insects

Article Molecular Characterization of Neoseiulus barkeri Vitellogenin Genes and Vitellogenin Receptor during Reproductive Diapause

Junqi Jiang †, Ying Zhang †, Lei Ma, Tingting Niu, Tingting Dong, Ruirui Sheng, Ling Li, Yeyu Xu, Lingyu Xi and Guiting Li * Department of Entomology, College of Plant Protection, Anhui Agricultural University, Hefei 230036, China; [email protected] (J.J.); [email protected] (Y.Z.); [email protected] (L.M.); [email protected] (T.N.); [email protected] (T.D.); [email protected] (R.S.); [email protected] (L.L.); [email protected] (Y.X.); [email protected] (L.X.) * Correspondence: [email protected]; Tel.: +86-1395-606-2545 Contributed equally to this work. †  Received: 30 January 2020; Accepted: 24 March 2020; Published: 26 March 2020 

Abstract: The relationship between reproductive diapause and the genes related to vitellogenin (Vg) and its receptor (VgR) in insectoid ovarian development is still unclear. Accordingly, in the present study, we used hematoxylin and eosin staining to study the ovarian structure in the predatory Neoseiulus barkeri, a species that shows promise as a biological pest control agent. Staining revealed the presence of oocytes on ovary surfaces, and the oocytes were deposited as yolk granules through the intake of Vg and other nutrients with the development of the ovary. Development of the ovary stopped at the oocyte stage in diapausing adult , and this stage presented the same characteristics as the first day of adulthood in non-diapause female adults, where oocytes with nutrient cells, but no yolk granules are observed. In order to further explore the effects of the Vg gene and its receptor on reproduction, the sequences of the N. barkeri vitellogenin genes NbVg1, NbVg2, NbVg3, and NbVgR were analyzed using bioinformatics, and the expression levels of the NbVgs and the VgR at different developmental stages were determined by quantitative polymerase chain reaction (qPCR). The results showed that the NbVgs and NbVgR have complete domains and that the positions of many conservative regions and conservative motif are consistent. The expression levels of the NbVgs and NbVgR were highest in the ovipositional period, followed by those in the preovipositional period. The expression levels of the NbVgs and the VgR in non-diapause female adult mites were significantly higher than those in reproductive diapause female adult mites.

Keywords: diapause; expression levels; Neoseiulus barkeri; ovary development; vitellogenin; vitellogenin receptor

1. Introduction Neoseiulus barkeri (: ) is a polyphagous Phytoseiid mite that preys on a wide range of targets, such as thrips, whiteflies, spider mites, and eriophyoid mites [1–3]. It is widely distributed in the United States, Europe, Israel, and Japan, while in China, it is mainly distributed in Jiangxi, Hunan, Guangdong, and Yunnan. It has a short life span, it is easily cultivated, and it is prolific. Accordingly, it is considered to be a highly promising biological control agent [4]. For instance, it has been used in the biological control of Frankliniella occidentalis on agricultural crops [5]. However, high winter mortality negatively affects the ability of Phytoseiidae mites to control target species [6]. In colder climates, N. barkeri overwinters by means of female reproductive diapause. Moreover, Phytoseiid mites must mate in order to lay eggs. N. barkeri show mating behavior in the deutonymph

Insects 2020, 11, 203; doi:10.3390/insects11040203 www.mdpi.com/journal/insects Insects 2020, 11, 203 2 of 16 period. Our earlier studies revealed that the mites enter the reproductive diapause stage when the temperature falls to 18 ◦C and an 8L/16D light-cycle occurs. However, the involvement of the yolk-precursor protein vitellogenin (Vg) and its receptor (VgR) in ovarian development and reproductive diapause in N. barkeri is unclear. There are currently limited data on ovarian development during reproductive diapause in Phytoseius mites, which are a subfamily of mites in the Phytoseiidae family. Several studies have reported that the main characteristic of reproductive diapause in Phytoseius mites is that they do not lay eggs, or, more accurately, that mites in reproductive diapause have longer preoviposition periods [7]. However, very few physiological and molecular studies on the cessation of reproductive activity in diapause Acarine species have been reported [8]. As limited examples, Swirski and Wysoki studied the ovarian development of Phytoseius mites under low temperature and short-light cycles. In that study, female adult mites raised in a short-daylight cycle (L/D = 8:16) had undeveloped eggs in their ovaries, much like female mites found in natural conditions in December and January, when they are dormant. However, the female mites raised in a long-daylight cycle (L/D = 16:8) had ovaries that developed normally and produced hatchable eggs, much like female mites found in natural conditions in March. In addition to reproductive diapause, overwintering female adults were found to have flattened dorsal abdomens during the ovipositional period. Conversely, the abdomens of fertile females were round and enlarged, and round eggs were observed. In another study, significant differences coloration between the two types of female mites was observed [9]. Moreover, female adult Thuja occidentalis in diapause are milky white with a granular and uniform texture throughout their body cavities, whereas the non-diapause female adults are pink to light red in color, have slightly reddened backs, and have developed eggs that can be observed in the ventral area of the terminal body [10]. In many vertebrates and invertebrates, Vg plays an important role in promoting growth and development [11]. Vg is mainly synthesized in the fat body and then secreted into the hemolymph, finally entering developing oocytes through endocytosis mediated by their VgRs. Receptor-mediated endocytosis is essential for normal eukaryotic cellular functions, including the uptake of nutrients (such as low-density lipoproteins or transferrin) and the recycling of membrane proteins [12,13]. Vg is stored as vitellin and releases synthesized amino acids during embryogenesis, providing material and energy for the development of embryos and ovaries [14]. Recent studies have shown that Vg is polygenic and that some species express one or more Vg genes. For example, Nilaparvata lugens has only one Vg gene [15]; Gallus gallus has three Vg genes [16]; Xenopus laevis has four [17]; Tetranychus urticae has four [18]; and Caenorhabditis elegans has six Vg genes [19]. It has also been demonstrated that Vg is evolutionarily conserved with similar domains and sequences, such as the lipoprotein N-terminal domain (LPD_N), unknown functional domain (DUF1943), Von Willebrand factor D domain (VWD), cleavage site (R/KXXR), and C-terminal GL/ICG motif [20]. VgR is a member of the low-density lipoprotein receptor (LDLR) superfamily and a key receptor for Vg uptake. It also plays an important role in oocyte maturation [21]. Lipoproteins are transported into their target cells by VgR, providing a variety of nutrients to support oocyte development [22]. Members of the LDLR superfamily have many common structural elements, including (i) epidermal growth factor-like repeats (EGF-R); (ii) ligand binding domains that consist of cysteine repeats (LBDs); (iii) the transmembrane domain (TMD); (iv) the internalization signal; and (v) the O-glycan domain in the C-terminal cytoplasmic tail. Studies have shown that VgRs are not only found in invertebrates such as insects, mites, , shrimps, crabs, and nematodes, but also in vertebrates, such as fish, frogs, and chickens [23]. Insects 2020, 11, 203 3 of 16

2. Materials and Methods

2.1. Source of Tested Insects Tyrophagus putrescentiae were used as an alternative prey for the large-scale breeding of N. barkeri. They were provided by the Key Laboratory of Biological Control, Ministry of Agriculture, Institute of Plant Protection, Chinese Academy of Agricultural Sciences. They were fed wheat bran in an artificial climate box (MGC-300H, Blue pard, China) at 25 1 C, relative humidity (RH) 80% 5%, ± ◦ ± and L/D = 14:10 h. N. barkeri were provided by the Key Laboratory of Biological Control, Ministry of Agriculture, Institute of Plant Protection, Chinese Academy of Agricultural Sciences and fed T. putrescentiae as raised above. The conditions in the artificial climate chambers were 25 1 C, RH 80% 5%, and L/D = 14:10 h ± ◦ ± for non-diapause N. barkeri, and 18 1 C, RH 80% 5%, and L/D = 8:16 h for diapause N. barkeri. ± ◦ ± 2.2. Collection of Insects A collection device was constructed using a 40 W incandescent lamp, a wire, a funnel (7 cm in diameter), gauze, and a test tube rack, as shown in Figure S1. A water isolation platform was constructed from a culture dish (9 cm in diameter), a filter paper, a black plastic film, and a sponge. A mixture containing wheat bran, T. putrescentiae, and N. barkeri was spread on the funnel, and a layer of gauze was laid at the upper opening of the funnel. An incandescent lamp was pointed at the top of the funnel and the water isolation platform was placed at the bottom. The mites move towards the water isolation platform. In this manner, the light, heat, and water tropisms of the mites were exploited to select mites in different developmental states. An anatomical mirror was then used to select the target mites, which were isolated by careful use of a writing brush or soft brush. The selected mites were placed on a new water isolation platform for future use.

2.3. Developmental Period of N. barkeri under Diapause and Non-Diapause Conditions A transparent plexiglass block (30 mm 20 mm 2 mm) with a circular hole (r = 5 mm) in the × × middle was sealed with parafilm on one side (30 mm 20 mm) and covered with a plexiglass block × (30 mm 20 mm 1.5 mm) on the other side. A foldback clip was used to fix the two glass blocks to × × prevent the mites from escaping. A mite was placed in each feeding room, and the food was changed every day to ensure its survival.

2.4. Abdominal Morphology of Female Adult Mites Diapause and non-diapause female adult N. barkeri in different ovary-development states were selected and gently swept into a 1.5 mL centrifuge tube with a writing brush, soaked in 1 mL lactic acid solution (Sinopharm, Shanghai, China), and then left at room temperature. The next day, an appropriate N. barkeri was selected and placed on a concave glass slide. Then, 100 µL of 50% lactic acid solution was dripped into it. This was heated with an alcohol lamp to make the lactic acid solution boil slightly, and then placed at room temperature. Under an anatomical mirror (ES-18BZL, Motic, China), the mite was flattened, covered lightly with a glass slide, and its abdominal morphology was observed under a trinocular stereo microscope (AE2000Met, Motic, China).

2.5. Ovarian Section Female adult mites at different stages were fixed at room temperature for 24 h, and then washed and dehydrated with increasing concentrations of alcohol. The tissues were embedded in paraffin wax blocks. The wax blocks were cut into 4 micron thick slices by a slicing machine (EM UC7, Leica, Germany) and stained with hematoxylin and eosin (H&E). After dewatering and sealing, they were observed under a transmission electron microscope (HT7700, Hitachi, Japan). Casevier 2.0 software (3DHISTECH, Budapest, Hungary) was used for processing the images. Insects 2020, 11, 203 4 of 16

2.6. RNA Extraction and cDNA Synthesis Total RNA of N. barkeri from different-aged mites was extracted using Trizol reagent (Invitrogen, CA, USA) following the manufacturer’s recommended procedure, and stored at 80 C for subsequent − ◦ tests. Then, 1 and 3 µL aliquots of the isolated RNAs were separately analyzed on 1.2% agarose gels by staining with ethidium bromide to determine the integrity of the RNA bands. The OD280 and OD260 values for the RNA samples were obtained using a Nanodrop 2000 Ultramicro spectrophotometer (Thermo Fisher, New York, USA). The typical A260/A280 absorbance ratios of the total RNA fell in the range of 1.8–2.0. A PrimeScript RT reagent kit with a gDNA Eraser (Takara, Dalian, China) was used to isolate the total RNA for the first chain of the cDNA.

2.7. Bioinformatics Analyses The gene sequences used in the bioinformatics analysis were obtained from the National Center of Biotechnology information (NCBI) (https://www.ncbi.nlm.nih.gov/). SMART (http://smart.embl- heidelberg.de/) was used to identify structural domains, and SIGNALP 3.0 (http://www.dbs.dtu. dk/service/SIGNALP) was used to predict signal peptides. The COMPUTE PI/MW program (http: //web.expasy.org/compute_pi/) was used to predict the molecular weight and isoelectric points (pI) of the derived protein sequences. ClustalX2 was used to edit amino acid sequences, and a phylogenetic tree was constructed using MEGA 7.0 software (Tempe, AZ, USA) by the neighbor-joining method with 1000 bootstrap replications.

2.8. Quantitative Real-Time Polymerase Chain Reaction (PCR) Using the Trizol reagent method (Takara, Dalian, China), RNA was extracted from female N. barkeri of different ages. Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) analysis was performed on a BIO-RAD CFX96 touch q-PCR system (BIO-RAD, Hercules, California, USA) using SYBR® Premix ExTaqTM II (Tli RNaseH Plus) (Takara). The 20 µL reaction volume contained 10 µL SYBR Premix Ex Taq II, 0.5 µM of each primer, 20 ng cDNA template, and nuclease-free water. The thermal conditions were as follows: one cycle of 95 ◦C for 30 s, 40 cycles of 95 ◦C for 5 s, and 55 ◦C for 20 s. Three replicates were performed for each sample, and the relative levels of gene ∆∆Ct expression among the different samples were measured by the 2− method. Expression of the actin gene of N. barkeri was used as an endogenous control for the normalization of the expression data [24]. The primers used in this section are listed in Table1.

Table 1. Primers used for quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR). Vg, vitellogenin; VgR, vitellogenin receptor.

Primer Sequence (50–30) Product Size (bp) NbVg1-qF CGACATTCCCATCTACGC 155 NbVg1-qR AACCTTCCTGCTCCTTACC NbVg2-qF CGCCAGGAAGAAGGTATC 187 NbVg2-qR TGTGAGTGGGGCAAACG NbVg3-qF GCCAGCGACCAACAGAT 177 NbVg3-qR GCGGAAGCAAGGGTAAT NbVgR-qF CACAAGAGGGCGAAGAGC 117 NbVgR-qR CCATCGGAGCAGAGTCAAG NbActin-qF TCAGCGATGTCAGTTTGAGG 103 NbActin-qR CCTCCTCTCGCAATGAGAAC

2.9. Statistical Analysis The relative levels of gene expression among the N. barkeri diapause and non-diapause strains were ct calculated by the 2−44 method, and three replicates were performed for each sample. Data Processing System (DPS) software version 7.5 was used to analyze the qPCR results for the NbVgs and NbVgR at Insects 2020, 11, x 5 of 16

The relative levels of gene expression among the N. barkeri diapause and non-diapause strains were calculated by the 2−△△ct method, and three replicates were performed for each sample. Data Processing System (DPS) software version 7.5 was used to analyze the qPCR results for the NbVgs Insectsand2020 NbVgR, 11, 203 at different developmental stages [25]. Differences among multiple samples5 ofwere 16 compared by one-way analysis of variance (ANOVA) with the Duncan new multiple range method, and the level of significance was set at p < 0.05. All figures were prepared using GraphPad Prism 5 di(GraphPadfferent developmental Software Inc., stages San[ 25Diego,]. Diff CA,erences USA). among multiple samples were compared by one-way analysis of variance (ANOVA)with the Duncan new multiple range method, and the level of significance was3. Results set at p < 0.05. All figures were prepared using GraphPad Prism 5 (GraphPad Software Inc., San Diego, CA, USA). 3.1. Developmental Periods of N. barkeri under Diapause and Non-Diapause Conditions 3. Results Under non-diapause conditions, N. barkeri undergo three molts, the time intervals of which are 3.1.as Developmentalshown in Figure Periods 1A, and of N. the barkeri total immatureunder Diapause period and is Non-Diapause 8.42 d. N. barkeri Conditions can develop into adults at 18 °C.Under The non-diapause development conditions, period of N.the barkeri diapauseundergo mite threeis slightly molts, prolonged, the time intervals and the of total which immature are as period is 18.57 d (Figure 1B). The molting time is delayed with decreasing temperature. The number shown in Figure1A, and the total immature period is 8.42 d. N. barkeri can develop into adults at 18 ◦C. Theof moltings development does period not change of the over diapause the whole mite development is slightly prolonged, period. and the total immature period is 18.57 d (Figure1B). The molting time is delayed with decreasing temperature. The number of moltings does not change over the whole development period.

A

B

Figure 1. N. barkeri development. (A) Developmental period of N. barkeri under non-diapause Figure 1. N. barkeri development. (A) Developmental period of N. barkeri under non-diapause conditions. (B) Developmental period of N. barkeri under diapause conditions. D = days, long blue conditions. (B) Developmental period of N. barkeri under diapause conditions. D = days, long blue barsbars denote denote life-stage, life-stage, short short yellow yellow bars bars denote denote hatching, hatching, and and short short red red bars bars denote denote molting. molting.

3.2.3.2. Analysis Analysis of of Ovarian Ovarian Development Development TheThe abdominal abdominal morphologies morphologies of lactic-acid-treated of lactic-acid-treated non-diapause non-diapause female female adult and adult diapause and diapause female N.female barkeri N.were barkeri observed. were Onobserved. the first On day the of thefirst female day of adult the stage,female the adult ovaries stage, of thethe non-diapauseovaries of the femalenon-diapause mites have female just begunmites tohave develop just begun (Figure to2 A).develop On the (Figure second 2A). day, On the the ovaries second of day, non-diapause the ovaries femaleof non-diapause ovaries show female obvious ovaries enlargement show obvious (Figure2 B).enlargement On the third (Figure day of 2B). the femaleOn the adult third mite day stage, of the thefemale ovaries adult of the mite non-diapause stage, the ovaries female mitesof the are non-diapause well developed, female egg mites outlines are arewell visible, developed, and yolk egg particlesoutlines are are deposited visible, and (Figure yolk2C). particles On the are fifth deposi day ofted the (Figure diapause 2C). female On the adult fifth mite day stage, of the there diapause are nofemale mature adult ovaries, mite and stage, no yolkthere particles are no mature are deposited ovaries, (Figure and 2noD). yolk particles are deposited (Figure 2D).The ovaries of non-diapause and diapause female adult N. barkeri (mated) were observed. On the first dayThe of ovaries the non-diapause of non-diapause female and adult diapause mite stage, female oocytes adult with N. barkeri one nucleus (mated) and were nurse observed. cells with On twothe nuclei first day are of observed, the non-diapause but no yolk female granules adult are mite observed stage, oocytes (Figure with3A). Onone thenucleus second and day nurse of the cells non-diapausewith two nuclei female are adultobserved, mite but stage, no theyolk yolk granules cells develop are observed into yolk (Figure granules 3A). inOn the the ovary. second At day this of stage,the non-diapause the eggs of the female female adult adult mite mites stage, are incompletely the yolk cells developed develop into and yolk appear granules wrinkled in the (Figure ovary.3B). At Onthis the stage, third daythe ofeggs the of non-diapause the female femaleadult mites adult are mite incompletely stage, the ovaries developed of the non-diapauseand appear wrinkled female mites(Figure are 3B). filled On with the yolk third granules day of andthe havenon-diapause distinct dividing female adult lines. mite The eggsstage, are the surrounded ovaries of bythe eggshellnon-diapause and separated female mites from theare ovariesfilled with (Figure yolk3 C).granules On the and fifth have day distinct of the female dividing adult lines. mite The stage, eggs theare ovaries surrounded of the by diapause eggshell female and separated mites shrink from and the there ovaries are two(Figure nurse 3C). cell On nuclei the fifth in the day ovaries of the without yolk granules (Figure3D). Insects 2020, 11, x 6 of 16 Insects 2020, 11, x 6 of 16

femaleInsects 2020 adult, 11, mite 203 stage, the ovaries of the diapause female mites shrink and there are two nurse6 cell of 16 female adult mite stage, the ovaries of the diapause female mites shrink and there are two nurse cell nuclei in the ovaries without yolk granules (Figure 3D). nuclei in the ovaries without yolk granules (Figure 3D).

Figure 2. Abdomen morphologies and the ovaries of non-diapause N. barkeri adults reared at 25 °C FigureFigure 2. Abdomen 2. Abdomen morphologies morphologies and and the theovaries ovaries of non-diapause of non-diapause N. barkeriN. barkeri adultsadults reared reared at 25 at °C 25 C (A–C) and those of diapause adults reared at 18 °C (D). (A) Day 1 of the non-diapause female adult◦ (A–(CA)– andC) and those those of diapause of diapause adults adults reared reared at 18 at °C 18 (DC(). D(A).) (DayA) Day 1 of 1 the of thenon-diapause non-diapause female female adult adult mite stage. (B) Day 2. (C) Day 3. (D) Day 5 (diapause◦ stage). Scale bar = 100 µm. mitemite stage. stage. (B) (DayB) Day 2. (C 2.) (DayC) Day 3. (D 3.) (DayD) Day 5 (diapause 5 (diapause stage). stage). Scale Scale bar = bar 100= µm.100 µm.

FigureFigure 3. 3. LongitudinalLongitudinal sections sections of of th thee ovaries ovaries of of non-diapause non-diapause N.N. barkeri barkeri adultsadults reared reared at at 25 25 °CC Figure 3. Longitudinal sections of the ovaries of non-diapause N. barkeri adults reared at 25 °C ◦ ((AA––CC)) and and those those of of diapause diapause adults adults reared reared at at 18 18 °CC( (DD).). ( (AA)) Day Day 1 1 of of non-diapause non-diapause female female adult adult mite mite (A–C) and those of diapause adults reared at 18 °C (◦D). (A) Day 1 of non-diapause female adult mite stage.stage. (B (B) Day) Day 2. 2.(C) ( CDay) Day 3. ( 3.D) (DayD) Day 5 (diapause 5 (diapause stage). stage). OC, oocyte; OC, oocyte; NC, nurse NC, nursecell; OV, cell; ovum. OV, ovum.Scale stage. (B) Day 2. (C) Day 3. (D) Day 5 (diapause stage). OC, oocyte; NC, nurse cell; OV, ovum. Scale barScale = 50 bar µm.= 50 µm. bar = 50 µm. TheThe time time course course of of ovarian ovarian development development in inthe the non-diapause non-diapause female female adult adult mites mites shows shows that thatthe The time course of ovarian development in the non-diapause female adult mites shows that the nymphthe nymph is equivalent is equivalent to the to 0 thestage 0 stageafter aftermaturation maturation (i.e., it (i.e., has itnot has yet not developed yet developed into a intofemale a female adult nymph is equivalent to the 0 stage after maturation (i.e., it has not yet developed into a female adult mite).adult mite).Ovarian Ovarian development development is in isphase in phase I on Ithe on thefirst first day day for for adult-female-stage adult-female-stage mites, mites, the the ovaries ovaries mite). Ovarian development is in phase I on the first day for adult-female-stage mites, the ovaries havehave just just begun begun to to develop, develop, and and oocytes oocytes and and nutrient nutrient cells cells have have appeared. appeared. On On the the second second day, day, have just begun to develop, and oocytes and nutrient cells have appeared. On the second day, ovariesovaries have have begun begun to develop in moremore thanthan halfhalf ofof thethe female female mites, mites, and and most most are are in in stage stage II. Ⅱ By. By the ovaries have begun to develop in more than half of the female mites, and most are in stage Ⅱ. By thethird third day, day, all the all femalethe female adult adult mites mites have have laid eggs.laid eggs. The ovariesThe ovaries have have matured matured and the and ovaries the ovaries are in the third day, all the female adult mites have laid eggs. The ovaries have matured and the ovaries arestage in stage III (Figure Ⅲ (Figure4A). 4A). are in stage Ⅲ (Figure 4A). Monitoring the time course of ovarian development in the diapause female adult mites Monitoring the time course of ovarian development in the diapause female adult mites revealed that all the female adult mites have immature ovaries. The ovarian development of revealed that all the female adult mites have immature ovaries. The ovarian development of Insects 2020, 11, x 7 of 16 diapause female adult mites stops at stage I, and they present ovarian characteristics that correspond to those in non-diapause female adult mites on the first day of the adult stage (Figure Insects 2020, 11, 203 7 of 16 4B).

Figure 4. Time course of ovarian development in the non-diapause adults of N. barkeri reared at 25 ◦C Figure 4. Time course of ovarian development in the non-diapause adults of N. barkeri reared at 25 °C (A) and the stages in ovarian development in the diapause adults reared at 18 ◦C(B). The sample size for(A) each and histogramthe stages isin 30.ovarian development in the diapause adults reared at 18 °C (B). The sample size for each histogram is 30. Monitoring the time course of ovarian development in the diapause female adult mites revealed that3.3. allBioinformatics the female adultAnalysis mites have immature ovaries. The ovarian development of diapause female adult mites stops at stage I, and they present ovarian characteristics that correspond to those in non-diapause3.3.1. Bioinformatic female Analysis adult mites of Vgs on the first day of the adult stage (Figure4B). The N. barkeri Vg genes include NbVg1, NbVg2, and NbVg3 (GenBank Accession Numbers are 3.3. Bioinformatics Analysis ASB34115.1, ASB34116.1, and ASB34117.1, respectively). The length of the open reading frame 3.3.1.(ORF) Bioinformatic for NbVg1 is Analysis 5571 bp, of encoding Vgs 1856 amino acids (Figure 5A); for NbVg2, the length of the ORF is 5532 bp, encoding 1843 amino acids (Figure 5B); and for NbVg3, the length of the ORF is 4728The bp, N.encoding barkeri Vg1575 genes amino include acids NbVg1,(Figure NbVg2,5C). Like and those NbVg3 of other (GenBank mites, Accessionthe NbVg1, Numbers NbVg2, and are ASB34115.1,NbVg3 sequences ASB34116.1, of N. andbarkeri ASB34117.1, all contain respectively). three conserved The lengthregions, of LPD_N, the open DUF reading 1943, frame and (ORF)VWD for(Figure NbVg1 5). isHowever, 5571 bp, encodingNbVg1 also 1856 has amino a small acids domain, (Figure5 A);Activator_LAG_3, for NbVg2, the which length ofis theinvolved ORF isin 5532regulating bp, encoding growth 1843and aminodevelopment. acids (Figure NbVg1,5B); NbVg2, and for and NbVg3, NbVg3 the lengthall have of thesignal ORF peptides. is 4728 bp,The encodingsignal peptides 1575 amino for NbVg1 acids (Figureand NbVg25C). Likeare cleaved those of between other mites, amino the acids NbVg1, 16 and NbVg2, 17, and and the NbVg3 signal sequencespeptides for of N.NbVg3 barkeri areall cleaved contain between three conserved amino acids regions, 30 and LPD_N, 31. The DUF molecular 1943, and weights VWD (Figureof NbVg1,5). However,NbVg2, and NbVg1 NbVg3 also hasare a212, small 211, domain, and Activator_LAG_3,179 kDa, and their which pI values is involved are in8.61, regulating 8.98, and growth 6.88, andrespectively development. (Table NbVg1, 2). NbVg2, and NbVg3 all have signal peptides. The signal peptides for NbVg1 and NbVg2 are cleaved between amino acids 16 and 17, and the signal peptides for NbVg3 are cleaved between amino acids 30 and 31. The molecular weights of NbVg1, NbVg2, and NbVg3 are 212, 211, and 179 kDa, and their pI values are 8.61, 8.98, and 6.88, respectively (Table2). Insects 2020, 11, 203 8 of 16 Insects 2020, 11, x 8 of 16

Figure 5. Schematic comparison of the primary protein structures of N. barkeri vitellogenin (Vg) Figure 5. Schematic comparison of the primary protein structures of N. barkeri vitellogenin (Vg) with with those of other mite or species. (A) Comparison of NbVg1 gene domains against those of those of other mite or tick species. (A) Comparison of NbVg1 gene domains against those of other other mite Vg genes. (B) Comparison of NbVg2 gene domains against those of other mite or tick mite Vg genes. (B) Comparison of NbVg2 gene domains against those of other mite or tick Vg genes. Vg genes. (C) Comparison of NbVg3 gene domains against those of other mite or tick Vg genes. (C) Comparison of NbVg3 gene domains against those of other mite or tick Vg genes. VWD, Von VWD, Von Willebrand factor D domain; DUF, unknown functional domain; LPD_N, Lipoprotein Willebrand factor D domain; DUF, unknown functional domain; LPD_N, Lipoprotein N-terminal N-terminal domain domain Table 2. Primary structure analysis of NbVg1, NbVg2, NbVg3, and NbVgR. Table 2. Primary structure analysis of NbVg1, NbVg2, NbVg3, and NbVgR. Theoretical Protein Amino Acids Molecular Weight (kDa) Protein Amino acids Molecular weight (kDa) pI Theoretical pI NbVg1Nb Vg1 18561856 212 212 8.61 8.61 NbVg2Nb Vg2 18431843 211 211 8.98 8.98 NbVg3Nb Vg3 15751575 179 179 6.88 6.88 NbVgR 1702 191 5.53 NbVgR 1702 191 5.53

A phylogenetic tree was constructed using MEGA 7.0 software (Tempe, AZ, USA) [26] and the A phylogenetic tree was constructed using MEGA 7.0 software (Tempe, AZ, USA) [26] and the neighbor-joining method was used to analyze the relationships between NbVg1, NbVg2, NbVg3, neighbor-joining method was used to analyze the relationships between NbVg1, NbVg2, NbVg3, and the Vgs of other Acarine species with those of N. barkeri. Figure 6 shows that the sequences of and the Vgs of other Acarine species with those of N. barkeri. Figure6 shows that the sequences of NbVg1, NbVg2, and NbVg3 for N. barkeri are clustered with those of , NbVg1, NbVg2, and NbVg3 for N. barkeri are clustered with those of Neoseiulus cucumeris, Galendromus Galendromus occidentalis, Varroa destructor, Dermanyssus gallinae, Hemicordulia flava, Planococcus citri, occidentalis, Varroa destructor, Dermanyssus gallinae, Hemicordulia flava, Planococcus citri, and Tetranychus and Tetranychus urticae (Table S1–S3). The amino acid sequence of NbVg1 has a relatively high urticae (Tables S1–S3). The amino acid sequence of NbVg1 has a relatively high homology with homology with those of the G. occidentalis and N. cucumeris Vgs. The amino acid sequence of NbVg2 those of the G. occidentalis and N. cucumeris Vgs. The amino acid sequence of NbVg2 is highly is highly homologous to those of the Vgs of N. cucumeris and V. destructor. The amino acid sequence homologous to those of the Vgs of N. cucumeris and V. destructor. The amino acid sequence of NbVg3 is of NbVg3 is highly homologous to that of the Vgs in G. occidentalis. In addition, Arachnida and highly homologous to that of the Vgs in G. occidentalis. In addition, Arachnida and Insecta Vgs form Insecta Vgs form separate branches. separate branches. Insects 2020, 11, 203 9 of 16 Insects 2020, 11, x 9 of 16

Figure 6.6. PhylogeneticPhylogenetic tree tree of ofN. N. barkeri barkeriVgs Vgs constructed constructed using using the neighbor-joining the neighbor-joining method method in MEGA in softwareMEGA software (version: (version: 7.0). Each 7.0). branch Each shows branch bootstrap shows bootstrap values from values 1000 from replications. 1000 replications. Tables S1–S3 Table list theS1–S3 Vgs list sequences the Vgs sequences used in this used analysis. in this analysis. 3.3.2. VgR Bioinformatics Analysis 3.3.2. VgR Bioinformatics Analysis The full length of the VgR (GenBank Accession Number XP_028968361.1) gene sequence The full length of the VgR (GenBank Accession Number XP_028968361.1) gene sequence in N. in N. barkeri is 5335 bp, and the length of the ORF is 5109 bp, encoding 1702 amino acids. barkeri is 5335 bp, and the length of the ORF is 5109 bp, encoding 1702 amino acids. The theoretical The theoretical molecular weight is 191.14 kDa, while the pI is 5.53 (Table2), and there are eight molecular weight is 191.14 kDa, while the pI is 5.53 (Table 2), and there are eight putative putative glycosylation sites (N-X-S or N-X-T). The presumed signal peptide is cleaved at amino acid glycosylation sites (N-X-S or N-X-T). The presumed signal peptide is cleaved at amino acid 19. 19. Predicted conserved domain analysis of the VgR amino acid sequence from N. barkeri is shown Predicted conserved domain analysis of the VgR amino acid sequence from N. barkeri is shown in in Figure7. The NbVgR gene encodes four classical functional domains: LBDs, theEGF precursor Figure 7. The NbVgR gene encodes four classical functional domains: LBDs, theEGF precursor homology domain, the transmembrane domain, and the intracellular domain. NbVgR has two LBDs, homology domain, the transmembrane domain, and the intracellular domain. NbVgR has two LBDs, containing four and eight class a repeat domains, respectively. The EGF precursor domain is located containing four and eight class a repeat domains, respectively. The EGF precursor domain is located C-terminal to the ligand binding domain, and includes an EGF-like repeat domain and a YWTD C-terminal to the ligand binding domain, and includes an EGF-like repeat domain and a YWTD motif (Tyr-Trp-Thr-Asp sequence) used to form a β-propeller domain. The NbVgR has two EGF motif (Tyr-Trp-Thr-Asp sequence) used to form a β-propeller domain. The NbVgR has two EGF precursor homology domains, each containing four EGF-like repeat domains. However, unlike the precursor homology domains, each containing four EGF-like repeat domains. However, unlike the proteins encoded by the corresponding genes for Haemaphysalis longicornis, Dermacentor variabilis, proteins encoded by the corresponding genes for Haemaphysalis longicornis, Dermacentor variabilis, and Amblyomma hebraeum, NbVgR does not contain the YWTD motif in the first LBD. NbVgR and that and Amblyomma hebraeum, NbVgR does not contain the YWTD motif in the first LBD. NbVgR and of Haemaphysalis longicornis do not contain O-connected structural regions. that of Haemaphysalis longicornis do not contain O-connected structural regions. Insects 2020, 11, x 10 of 16

InsectsInsects 20202020, 11,, 11x , 203 10 of10 16 of 16

Figure 7. Schematic comparison of primary protein structures for the N.barkeri VgR with those of otherFigure mite 7. species.Schematic comparison of primary protein structures for the N.barkeri VgR with those of other Figuremite 7. species. Schematic comparison of primary protein structures for the N.barkeri VgR with those of otherFrom mite the species. phylogenetic trees of NbVgR and the VgR for other species, we can see that From the phylogenetic trees of NbVgR and the VgR for other species, we can see that , arachnids, insects, crustaceans, and vertebrates form independent VgR branches. Among them, insects,From crustaceans,the phylogenetic and vertebrates trees of formNbVgR independent and the VgRVgR branches.for other Amongspecies, them, we NbVgRcan see belongsthat NbVgR belongs to Phytoseidae, Tetranychuidae, and , which form three separate groups. arachnids,to Phytoseidae, insects, Tetranychuidae, crustaceans, and and vertebrates Ixodidae, which form formindependent three separate VgR groups.branches. This Among shows them, that the NbVgRThisVgR shows is belongs highly that evolutionarilyto the Phytoseidae, VgR is highly conserved Tetranychuidae, evolutionarily (Figure8 and). cons Ixodidae,erved (Figure which 8).form three separate groups. This shows that the VgR is highly evolutionarily conserved (Figure 8). DvaVgR AAZ31260.3 53 DvaVgR AAZ31260.3 96 53 HloVgR BAG14342.1

96 HloVgR BAG14342.1 100 RmiVgR AUQ44344. 100 RmiVgR AUQ44344. 100 AheVgR AGQ57038.1 100 AheVgR AGQ57038.1 61 IscVgR EEC20133.1 61 IscVgR EEC20133.1 TciVgR ANS13820.1 TciVgR ANS13820.1 100 93 100 PciVgR AHN48901.1 93 PciVgR AHN48901.1 AceVgR XP 028521104.1 68 AceVgR XP 028521104.1 68 AmeVgR XP 026295652.1 AmeVgR XP 026295652.1 100 100 AflVgR XP 012350206.1 AflVgR XP 012350206.1

TmeVgR OQR73944.1 TmeVgR OQR73944.1

NbVgR NbVgR XP 028968361.1 XP 028968361.1

0.050.05

FigureFigureFigure 8. 8. Phylogenetic 8.PhylogeneticPhylogenetic tree tree tree for for the the VgR VgRVgR in in N.inN. N.barkeri barkeri barkeri constructedconstructed constructed using using using the the neighbor-joiningthe neighbor-joining neighbor-joining method method method in inin MEGA MEGA software software (version: (version: 7.0) 7.0). 7.0). Each . Each branch branch shows shows bootstrap bootstrap va valueslues values from from 1000 1000 replications. replications. Table Table S4 S4S4 lists lists the the VgsVgs Vgs sequencessequences sequences usedused used inin in thisthis this analysis.analysis. analysis. Insects 2020, 11, 203 11 of 16 Insects 2020, 11, x 11 of 16

3.3.3.3.3.3. Transcriptional Transcriptional Expression of the NbVgs and NbVgR FigureFigure 99 showsshows thatthat therethere areare significantsignificant didifferenfferencesces in in the the expression levels levels of of NbVg1, NbVg1, NbVg2, NbVg2, NbVg3,NbVg3, and and NbVgR NbVgR in in the eggs, larvae, nymphs, nymphs, preovipositional preovipositional female female mites mites (the (the first first day day of adultadult mites), mites), ovipositional ovipositional female female mites mites (the (the fourth fourth day day of adult of adult mites), mites), and andmale male adult adult mites. mites. The expressionThe expression levels levels for male for male mites mites are arethe the lowest, lowest, followed followed by by those those of of the the eggs. eggs. In In the the development development stage,stage, thethe expressionexpression levels levels for for female female adult adult mites mite ins thein preovipositionalthe preovipositional period period are significantly are significantly lower lowerthan those than ofthose female of female adult mitesadult inmites the ovipositionalin the ovipositional period. period. FigureFigure 1010 showsshows the the expression expression levels levels of diofff differenterent genes genes in the in non-diapausethe non-diapause female female adult N.adult barkeri N. barkeri(the fourth (the dayfourth of adulthood)day of adulthood) and the diapauseand the diapause female adult femaleN. barkeriadult (theN. barkeri fifth day (the of fifth adulthood). day of adulthood).The expression The levelsexpression of NbVg1, levels of NbVg2, NbVg1, NbVg3, NbVg2, and NbVg3, NbVgR and are NbVgR significantly are significantly different, different, and the andexpression the expression levels for levels diapause for diapause female adultfemale mites adult are mites down-regulated. are down-regulated.

FigureFigure 9. RelativeRelative expression expression of of N.N. barkeri barkeri VgsVgs and and the the VgR VgR in in non-diapau non-diapausese mites mites at at different different stages asas determined determined by by quantitative quantitative po polymeraselymerase chain chain reaction reaction (qPCR). (qPCR). (A) ( ARelative) Relative expression expression of Vg1. of Vg1. (B) Relative(B) Relative expression expression of Vg2. of Vg2. (C) Relative (C) Relative expression expression of Vg3. of Vg3.(D) Relative (D) Relative expression expression of the ofVgR. the Data VgR. Data are presented as the means of three replicates standard error (n = 3). Different lowercase letters are presented as the means of three replicates ± st±andard error (n = 3). Different lowercase letters indicateindicate significant significant differences differences (one-way (one-way analysis analysis of of variance variance (ANOVA) (ANOVA) with Duncan new multiple rangerange method, method, p < 0.05).0.05). InsectsInsects 20202020, ,1111, ,x 203 1212 of of 16 16

FigureFigure 10. 10. ExpressionsExpressions of N. barkeri VgsVgs andand the the VgR VgR in in non-diapause non-diapause and and diapause diapause periods. periods. Data Data are arepresented presented as the as meanthe meanstandard ± standard error (nerror= 3). (n Di =ff erent3). Different lowercase lowercase letters indicate letterssignificant indicate significant differences ± differences(one-way ANOVA (one-way with ANOVA Duncan with new Du multiplencan new range multiple method, rangep < method,0.05). p < 0.05). 4. Discussion 4. Discussion The main characteristic of diapause in Phytoseius mites is non-oviposition. The abdominal The main characteristic of diapause in Phytoseius mites is non-oviposition. The abdominal examination of diapause female adult N. barkeri mites showed that the ovaries inside the mite are examination of diapause female adult N. barkeri mites showed that the ovaries inside the mite are underdeveloped. No yolk granules are formed in the ovaries of diapause female adult mites at any underdeveloped. No yolk granules are formed in the ovaries of diapause female adult mites at any time, and the ovaries of diapause female adult mites remain at first-day development into adulthood time, and the ovaries of diapause female adult mites remain at first-day development into (this stage is equivalent to the first day of a non-diapause mated female adult mite, that is, the ovaries adulthood (this stage is equivalent to the first day of a non-diapause mated female adult mite, that have just begun to develop, oocytes and vegetative cells have appeared, but no yolk particles are is, the ovaries have just begun to develop, oocytes and vegetative cells have appeared, but no yolk observed), while the ovaries of non-diapause female adult mites have developed normally by the particles are observed), while the ovaries of non-diapause female adult mites have developed third day of adulthood (there are obvious yolk granules) and produced eggs. Therefore, this study normally by the third day of adulthood (there are obvious yolk granules) and produced eggs. demonstrates that the ovarian development of diapause female adult mites is halted in stage I. Therefore, this study demonstrates that the ovarian development of diapause female adult mites is Morphology and behavior are also used as criteria for defining reproductive diapause. The external halted in stage I. morphological characteristics of diapause female adult N. barkeri are a flattened dorsum and abdomen, Morphology and behavior are also used as criteria for defining reproductive diapause. The while in non-diapause females, the abdomen is rounded and enlarged and the ovary has rounded eggs. external morphological characteristics of diapause female adult N. barkeri are a flattened dorsum This feature is similar to other species of Phytoseid mites. For example, the external morphological and abdomen, while in non-diapause females, the abdomen is rounded and enlarged and the ovary characteristics of Phytoseius mites, such as G. occidentalis and Amblyseius fallacis, can be used to identify has rounded eggs. This feature is similar to other species of Phytoseid mites. For example, the reproductive diapause [27]. external morphological characteristics of Phytoseius mites, such as G. occidentalis and Amblyseius The ORFs of Vg1, Vg2, and Vg3 are 5571, 5532, and 4728 bp, respectively. Bioinformatic analysis fallacis, can be used to identify reproductive diapause [27]. showed that the amino acid sequences of NbVg1, NbVg2, and NbVg3 all have characteristic domains The ORFs of Vg1, Vg2, and Vg3 are 5571, 5532, and 4728 bp, respectively. Bioinformatic of Vg, such as LPD_N, DUF 1943, and VWD, which comprise three traditionally conserved analysis showed that the amino acid sequences of NbVg1, NbVg2, and NbVg3 all have regions. The N-terminal of NbVg2 contains the lipoprotein domain LPD_N. Studies have shown that characteristic domains of arthropod Vg, such as LPD_N, DUF 1943, and VWD, which comprise LPD_N may be involved in lipid interactions. The VWD protein is the carrier of coagulation factor three traditionally conserved regions. The N-terminal of NbVg2 contains the lipoprotein domain VIII [28]. LPD_N. Studies have shown that LPD_N may be involved in lipid interactions. The VWD protein is The ORF length of the VgR is 5109 bp, encoding 1702 amino acids. The VgR belongs to the LDLR the carrier of coagulation factor VIII [28]. superfamily. As expected, like other members of the LDLR family, the highly conserved domains The ORF length of the VgR is 5109 bp, encoding 1702 amino acids. The VgR belongs to the of NbVgR include the following: (i) the class A cysteine enrichment repeat (ligand binding repeat); LDLR superfamily. As expected, like other members of the LDLR family, the highly conserved (ii) the class B cysteine-rich repeat sequence (EGF repeat sequence); (iii) the transmembrane domain; domains of NbVgR include the following: (i) the class A cysteine enrichment repeat (ligand binding and (iv) the intracellular domain. NbVgR does not contain O-linked sugar domains, similar to H1VgR repeat); (ii) the class B cysteine-rich repeat sequence (EGF repeat sequence); (iii) the transmembrane in H. longicornis and PcVgR in P. citri. O-linked sugar domains represent oocyte-specific transcripts, domain; and (iv) the intracellular domain. NbVgR does not contain O-linked sugar domains, and the existence of this domain is not common in the LDLR family [29]. similar to H1VgR in H. longicornis and PcVgR in P. citri. O-linked sugar domains represent We measured the transcriptional expression levels of Vg1, Vg2, Vg3, and the VgR in different oocyte-specific transcripts, and the existence of this domain is not common in the LDLR family [29]. developmental stages (eggs, young mites, nymphs, preovipositional female adults, ovipositional We measured the transcriptional expression levels of Vg1, Vg2, Vg3, and the VgR in different female adults, diapause female adults, and male adults). For these genes, the expression levels for developmental stages (eggs, young mites, nymphs, preovipositional female adults, ovipositional adult mites are significantly higher than those for the other developmental stages, which is consistent female adults, diapause female adults, and male adults). For these genes, the expression levels for with the trend of other Acarine species, such as P. citri, H. longicornis, and A. hebraeum [30]. adult mites are significantly higher than those for the other developmental stages, which is Insects 2020, 11, 203 13 of 16

The expression levels of Vg1, Vg2, Vg3, and the VgR for mated female adult mites in diapause are significantly lower at similar developmental time points for non-diapause mated female adult mites, because the Vg content in ovaries is affected by Vg biosynthesis and VgR uptake to female germ cells. The decrease of VgR content leads to the inability of oocytes to absorb enough Vg, resulting in reproductive diapause and an inability to lay eggs. NbVgR has a similar expression pattern to the expression of VgR for other insects. Its expression is very low at the beginning of the pre-ovarian stage and significantly increases before the full yolk stage. For example, the highest expression level of B. dorsalis (GenBank Accession Number JX469118) VgR appears on the seventh day, and the expression level in the early stage is significantly lower than that on the seventh day [31]. The expression trend of Vg and VgR in diapause and non-diapause N. barkeri to some extent reflects its physiological function, that is, Vg and VgR genes are involved in yolk deposition into ovarian oocytes, providing Vg as a nutrient reserve material for the maturation of oocytes [32,33]. Vg and VgR genes are responsible for oviposition in N. barkeri and other oocytes. This process is crucial. The results of real-time fluorescent quantitative PCR showed that the relative expression of the Vg gene is significantly higher in sexually mature and ovipositing females than in immature females. Liu et al. reported that Vg gene mRNA is detected in Chrysopa pallens at the age of four days and is most accumulated in C. pallens at the age of 10 days, after which its Vg gene mRNA expression begins to decrease [34]. After silencing the Vg gene of C. pallens with RNAi technology, the number of eggs laid decreases significantly, and the hatchability of the eggs also decreases significantly. This indicates that the Vg gene plays an important role in the laying and hatching of C. pallens. In addition, the transcription levels of Vg and the VgR have also been measured in male adult mites, revealing that low levels of Vg and the VgR are also expressed in some insect males, such as Apis mellifera [35]. However, Vg is not expressed in the spider mite (T. urticae)[36]. Thus, the role of Vg and the VgR in males is unclear and needs further study. The VgR plays an important role in the development of eggs and is generally considered to be an effective factor in evaluating female reproductive capacity. At present, the synthesis process for Vg is still controversial. There are two main ways to synthesize Vg: (a) It is synthesized in extraovarian tissues, secreted into the circulatory system, and reaches the oocyte. It then enters the ovary through endocytosis mediated by the VgR and provides nutrients for the development of embryos and larvae [37]. (b) The endogenous synthesis of Vg, where Vg is produced by the oocyte itself with the participation of relevant organelles [38]. There is a certain correlation between the expression of Vg and the number of eggs laid by insects. For example, the overexpression of Vg and its receptors in Tetranychus cinnabar may cause increased fecundity [39].

5. Conclusions In summary, histological observation revealed marked differences between the ovaries of non-diapause females and those in reproductive diapause. Specifically, the abdomens of fertile females are enlarged and round, while those of females in reproductive diapause are flat. Moreover, the boundaries of eggs can be clearly seen in the ovaries of non-diapause females, along with yolk particles. Conversely, the ovaries of females in reproductive diapause are not completely developed, thus they lack eggs. Given the fact that Vg and the VgR play important roles in reproduction and oviposition, it is not surprising that the expression of Vg and the VgR is significantly higher in fertile females than those in reproductive diapause. This indicates that, like that of Drosophila [40], the reproductive capacity of N. barkeri is closely related to Vg and the VgR. Specifically, upregulation of Vg and the VgR in mites leads to an increase in Vg and VgR protein content in the fat body and ovaries, providing more nutrients for oviposition. However, the specific mechanism of this process requires further study. Insects 2020, 11, 203 14 of 16

Supplementary Materials: The following are available online at http://www.mdpi.com/2075-4450/11/4/203/s1, Figure S1: Collection device of Neoseiulus barkeri; Table S1: BLAST alignment of the Vg1 gene of Neoseiulus barkeri with other species in the NCBI database; Table S2: BLAST alignment of the Vg2 gene of Neoseiulus barkeri with other species in the NCBI database; Table S3: BLAST alignment of the Vg3 gene of Neoseiulus barkeri with other species in the NCBI database; Table S4: BLAST alignment of the VgR gene of Neoseiulus barkeri with other species in the NCBI database. Author Contributions: Conceptualization, Y.Z. and J.J.; methodology, Y.Z., J.J. and G.L.; formal analysis, Y.Z., J.J., L.M. and T.N.; investigation, Y.Z., J.J., L.M., T.N., T.D., R.S., L.L., Y.X. and L.X.; resources, J.J. and G.L.; writing—original draft preparation, Y.Z.; writing—review and editing, Y.Z. and J.J.; supervision, J.J. and G.L.; funding acquisition, J.J. and G.L. All authors have read and agreed to the published version of the manuscript. Funding: This study was supported by the National Key R&D Program of China Grant/Award Numbers: 2017YFD0201000 and 2016YFD0200500, and this study was also supported by the graduate innovation fund of Anhui Agricultural University. Conflicts of Interest: The authors declare no conflict of interest.

References

1. Hessein, N.A.; Parrella, M.P. Predatory mites help control thrips on floriculture crops. Calif. Agric. 1991, 44, 19–21. [CrossRef] 2. Grafton Cardwell, E.E.; Ouyang, Y.; Striggow, R.A. Predacious mites for control of citrus thrips, Scirtothrips citri (Thysanoptera: Thripidae) in nursery citrus. Biol. Control 1999, 14, 29–36. [CrossRef] 3. Nomikou, M.; Janssen, A.; Schraag, R.; Sabelis, W. Phytoseiid predators as potential biological control agents for Bemisia tabaci. Exp. Appl. Acarol. 2001, 25, 271–291. [CrossRef] 4. Fernando, L.C.P.; Waidyarathne, K.P.; Perera, K.F.G.; Silva, P.H.P.R.D. Evidence for suppressing coconut mite, Aceria guerreronis by inundative release of the predatory mite, Neoseiulus baraki. Biol. Control 2010, 53, 108–111. [CrossRef] 5. Endong, W.; Xuenong, X.; Shengyong, W. Control effects of Amblyseius barkeri on Frankliniella occidentalis on the eggplants and their natural enemy Orius sauteri in the greenhouse. Plant Prot. 2010, 36, 101–104. 6. Xia, B.; Zou, Z.; Li, P.; Lin, P. Effect of temperature on development and reproduction of Neoseiulus barkeri (acari: Phytoseiidae) fed on aleuroglyphus ovatus. Exp. Appl. Acarol. 2012, 56, 33–41. [CrossRef] 7. Prestwich, G.D. Comprehensive insect physiology, biochemistry, and pharmacology. Int. J. Biochem. 1985, 17, 1282–1283. [CrossRef] 8. Gotoh, T. Annual Life Cycle of the Two-Spotted Spider Mite, Tetranychus urticae KOCH (Acarina: Tetranychidae), on Ribes rubrum L. in Sapporo: The Presence of Non-Diapausing Individuals. Appl. Entomol. Zool. 1986, 21, 454–460. [CrossRef] 9. Wysoki, M. Studies on diapause and the resistance to low temperatures of a predacious mite, Phytoseius finimus (, phytoseiidae). Eniomol. Exp. Appl. 1974, 17, 22–30. [CrossRef] 10. Croft, B.A. Comparative Studies on Four Strains of Typhlodromus occidentalis (Acarina: Phytoseiidae). V. Photoperiodic Induction of Diapause. Ann. Entomol. Soc. Am. 1971, 64, 962–964. [CrossRef] 11. Lu, K.; Shu, Y.H.; Zhou, J.L.; Zhang, X.Y.; Zhang, X.Y.; Chen, M.X.; Yao, Q.; Zhou, Q.; Zhang, W.Q. Molecular characterization and RNA interference analysis of vitellogenin receptor from Nilaparvata lugens. J. Insect Physiol. 2015, 73, 20–29. [CrossRef][PubMed] 12. Tufail, M.; Takeda, M. Molecular characteristics of insect vitellogenins. J. Insect Physiol. 2008, 54, 1447–1458. [CrossRef][PubMed] 13. Sappington, T.W.; Raikhel, A.S. Molecular characteristics of insect vitellogenins and vitellogenin receptors. Insect Biochem. Mol. Biol. 1998, 28, 277–300. [CrossRef] 14. Huo, Y.; Liu, W.W.; Zhang, F.J.; Chen, X.Y.; Li, L.; Liu, Q.F.; Zhou, Y.J.; Wei, T.Y.; Fang, R.X.; Wang, X.F. Transovarial transmission of a plantvirus is mediated by vitellogen of its insect vector. PLoS Pathog. 2014, 10, e1003949. [CrossRef][PubMed] 15. Ge, L.Q.; Wu, J.C.; Zhao, K.F.; Chen, Y.; Yang, G.Q. Induction of Nlvg and suppression of Nljhe gene expression in Nilaparvata lugens (Stål) (hemiptera: Delphacidae) adult females and males exposed to two insecticides. Pestic. Biochem. Physiol. 2010, 98, 269–278. [CrossRef] Insects 2020, 11, 203 15 of 16

16. Schip, F.D.V.H.; Samallo, J.; Broos, J.; Ophuis, J.; Mojet, M.; Gruber, M.; Geert, A.B. Nucleotide sequence of a chicken vitellogenin gene and derived amino acid sequence of the encoded yolk precursor protein. J. Mol. Biol. 1987, 196, 245–260. [CrossRef] 17. Wahli, W.; Dawid, I.B.; Ryffel, G.U.; Wyler, T.; Jaggi, R.B.; Weber, R. Vitellogenin in Xenopus laevis is encoded in a small family of genes. Cell 1979, 16, 535–549. [CrossRef] 18. Kawakami, Y.; Goto, S.G.; Ito, K.; Numata, H. Suppression of ovarian development and vitellogenin gene expression in the adult diapause of the two-spotted spider mite Tetranychus urticae. J. Insect Physiol. 2009, 55, 70–77. [CrossRef] 19. Blumenthal, T.; Squire, M.; Kirtland, S.; Cane, J.; Donegan, M.; Spieth, J.; Sharrock, W. Cloning of a yolk protein gene family from Caenorhabditis elegans. J. Mol. Biol. 1984, 174, 1–18. [CrossRef] 20. Sappington, T.W.; Raikhel, A.S. Insect vitellogenin/yolk protein receptors. Reprod. Biol. Invertebr. 2005, 229–264. 21. Okabayashi, K.; Shoji, H.; Nakamura, T.; Hashimoto, O.; Asashima, M.; Sugino, H. cDNA cloning and expression of the Xenopus laevis vitellogenin receptor. Biochem. Biophys. Res. Commun. 1996, 224, 406–413. [CrossRef][PubMed] 22. Raikhel, A.S.; Dhadialla, T.S. Accumulation of yolk proteins in insect oocytes. Annu. Rev. Entonzol. 1992, 37, 217–251. [CrossRef] 23. Zhong, R.; Ding, T.B.; Niu, J.Z.; Xia, W.K.; Liao, C.Y.; Dou, W.; Wang, J.J. Molecular characterization of vitellogenin and its receptor genes from citrus red mite, Panonychus citri (McGregor). Int. J. Mol. Sci. 2015, 16, 4759–4773. [CrossRef][PubMed] 24. Ding, L.; Chen, F.; Luo, R.; Pan, Q.; Wang, C.; Yu, S.; Cong, L.; Liu, H.; Li, H.; Ran, C. Gene cloning and difference analysis of vitellogenin in Neoseiulus barkeri (Hughes). Bull. Entomol. Res. 2018, 108, 141–149. [CrossRef][PubMed] 25. Tang, Q.Y.; Zhang, C.X. Data Processing System (DPS) software with experimental design, statistical analysis and data mining developed for use in entomological research. Insect Sci. 2013, 20, 254–260. [CrossRef] 26. Kumar, S.; Stecher, G.; Tamura, K. Mega7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 2016, 33, 1870–1874. [CrossRef][PubMed] 27. Veerman, A. Diapause in phytoseiid mites: A review. Exp. Appl. Acarol. 1992, 14, 1–60. [CrossRef] 28. Shikina, S.; Chen, C.J.; Chung, Y.J.; Shao, Z.F.; Liou, J.Y.; Tseng, H.P.; Lee, Y.H.; Chang, C.F. Yolk formation in a stony coral Euphyllia ancora (Cnidaria, Anthozoa): Insight into the evolution of vitellogenesis in nonbilaterian . Endocrinology 2013, 154, 3447–3459. [CrossRef] 29. Alexander, D.S.; Kaufman, W.R. Molecular characterization of the vitellogenin receptor from the tick, Amblyomma hebraeum (Acari: Ixodidae). Insect Biochem. Mol. Biol. 2014, 5, 821–833. 30. Chippendale, G.M.; Yin, C.M. Endocrine interactions controlling the larval diapause of the southwestern corn borer, Diatraea grandiosella. J. Insect Physiol. 1976, 22, 989–995. [CrossRef] 31. Lin, C.; Yang, W.J.; Jiang, X.Z.; Niu, J.Z.; Shen, G.M.; Ran, C.; Wang, J.J. The Essential Role of Vitellogenin Receptor in Ovary Development and Vitellogenin Uptake in Bactrocera dorsalis (Hendel). Int. J. Mol. Sci. 2015, 16, 18368–18383. 32. Taylor, D.; Chinzei, Y.; Miura, K.; Ando, K. Vitellogenin synthesis, processing and hormonal regulation in the tick, Ornithodoros parkeri (Acari: ). Insect Biochem. 1991, 21, 723–733. [CrossRef] 33. Qian, C.; Fu, W.W.; Wei, G.Q.; Wang, L.; Liu, Q.N.; Dai, L.S.; Sun, Y.; Zhu, B.J.; Liu, C.L. Identification and expression analysis of vitellogenin receptor from the Wild Silkworm, Bombyx mandarina. Arch. Insect Biochem. Physiol. 2015, 89, 181–192. [CrossRef][PubMed] 34. Liu, C.; Mao, J.; Zeng, F. Chrysopa septempunctata (Neuroptera: Chrysopidae) Vitellogenin Functions through Effects on Egg Production and Hatching. J. Econ. Entomol. 2015, 108, 2779–2788. [CrossRef][PubMed] 35. Guidugli-Lazzarini, K.R.; Nascimento, A.M.D.; Tanaka, E.D.; Piulachs, M.D.; Hartfelder, K.; Márcia, G.B.; Paulino, L.Z.S. Expression analysis of putative vitellogenin and lipophorin receptors in honey bee (Apis mellifera L.) queens and workers. J. Insect Physiol. 2008, 54, 1138–1147. [CrossRef] 36. Kawakami, Y.; Numata, H. Effects of a Pyrethroid on Ovarian Development in Diapause Females of the Two Spotted Spider Mite. J. Acarol. Soc. Jpn. 2013, 22, 45–47. [CrossRef] 37. Okumura, T.; Yamano, K.; Sakiyama, K. Vitellogenin gene expression and hemolymph vitellogenin during vitellogenesis, final maturation, and oviposition in female kuruma prawn, Marsupenaeus japonicus. Comp. Biochem. Physiol. Part A Mol. Integr. Physiol. 2007, 147, 1028–1037. [CrossRef] Insects 2020, 11, 203 16 of 16

38. Avarre, J.C.; Michelis, R.; Tietz, A.; Lubzens, E. Relationship between vitellogenin and vitellin in a marine shrimp (Penaeus semisulcatus) and molecular characterization of vitellogenin complementary DNAs. Biol. Reprod. 2003, 69, 355–364. [CrossRef] 39. Liu, X.; Shen, G.; Xu, H.; He, L. The fenpropathrin resistant Tetranychus cinnabarinus showed increased fecundity with high content of vitellogenin and vitellogenin receptor. Pestic. Biochem. Physiol. 2016, 134, 31–38. [CrossRef] 40. Schonbaum, C.P.; Perrino, J.J.; Mahowald, A.P. Regulation of the vitellogenin receptor during Drosophila melanogaster oogenesis. Mol. Biol. Cell 2000, 11, 511–521. [CrossRef]

© 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).