fragment promotes H. axyridis reproduction.
The Vg fragment, VWD, stimulates the egg production may due to the increase in egg related gene expression and addition of nutrients for egg development. Our hypothesis is that the Vg fragment stimulates the egg production may due to the increase in egg related gene expression in H. axyridis and additional nutrients for egg development. The qRT-PCR analysis of Vg gene expression levels during the different developmental stages of H. axyridis support this hypothesis. Our data show that Vg gene expression is increased upon treatment with the feeding of VWD. Vg gene expression in female adults increased by day 9 (Fig. 6 and Supplementary information Fig. 2) after emergence, and it reached a maximum level on day 18, at which point it decreased gradually until day 32 after emergence. This trend is consistent with egg production, and the number of eggs laid by H. axyridis female adults was higher for animals in the treatment groups compared with those of the control groups. The Vg protein can be detected 1 day after Vg mRNA expression begins [36][37][38][39][40] and is a main nutritional source stored in ovary. As such, high Vg gene expression promoted by the VWD leads to increased protein levels of Vg in the ovaries. Therefore to promote the Vg expression will help egg production. Moreover, we have calculated and compared the percentage of each of the 20 amino-acids in Vg, the VWD fragment and BSA (Supplementary information Table 1). The results indicate that the percentage of amino-acids, Gly, Thr, Tyr and Val were higher in VWD fragment were much higher compared with those of BSA. This may be the reason of the increase of egg production in the VWD treatment, the treatment may provide more nutrients than the BSA treatment for egg development and production.
In addition, the amount of egg production is closely related to the nutrients for egg development. Many researchers have showed that Vg is one of important source of nutrition for embryo development (Tufail and Takeda) 24 , and Vg level is related to the growth and development of oocytes and egg production (Zeng et al., 1997 41 ;2000 29 ). Like all oviparous animals, insects provision their eggs with proteins, lipids, carbohydrates, and other resources for the sustenance of the developing embryo (Sappington et al. 42 ). The vitelloenin promotes the transport of carbohydrates, lipids and hormones, these nutrients can play a certain role in insect vitellogenin 37 . The treatment (feeding of VWD) increases the Vg gene expression, produces more Vg in H. axyridis female adults and results in an increase in more nutrients for egg development, so feeding of a Vg fragment can stimulate oogenesis and egg production. This may explain that the female feeding of VWD laid more eggs than the control. The results of the study suggest that the protein concentration of Vg in female adults might be used as a molecular marker to predict the fecundity of H. axyridis.
Furthermore, the results of this study also reveal that trypsin and lipase activities in H. axyridis were significantly higher in the treatment groups compared with those of the control groups ( Fig. 7 and Supplementary information Fig. 3). This is the same as those reported by other researchers that food quality affects insect biochemistry 43 . Zeng and Cohen 44 found that certain food can induce enzyme activity. The different component of artificial diets may result in differed activity of digestive enzymes; however, the most important reason for this may be that the Vg fragment stimulates the nutritional related gene expression and then simulates more trypsin and lipase activities.
In summary, the results from current study show that Vg fragment in artificial diets significantly alters the physiology of H. axyridis and increases egg production in the experimental insects. These results suggest that the Vg fragment increases the insects' food quality. The techniques developed in this study have potential applications for increasing the population of beneficial insects.  (Takara, Dalian, China) for sequencing. The overlapping sequences of the above PCR fragments were assembled to obtain the full-length sequences. The sequence of the cloned Vg gene was subjected to a homology search using the NCBI's Basic Local Alignment Search Tool database: http://www.ncbi.nlm.nih.gov/. The signal peptide was predicted using the SignalP 4.0 Server: http://www.cbs.dtu.dk/services/SignalP/. Protein molecular weight and isoelectric point were calculated using the ExPASy: http://www.expasy.org/tools/pi_tool.html. The conserved domain structure was predicted by the Conserved Domain Database (CCD) search: http://www.ncbi.nlm.nih.gov/Structure/cdd/cdd. shtml.

Methods
Third, the VWD domain was expressed and collected. The specific primers of HaVg3 were designed based on the known sequence of Vg, with the first strand of H. axyridis cDNA used as a template for PCR amplification. The amplification resulted in a PCR product containing a 543-bp fragment, which was then cloned into a pEASY-T1 vector (TransGen Biotech, Beijing, China) to produce the pEASY-Vg plasmid. The pEASY-Vg and pET-28 plasmids were double digested with BamHI and NheI restriction endonucleases, respectively. After linking the double-digested pEASY-Vg and pET-28a, the recombinant expression plasmid pET28a-HaVg was sequenced. The pET28a-HaVg plasmid was then transformed into E. coli BL21 cells (TransGen Biotech, Beijing, China), which were grown at 37 °C in 1,000 mL of LB medium with 100 μg/mL kanamycin until an OD 600 value of 0.6-0. The inclusion bodies were future re-suspended in 20 mL buffer B (50 mM Tris-HCl, 100 mM NaCl, 0.5 mM EDTA, 2 M urea and 1% Triton X-100, pH 8.0), room temperature in 0.5 hours and then centifuged at 12,000 × g at 4 °C for 30 min. The pellets were washed twice with buffer C (100 mM Tris-HCl, 500 mM NaCl, 6 M urea, pH 7.4) with 10 mM imidazole, and keep in 4 °C for 24 h and centifuged at 12,000 × g at 4 °C for 30 min. The supernatant was filtered through 0.22 μm filter membrane, and used a Ni-NTA-Sepharose Column (GE Healthcare). The column was washed with buffer D (buffer C with 250 mM imidazole). Finally, The purified proteins were dialyzed at 4 °C by dialysis buffers in different gradient 45 . The concentration of the purified Vg fragment was determined using a BCA protein assay kit (Pierce, Rockford, IL); BSA was used as the standard, and the concentration of the target protein sample was calculated against the standard curve.
Effects of Vg fragment on the reproduction. The purified soluble Vg fragment was included as part of an artificial diet, which was fed at different concentrations (30 μg/mL and 60 μg/mL) to adult H. axyridis animals. The diets of control groups were supplemented with equivalent concentrations of BSA proteins (Jiangchen, Beijing, China). Male and female adults were paired within 1 day after eclosion and placed in a Petri-dish (Jiangchen, Beijing, China), and the artificial diet was replaced every day with fresh food. At least 15 pairs of adults were used for each experiment. The total amount of eggs produced within a one-month period by each pair of adults in the treatment or control groups was recorded, and the egg hatching rates were also determined. Animals were randomized to 4 different experimental groups, and 4 replications were done for each group.
Vg mRNA expression in female adults. The expression level of PmVg and 18S transcripts at different development stages is shown Table 1. Total RNA was isolated from female adults and treated with RNase-free DNase I at 37 °C for 30 min using the DNase I kit (Takara, Dalian, China). A reaction volume of 20 μL was used: 0.5 μL forward and reverse primers, 1 μL cDNA, 8 μL nuclease-free water, and 10 μL 2X iTaq universal SYBR Green supermix (BIO-RAD, California, American). This real-time PCR reaction produced 200 ng total RNA, which was analyzed by a 7500 real-time system (Applied Biosystems, California, USA). The qPCR reaction conditions were as follows: 95 °C for 2 min, 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The housekeeping gene 18 S was used for comparison in the 2 −ΔΔCt qPCR method. Means and standard errors for each time point were obtained from the average of four independent samples. Individual animals were randomized into 2 treatment groups, and 3 replications were performed for each treatment.
Effects of the Vg fragment on lipase and trypsin activities. Trypsin activity was determined according to the method described by Erlanger et al. 46 . BAPNA and BTPNA were used as chromogenic substrates. A total of 20 μL of extract was mixed with 150 μL chromogenic substrate and incubated at 37 °C for 45 min. Crude extract samples (20 μL) and substrate were mixed using a spectrophotometer (Flexstation 3, California, USA), and the absorbance was read at 410 nm. Trypsin activity was recorded as absorbance units per U/mg of protein, and the measurements were repeated three times for each sample. Individual animals were randomized into 2 treatment groups, and 3 replications were performed for each treatment. This same method was used for measuring lipase activity, but nitro phenyl palmitate (p-NPP) served as the chromogenic substrate.