International Journal of
Molecular Sciences Article
Nutritional Signaling Regulates Vitellogenin Synthesis and Egg Development through Juvenile Hormone in Nilaparvata lugens (Stål) Kai Lu 1,2 , Xia Chen 1 , Wen-Ting Liu 1 , Xin-Yu Zhang 2 , Ming-Xiao Chen 2 and Qiang Zhou 2, * 1 2
*
College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China;
[email protected] (K.L.);
[email protected] (X.C.);
[email protected] (W.-T.L.) State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, China;
[email protected] (X.-Y.Z.);
[email protected] (M.-X.C.) Correspondence:
[email protected]; Tel.: +86-20-8411-2259; Fax: +86-20-8411-2297
Academic Editor: Lee A. Bulla Received: 22 December 2015; Accepted: 14 February 2016; Published: 26 February 2016
Abstract: Insect female reproduction which comprises the synthesis of vitellogenein (Vg) in the fat body and its incorporation into developing oocytes, needs a large amount of energy and food resources. Our previous studies found that juvenile hormone (JH) regulates vitellogenesis in the brown planthopper, Nilaparvata lugens. Here, we report on the role of JH in nutrient-regulated Vg synthesis and egg development. We first cloned the genes coding for juvenile hormone acid methyltransferase (JHAMT) which is involved in JH biosynthesis and methoprene-tolerant (Met) for JH action. Amino acids (AAs) induced the expression of jmtN, while showing no effects on the expression of met using an artificial diet culture system. Reduction in JH biosynthesis or its action by RNA interference (RNAi)-mediated silencing of jmtN or met led to a severe inhibition of AAs-induced Vg synthesis and oocyte maturation, together with lower fecundity. Furthermore, exogenous application of JH III partially restored Vg expression levels in jmtN RNAi females. However, JH III application did not rescue Vg synthesis in these met RNAi insects. Our results show that AAs induce Vg synthesis in the fat body and egg development in concert with JH biosynthesis in Nilaparvata lugens (Stål), rather than through JH action. Keywords: Juvenile hormone; vitellogenesis; nutritional signaling; RNA inteference; Nilaparvata lugens
1. Introduction Nutrition and reproduction are important physiological processes in insects and nutrients have long been known to play key roles in the regulation of reproduction [1,2]. Egg maturation and successful reproduction requires an input of energy-rich food resources to achieve a nutritional status [3,4]. Vitellogenesis, an important process in insect reproduction, is a nutrient-dependent process. Initiation of vitellogenesis by feeding is a crucial event in the reproductive cycle of the anautogenous insects [5,6]. The free amino acids (AAs) concentration in the hemolymph increases dramatically after a blood meal in Aedes aegypti [7] and Culex pipiens [8]. Furthermore, AAs derived from the blood meal have been demonstrated to be the key nutritional signals to stimulate Vg gene transcription and Vg protein synthesis in the fat body of mosquito [9–11]. Although the key role of nutrients in regulation of reproduction has been demonstrated in many insect species, very little is known about its molecular mechanism. Recently, the hormonal control of vitellogenesis has been described in many insects, and hormones are suggested to be involved in nutritionally dependent reproduction [2,12,13].
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Juvenile hormone (JH), a sesquiterpenoid hormone synthesized and secreted by the corpus allatum (CA), regulates many aspects of insect reproduction including vitellogenesis, oocyte maturation and ovarian growth [14,15]. JH-regulated vitellogenesis has been reported in different kinds of insects, including Dictyoptera [16], Hymenoptera [17,18], Coleoptera [19], Orthoptera [20] and Hemiptera [21] including mosquitoes [22]. JH has been shown as part of a transduction system that balances nutritional status and controls reproduction in mosquitoes [13,23]. The nutrient-deficient A. aegypti developed into smaller adults and showed lower JH titers. Topical application of JH to these small mosquitoes could enable them to complete their first gonotrophic cycle after a single blood meal [24]. In starved adult female Blattella germanica, JH synthesis and Vg production are low [6]. Starvation of female Tribolium castaneum also led to inhibition of Vg synthesis whereas feeding after starvation stimulated Vg synthesis and egg maturation. However, in the starved beetles where genes coding for protein involved in JH biosynthesis (juvenile hormone acid methyltransferase, JHAMT) was disrupted by RNAi, Vg expression was severely inhibited even after feeding, suggesting that JH is indispensable for the nutrient-dependent Vg synthesis [12]. However, the mechanism of JH action in regulating nutrient-induced vitellogenesis remains obscure. This report attempts to find out the role of JH III in nutrient-regulated vitellogenesis using the brown planthopper, Nilaparvata lugens (Hemiptera), which is an ideal research model to address this question. Additionally, robust RNAi and rapid JH response can be achieved in the adult females [25–27]. Using a newly developed artificial diet culture system, we found that nutrients (AAs) are necessary for the expression of jmtN which is involved in JH biosynthesis [28,29]. Depletion of jmtN and met (Methoprene-tolerant, JH receptor) show that JH is required for AAs-induced Vg synthesis, and knockdown of jmtN or met severely inhibited ovarian development and reduced fecundity. Our results suggest that the JH pathway mediates AAs-induced Vg synthesis in the fat body playing an important role in ovarian growth and development. 2. Results 2.1. Identification of Genes Involved in JH Signaling and Phylogenetic Analysis The cDNA sequence of met (KJ690934) is 2895 bp with an open reading frame (ORF) of 2391 bp. Met ORF encodes a protein of 796 amino acids with a predicted molecular mass of 89 kDa and an isoelectric point (pI) of 5.53. A basic Helix-Loop-Helix (bHLH) domain and two highly conserved Pert-Arnt-Sim (PAS) domains, which were shown to be sufficient for JH binding, were identified in Met. The complete ORF of jmtN (KP769805) is 840 bp in length encoding a 279-amino acid polypeptide with a calculated molecular weight of 31.76 kDa and a theoretical pI of 5.46. N. lugens JHAMT has a highly conserved S-adenosylmethionine (SAM) binding motif IMDVGCGPG [29]. To reveal the evolutionary relationship of Met orthologs, a comprehensive phylogenic tree was constructed based on 17 Met protein sequences from five insect orders with 1000 bootstrap replicates. The phylogenetic analysis indicated that N. lugens Met is closely related to two orthologs of Hemiptera: Pyrrhocoris apterus and Rhodnius prolixus (Figure 1A). The phylogenetic relationship of JHAMTs derived from 24 insect species was similarly investigated showing that N. lugens JHAMT is a monophyletic branch with a large genetic distance from other insects in the phylogenetic tree. The most closely related protein sequence to JHAMT (34% identity) is from a Hymenoptera insect: Solenopsis invicta (EFZ09595) (Figure 1B).
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Figure 1. Phylogenetic analysis of Met (A) and JHAMT (B) from different insect species based on the
Figure 1. Phylogenetic analysis of Met (A) and JHAMT (B) from different insect species based on the amino acid sequences alignment. Protein sequences were downloaded from the GenBank database amino acid sequences alignment. Protein sequences were downloaded from the GenBank database and and the accession numbers are shown. The phylogenetic tree was generated by MEGA6 using the theMaximum accession numbers are shown. The phylogenetic tree was generated by MEGA6 using the Maximum Likelihood method. Bootstrap values (1000 replicates) are shown in the cladogram. The Likelihood method. Bootstrap genetic distance is drawn to values scale. (1000 replicates) are shown in the cladogram. The genetic distance is drawn to scale.
2.2. Tissue-Specific Expression Profiles of Met and JmtN Examined by qRT-PCR
2.2. Tissue-Specific Expression Profiles of Met and JmtN Examined by qRT-PCR
Met and jmtN mRNA expression levels in the brain, corpora allata, fat body, ovary, Malpighian tubules andjmtN epidermis from three-day-old females analyzed byfat qRT-PCR. Met transcripts Met and mRNA expression levelsadult in the brain,were corpora allata, body, ovary, Malpighian were detected in all tissues and were primarily expressed in the fat body, with relatively lower tubules and epidermis from three-day-old adult females were analyzed by qRT-PCR. Met transcripts expression in the ovary and epidermis (Figure 2A). JmtN mRNA expression was almost exclusively were detected in all tissues and were primarily expressed in the fat body, with relatively lower located ininthe allata. Trace amounts of 2A). jmtNJmtN mRNA were present in the brain, fat body and expression thecorpora ovary and epidermis (Figure mRNA expression was almost exclusively ovary (Figure 2B). Since expression of met was highest in the fat body and transcripts of jmtN were located in the corpora allata. Trace amounts of jmtN mRNA were present in the brain, fat body and mostly produced in theexpression corpora allata, the fat body was used tofat investigate thetranscripts changes of of metjmtN mRNA ovary (Figure 2B). Since of met was highest in the body and were and the corpora allata was used to assess the changes in jmtN transcript levels.
mostly produced in the corpora allata, the fat body was used to investigate the changes of met mRNA and the corpora allata was used to assess the changes in jmtN transcript levels.
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Figure 2. Tissue-specific expression profiles of met (A) and jmtN (B). All tissues were dissected from
Figure 2. Tissue-specific expression profiles of met (A) and jmtN (B). All tissues were dissected from three-day-old adult females. BR: brain; CA: corpora allata; FB: fat body; OV: ovary; MG: Malpighian three-day-old adult females. BR: brain; CA: corpora allata; FB: fat body; OV: ovary; MG: Malpighian tubules; EP: epidermis. Values represent the mean of 7–8 independent pools of 30–50 females and tubules; EP: epidermis. Valuesnormalized represent the of(n7–8 independent pools of 30–50 females and three three technical replicates, withmean β-actin = 7–8). Vertical bars indicate standard errors. technical replicates, normalized withthe β-actin (nofindicate = met 7–8). bars standard errors. Different Figure 2.lowercase Tissue-specific profiles (A)Vertical and jmtN (B).indicate All tissues were dissected from Different lettersexpression above columns significant differences within different tissues three-day-old adultpthe females. BR: brain; CA:significant corpora allata; FB: fat body; OV: ovary; MG:tissues Malpighian (one-way ANOVA, < 0.05). lowercase letters above columns indicate differences within different (one-way tubules; EP: epidermis. Values represent the mean of 7–8 independent pools of 30–50 females and ANOVA, p < 0.05). three technical with β-actin (n = 7–8). Vertical bars indicate standard errors. 2.3. AAs Regulate Vgreplicates, Synthesis normalized in Adult Females Different lowercase letters above the columns indicate significant differences within different tissues (one-way ANOVA, p < 0.05).
2.3. AAs Regulate Vg Synthesis AdultasFemales To ascertain the effect in of AAs a stimulus activating Vg synthesis in the N. lugens females, we used an established chemically defined artificial diet culture system for the insect rearing. Newly
Toemerged ascertain the effect of12AAs as areared stimulus activating Vg synthesis in the N. lugens females, we h) were on artificial diets either containing (+AAs) or lacking AAs 2.3. AAs females Regulate(within Vg Synthesis in Adult Females used an established chemically defined artificial diet culture system fortothe insect rearing. (−AAs). In conjunction with the artificial culture system, qRT-PCR was used determine Vg mRNANewly To ascertain the effect of AAs as a stimulus activating Vg synthesis in the N. lugens females, we emerged females (within 12 h) were reared on artificial diets either containing (+AAs) or lacking levels and western blot was applied to measure Vg protein levels in the fat bodies in response to AAs used an established chemically defined artificial diet culture system for the insect rearing. Newly various AAs treatments. in Figure 3B,system, females qRT-PCR treated with depleted AAs for three days (´AAs). In conjunction with As theshown artificial culture was used to determine Vg mRNA emerged females (within 12 h) were reared on artificial diets eitherlevel containing (+AAs) or lacking AAs (AD3d) demonstrated approximate 90% reduction in Vg mRNA the control levels and western blot wasanapplied to measure Vg protein levels in theas fatcompared bodies inwith response to various (−AAs).reared In conjunction with the artificial culture system, qRT-PCR was at used to determine Vg in mRNA (AF3d, on AAs for three days). Vg protein was not present a detectable level the(AD3d) AAs treatments. As shown in Figure 3B,tofemales treated with depleted AAs for three days levels and western blotand was aapplied measure Vg was protein levelsininthe theAF3d fat bodies in response AAs-deprived females, large amount of Vg present control group. Toto demonstrated approximate 90% reduction in3B, Vgfemales mRNAtreated level as compared with thethree control various an AAs treatments. As shown in Figure depleted AAs for days(AF3d, determine whether AAs have any activating Vg expression effect,with females lacking AAs in the first rearedand on AAs for three days). Vg protein was not present at a detectable level in the AAs-deprived (AD3d) demonstrated approximate 90% AAs reduction Vg mRNA level as compared of with theyielded control second day (AD2d)an were supplied with in thein third day (AR3d). Supplement AAs on AAs three days). Vg protein not control present at a detectable level in the females, and areared large amount of Vg was present thewas AF3d Towith determine whether a(AF3d, significant increase in Vgfor mRNA levels, about ain 4.5-fold higher level as group. compared the females females, and a (AD2d). largeeffect, amount of Vg was present in control group. AAs have any activating Vg expression females lacking AAs in the theofAF3d first and second dayTo (AD2d) inAAs-deprived absence of AAs continuously Similarly, a considerable amount Vg protein was present determine whether AAs have anyday activating Vg Supplement expression effect, females lackinga AAs in the first after a supplement with (Figure 3B).(AR3d). were supplied with AAs in AAs the third of AAs yielded significant increase and second day (AD2d) were supplied with AAs in the third day (AR3d). Supplement of AAs yielded in Vg mRNA levels, about a 4.5-fold higher level as compared with the females in absence of AAs a significant increase in Vg mRNA levels, about a 4.5-fold higher level as compared with the females continuously (AD2d). a considerable amount of Vg protein was after supplement in absence of AAsSimilarly, continuously (AD2d). Similarly, a considerable amount ofpresent Vg protein wasa present with AAs (Figure 3B). with AAs (Figure 3B). after a supplement
Figure 3. AAs regulate Vg synthesis. (A) Diagram of experimental design. AFE, after adult female emergence. AD3d, three-day-old AAs-deprived females; AF3d, three-day-old AAs-fed females; AD2d, two-day-old AAs-deprived females; AR3d, two-day-old AAs-deprived females were fed on normal diet (+AAs) for another day; (B) Relative expression levels of Vg mRNA in the fat body were Figure 3. AAs regulate Vg β-actin synthesis. Diagram of experimental design. AFE, adult female qRT-PCR as a(A) were performed basedafter on independent Figuredetected 3. AAsby regulate Vgusing synthesis. (A)reference. DiagramReactions of experimental design. AFE, after adult female emergence. AD3d, three-day-old AAs-deprived females; AAs-fed females; RNA sample preparations and values were shown as mean ± AF3d, SE (n = three-day-old 8). Asterisk denoted significant emergence. AD3d, three-day-old AAs-deprived females; AF3d, three-day-old AAs-fed females; AD2d, AD2d, two-day-old AAs-deprived two-day-old AAs-deprived females were fedby on differences from controls (Student’s females; t-test, “*”AR3d, denotes p < 0.05). Vg protein levels were detected two-day-old females; AR3d, two-day-old AAs-deprived females were fed on normal normalAAs-deprived diet (+AAs) for another (B) was Relative levels of Vg mRNA in the fat body were western blot, an antibody againstday; β-actin usedexpression as a loading control. diet (+AAs) for another day; (B) Relative expression levels of Vg mRNA in the fat body were detected detected by qRT-PCR using β-actin as a reference. Reactions were performed based on independent by qRT-PCR using β-actin as a reference. were performed on independent RNA sample RNA sample preparations and valuesReactions were shown as mean ± SE (n based = 8). Asterisk denoted significant differences controls “*”˘denotes < 0.05). Vg protein levelssignificant were detected by preparations and from values were(Student’s shown ast-test, mean SE (n =p 8). Asterisk denoted differences western blot, an antibody against β-actin was used as a loading control. from controls (Student’s t-test, “*” denotes p < 0.05). Vg protein levels were detected by western blot,
an antibody against β-actin was used as a loading control.
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2.4. JmtN Expression Expression Is 2.4. JmtN Is Regulated Regulated by by AAs AAs Signaling Signaling To of the the met met and and jmtN jmtN in in the the N. N. lugens lugens females, females, To determine determine the the effect effect of of AAs AAs on on transcription transcription of the relative mRNA expression levels between the AAs-fed and deprived females were analyzed the relative mRNA expression levels between the AAs-fed and deprived females were analyzed by by qRT-PCR. qRT-PCR. The The mRNA mRNA expression expression levels levels of of met met in in the the fat fat body body remained remained constant constant without without significant significant differences differences among among the the different different AAs AAs treatments treatments (Figure (Figure 4A). 4A). However, However, the the jmtN jmtN mRNA mRNA level level in in the the corpora allata significantly dropped in the AAs-deprived females (AD3d) compared with their mRNA corpora allata significantly dropped in the AAs-deprived females (AD3d) compared with their expression levels in levels these AAs-fed (AF3d). Furthermore, jmtN expression successfully mRNA expression in thesefemales AAs-fed females (AF3d). Furthermore, jmtN was expression was induced after AAs supplement (AR3d) compared with its expression in the AAs-deprived females successfully induced after AAs supplement (AR3d) compared with its expression in the AAs-deprived (AD2d) females (Figure (AD2d)4B). (Figure 4B).
Figure 4. 4. Expression (A) and and jmtN jmtN (B) (B) mRNA mRNA in in AAs-deprived AAs-deprived females. females. Newly Newly emerged emerged Figure Expression of of met met (A) females (within (within 12 12 h) h) were were reared reared on on artificial artificial diets diets either either containing containing (+AAs) (+AAs) or or lacking lacking amino amino acids acids females (−AAs). Relative mRNA expression levels of met in the FB and jmtN in the CA were detected using (´AAs). Relative mRNA expression levels of met in the FB and jmtN in the CA were detected using qRT-PCR. Bars Bars represent representmean mean˘ ± SE qRT-PCR. SE of of 7–8 7–8 biologically biologically independent independent pools pools of of 30–50 30–50 females females and and three three technical replicates, replicates, normalized normalized with = 7–8, denotes pp > > 0.05 0.05 and and technical with β-actin β-actin (n (n = 7–8, Student’s Student’s t-test, t-test, “ns” “ns” denotes “*” denotes denotes pp 0.05 and “*” denotes p < 0.05). Vg protein levels were Figure Figure 6. 6. Effect Effect of of JH JH III III on onAAs-induced AAs-induced Vg Vg synthesis. synthesis. Newly Newly emerged emerged females females were were injected injected with with detected by western blot.
100 met, jmtN or or a nonspecific gfp and reared for two AfterAfter that, 100 ng ngof ofrespective respectivedsRNA dsRNAtargeting targeting met, jmtN a nonspecific gfp and reared for days. two days. the were were topically applied with with JH IIIJH (100 ng inng 100 or acetone (100 (100 nL) and fed that,insects the insects topically applied III (100 innL 100acetone) nL acetone) or acetone nL) and with artificial diets for another day. Relative mRNA levels of Vg in the FB were detected by qRT-PCR fed with artificial diets for another day. Relative mRNA levels of Vg in the FB were detected by (n = 7, Student’s t-test, “ns”t-test, denotes p denotes > 0.05 and < 0.05). Vg protein were detected qRT-PCR (n = 7, Student’s “ns” p >“*” 0.05denotes and “*”p denotes p < 0.05). Vglevels protein levels were by western detected byblot. western blot.
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2.6. Silencing Met or JmtN Inhibits Ovarian Development Development and and Reduction Reduction in in Fecundity Fecundity To To investigate investigate the the role role of of JH JH and and AAs AAs on on ovarian ovarian development development and fecundity, fecundity, newly newly emerged emerged females were injected injected with withdsRNA dsRNAofofmet, met,jmtN jmtN or gfp and reared on an artificial diet. Ovaries or gfp and reared on an artificial diet. Ovaries were were dissected day six control from control and dsRNA injected females, and photographed a dissected at dayatsix from and dsRNA injected females, and photographed using ausing stereo stereo microscope. The development of ovaries were dissectedfrom fromAAs-deprived AAs-deprived females females was microscope. The development of ovaries thatthat were dissected completely blocked, and no mature eggs eggs were were found. found. In the AAs-fed group, fully fully developed developed eggs eggs and matured matured ovaries ovaries were were observed observed in the gfp dsRNA-injected controls. However, However, knockdown knockdown of of met and jmtN jmtN transcript transcript levels by dsRNA dsRNA severely severely inhibited inhibited the the ovarian ovarian growth growth and and oocyte oocyte maturation maturation (Figure 7A). Those females that were injected with met or jmtN dsRNA and reared on (Figure 7A). Those injected jmtN on normal normal diet diet (+AAs) with an an average of 21.1 female respectively, a significant decrease (+AAs) laid laidfewer fewereggs, eggs, with average of and 21.1 19.8 andper 19.8 per female respectively, a significant (55.67% 58.4%, respectively) as compared with thewith number of eggs of laid by laid the control group decreaseand (55.67% and 58.4%, respectively) as compared the number eggs by the control (injected with gfpwith dsRNA), and no and eggsno were by laid the AAs-deprived femalesfemales (Figure (Figure 7B). group (injected gfp dsRNA), eggslaid were by the AAs-deprived 7B).
Figure 7. 7. Ovarian Ovarian growth growth and and fecundity fecundity in in the the RNAi, RNAi, AAs-deprived AAs-deprived and and fed fed females. females. (A) (A) AAs, AAs, met met Figure and jmtN jmtN are are required required for for ovary ovary development. development. Equal Equal amount amount of of dsRNA dsRNA for for JH JH pathway pathway genes genes and and and (control) was was injected injected into into newly newly emerged emerged females females (within (within 12 12 h). h). The The ovaries ovaries were were dissected dissected on on gfp (control) day 6 post adult emergence and photographed with stereo microscopy SMZ18 (Nikon, Tokyo, Japan). stereo microscopy SMZ18 (Nikon, Tokyo, Japan). Scale bars indicate 500 µm; μm; (B) (B) Fecundity Fecundity was was reduced reduced in in met met and and jmtN jmtN dsRNA-treated dsRNA-treated females. females. The The total number number of of eggs laid was counted for fifteen days. Since AAs-deprived females laid no eggs, only total egg numbers numbers derived derived from fromAAs-fed AAs-fedfemales femaleswere wereused usedfor forstatistical statisticaltest. test.Bars Barsrepresent representmean mean˘± SE SE of egg three independent independent experiments experiments of of ten ten females and asterisk indicates significant significant differences differences (Student’s (Student's three t-test, “*” “*” denotes denotes pp
