An adenylyl cyclase like-9 gene (NlAC9) influences growth and fecundity in the brown planthopper, Nilaparvata lugens (Stål) (Hemiptera: Delphacidae)

The cAMP/PKA intracellular signaling pathway is launched by adenylyl cyclase (AC) conversion of adenosine triphosphate (ATP) to 3', 5'-cyclic AMP (cAMP) and cAMP-dependent activation of PKA. Although this pathway is very well known in insect physiology, there is little to no information on it in some very small pest insects, such as the brown planthopper (BPH), Nilaparvata lugens Stål. BPH is a destructive pest responsible for tremendous crop losses in rice cropping systems. We are investigating the potentials of novel pest management technologies from RNA interference perspective. Based on analysis of transcriptomic data, the BPH AC like-9 gene (NlAC9) was up-regulated in post-mating females, which led us to pose the hypothesis that NlAC9 is a target gene that would lead to reduced BPH fitness and populations. Targeting NlAC9 led to substantially decreased soluble ovarian protein content, yeast-like symbiont abundance, and vitellogenin gene expression, accompanied with stunted ovarian development and body size. Eggs laid were decreased and oviposition period shortened. Taken together, our findings indicated that NlAC9 exerted pronounced effects on female fecundity, growth and longevity, which strongly supports our hypothesis.


Introduction
Many extracellular signals, such as hormones, peptides, biogenic amines and prostaglandins, are transduced into intracellular second messengers via G-protein coupled receptors (GPCRs). As the receptors bind to their specific ligands they undergo a conformational change that leads to activation of their coupled G proteins, the heterotrimeric guanine nucleotide-binding proteins. The G proteins activate any of several downstream effectors, one of which is adenylyl cyclase (AC) [1]. Activated AC converts ATP into cyclic adenosine 3',5'-monophosphate (cAMP), which stimulates cAMP-dependent protein kinase (PKA). PKA is one of the major kinases acting in reversible protein phosphorylation mechanisms in which protein functions PLOS  are regulated by adding one or more phosphate groups to proteins (by kinases) and by removing them (by phosphatases) [2]. The very broad significance of the PKA pathway is indicated by a list of over 100 physiological PKA substrates [3]. The number of PKA substrates draws attention to the significance of AC, which operates in most cells, including insect cells, first discovered over 30 years ago [4]. They have been documented in a wide range of insect tissues, including prothoracic glands of the tobacco hornworm, Manduca sexta [5] and silkworm, Bombyx mori [6] and brains of Drosophila melanogaster and honey bees, Apis mellifera [7]. ACs act in a very wide array of cellular functions. For a single example, AC/cAMP/PKA signaling acts in Drosophila behavioral changes during alcohol intoxication [8]. ACs also act in several aspects of reproduction, including spermatogenesis in Drosophila [8]. Because of their many, central signaling functions in reproduction and other aspects of insect biology, we raised the question of whether silencing AC would influence reproductive biology of insect pests.
The brown planthopper (BPH), Nilaparvata lugens Stål (Hemiptera: Delphacidae), is a major insect pest throughout Asian rice growing regions. It is responsible for very large reduction in rice yields (up to 60%), directly by feeding on plant fluids and indirectly as vectors of rice pathogenic viruses [9]. BPH damage is difficult to detect because significant yield loss occurs before plant damage can be registered [10]. The potentials for wide-spread crop losses are exacerbated by two patterns. First, pest resistance and resurgence due to long-term overuse of chemical insecticides and other agricultural chemicals is increasing. Second, BHP has evolved unexpected responses to agricultural chemicals: increased fecundity and populations [11,12]. These patterns amount to a case-study of the long called for development of novel insect pest management technologies [13].
One novel technology is the concept of regulating pest populations by silencing genes operating at crucial points in insect biology [14], which we have been developing for BHP in which gene-silencing constructs are ingested via the food stream [15][16][17]. Based on analysis of transcriptomic data, the BPH AC like-9 gene (NlAC9) was up-regulated in post-mating females (S1 Table). A central issue in this new technology is identification and selection of the most appropriate target genes. Because AC operates in a very wide range of cell signaling processes, including those directly involved in reproduction, we posed the hypothesis that AC9 is a target gene that would lead to reduced BPH fitness and populations. Here we report on the outcomes of experiments designed to test our hypothesis.

Rice variety and culture
Seeds of susceptible rice (Oryza sativa ssp. japonica) cv. Yangjing 805 were sown in cement tanks (height 60 cm, width 100 cm, and length 200 cm) containing standard rice-growing soil at the Yangzhou University Experimental Farm under natural outdoor conditions. Seedlings bearing six leaves were transplanted into 16 cm diameter plastic pots with four hills per pot, three plants per hill. All rice plants were grown to the tillering stage under natural condition for experiments Insect culture BPH used in the experiments were obtained from a stock population at the China National Rice Research Institute (Hangzhou, China), and maintained in a greenhouse under a 16L: 8D photoperiod at 26±2˚C, with 70-80% humidity in the ecological lab of Yangzhou University without insecticide application. Prior to the experiments, the insect colony was allowed to reproduce two generations in cement tanks (60×100×200 cm) under natural conditions.

dsRNA
We created NlAC9-specific primers and amplified a 579-bp (1437-2015bp) NlAC9 cDNA fragment (NLU001904) according to NlAC9 sequence (S2 Table and S1 File) using forward and reverse primers containing the T 7 primer sequence at the 5 0 -ends ( Table 1). The amplification program was 35 cycles of 95˚C for 40 s, 56˚C for 40 s and 72˚C for 1 min, with a final extension step at 72˚C for 10 min. The sequence was verified by sequencing (done by Invitrogen, Shanghai, China). We amplified a 688 bp fragment of GFP using primers detailed in Table 1 as control dsRNA (ACY56286; supplied by Zhang Chuan-xi, Institute of Insect Sciences, Zhejiang University). We used the T 7 RiboMAXTM Express RNAi System (Promega, Sunnyvale, CA) for dsRNA synthesis, following the Promega instructions. Sense and antisense dsRNAs were produced in separate 20 μL reaction volumes, annealed by mixing and incubating at 70˚C for 10 min, and then cooling to room temperature over 20 min. Two microliter RNase A solution (4 mg/ml) and 2 μL RNase-free DNase (1 u/μL) were added to the reaction tube and incubated in a 37˚C water bath for 30 min. The dsRNA was precipitated by adding 110 μL 95% ethanol and 4.4 μL 3 M sodium acetate (pH 5.2), then rinsed with 0.5 mL 70% ethanol, dried at room temperature and dissolved in 50 μL nuclease-free water. The purified dsRNAs were quantified by spectroscopy.
dsRNAs were orally delivered to nymphs via an artificial diet [18], with minor modifications to the rearing protocol. RNAi via dsRNA feeding is an effective protocol, inducing a rapid reduction in mRNAs encoding selected BPH genes [15]. In glass-cylinder chambers (15.0-cm high × 2.5-cm diameter), BPHs were fed with three concentrations of dsRNA (0.125, 0.0625, and 0.05) to select the optimal treatment concentration 0.0625μg/μL for this study based on dsRNA-treated silencing efficiency and nymphs mortality (S3 Table). The experimental diets (20 μL) were placed between two layers of stretched Parafilm M membrane enclosed at the two open ends of the chamber, which was replaced every second day. The cylinders were covered with light-tight black cloth, and the two ends loaded with artificial diet were exposed to light. Insects fed on the diets by piercing-sucking the inner Parafilm M membrane of the diet capsule. Experimental insects were transferred into chambers and reared on artificial diets for 24 h before a series of assays. Twenty third instar individuals collected from the trial field were placed in each chamber, and three chambers made up three independent biological replicates. The feeding experiments were carried out in a humidified climate cabinet (90% RH) at 26±2˚C with a 16L: 8D photoperiod. Mortality was recorded every other day.

Effects of dietary dsRNA on biological performance parameters
Our pre-experiments showed that the dsRNA treatments induced 95% mortality in second and third instar nymphs, therefore the third instar nymphs were used for all experiments [18]. When the nymphs reached fifth (final) instar (approximately 8 days), they were collected, released into a glass jar (12-cm high × 6-cm diameter) and maintained with rice plants at the tillering stage under standard conditions until emergence. We paired individual newly-emerged females with newly-emerged males, in three combinations: dsNlAC-treated female × untreated male (♀dsNlAC×♂control), dsGFP-treated female × untreated male (♀dsGFP × ♂control), and untreated female × untreated male (♀control × ♂control). Each pair (♂×♀) was introduced into a glass jar (diameter 6-cm, height 12-cm) with rice stems under standard conditions for oviposition. Fifteen mated females per replicate were maintained to record pre-oviposition period, oviposition period, adult longevity and fecundity. Rice stems were replaced daily during pre-oviposition period, every 2 days during the oviposition period, and every 3 days for female longevity until females died. Eggs inside each rice stem were scraped from the leaf sheaths and blades using a pin to count the number of eggs laid under a microscope. Fecundity of 15 mated pairs was recorded as the average number of total eggs laid. A total of 120 mated females (each treatment) at 2 days post emergence (2 d PE) were collected for determination of fresh body weight, soluble ovary protein content, soluble fat body protein content and ecdysone and JH titers. A part of 15 mated females were collected separately at 2, 3, 5 and 7 d PE to determine induced-NlAC9 transcript level and dsNlAC9-treated females silencing efficiency posting mating. Another 15 virgin females (each treatment) were recruited separately at 1, 3, 5 and 7 d PE to determine NlAC9 transcript accumulation and dsNlAC9-treated females silencing efficiency.

Protein extraction and determinations
Protein was extracted from fat bodies and ovaries as described [19] with slight modification. Ovaries and fat bodies were isolated from ten adults under a zoom stereomicroscope (model XTL20, Beijing Tech Instrument Co., Ltd., Beijing, China) in a cooled petri dish. Ovaries and fat bodies were separately placed in pre-weighed, ice-cold centrifuge tubes and then weighed using a Mettler-Toledo electronic balance (EC100 model; 1/10,000 g sensitivity

Numbers of yeast-like symbionts (YLS) in fat bodies
Numbers of YLS in fat bodies were measured with a hemacytometer (0.01 mm, 1/400 mm 2 ) (25×16 model, Shanghai Qiujing Biochemical Reagent Co., Shanghai, China following Ying and Hou [21]. A total of 45 adult females (15 for each treatment) were collected separately at 2 d post mating. Fat body was dissected from five females in experimental (dsNlAC9 and dsGFP) and control groups, and homogenized in 200 μL saline solution (0.9% NaCl) to generate a mixture from which YSL in 2 μL were counted. The numbers of YLS were counted from 80 mm 2 squares under a microscope. Each treatment and control was replicated three times.

Body weights and body size comparison
Replicates for each treatment had 15 females at 2 d PE frozen in liquid nitrogen. The insects were placed in pre-weighed centrifuge tubes and then weighed using a Mettler-Toledo electronic balance, n = 3. Body size from at least fifteen females for each group was photographed with a Leica DMR connected to a Fuji FinePix S2 Pro digital camera (Germany). Scale bar, 200μm.

JH and ecdysone titers
We followed the instructions from an insect double sandwich ELISA kit for JH and ecdysone determinations (Qiaodu biological technology Co., LTD, Shanghai, China). Each treatment and control was replicated three times (n = 3).

Ovary dissection
The  [16] of constitutive actin-1 (EU179846). After amplification, a melting curve analysis was performed in triplicate and the results were averaged. The values were calculated using three independent biological samples and the relative NlAC9 transcript level was analyzed by 2 -ΔΔCT method [22]. Each treatment was replicated three times (five females per replicate).

Statistical analysis
Before performing an analysis of variance (ANOVA), normality and homogeneity of variance were verified based on a Bartlett test. A one-way ANOVA was performed to analyze biological parameter data. Data presented in figures were expressed as mean ± S.E and the differences between three groups were analyzed using a t-test (significance level p < 0.05). All Statistical analysis was conducted manipulating DPS data processing system developed by Tang and Feng [23].

Effect of dietary dsNlAC9 on reproductive systems
In relation to untreated (Fig 5A, 5D and 5G) and dsGFP-treated controls (Fig 5B, 5E and 5H), dietary dsNlAC9 (Fig 5C, 5F and 5I) inhibited ovary development at 2 d PE, 4 d PE, and 6 d PE. The ovarioles within ovaries of dsGFP-treated and control females contained one or two ripe banana-shaped oocytes. The dietary dsNlAC9 treatments severely inhibited oocyte growth. No mature oocytes were observed at 2 d PE. We observed underdeveloped ovaries with fewer ovarioles and fewer developed eggs in the adults treated with dsNlAC9 (Fig 5F and  5I) at 4 and 6 d PE. In contrast, the control groups showed replete ovarioles and plentiful eggs (Fig 5D and 5G, 5E and 5H) at 4 and 6 d PE.
Dietary dsNlAC9 led to reduced expression of Nlvg mRNA

Discussion
The data set forth in this paper strongly support our hypothesis that NlAC9 is a target gene that would lead to reduced BPH fitness and populations. Several points apply. First, dietary dsNlAC9 led to severely decreased mRNAs encoding NlAC9. Second, the experimental treatments led to decreased ovarian protein contents, sizes and development. Third, dietary dsNlAC9 led to meaningful changes in circulating titers of development hormones, ecdysone (increased) and JH (decreased). Fourth, the treatments led to seriously reduced reproductive potentials, recorded as reduced egg laying, oviposition periods and mRNAs encoding the BPH vitellogenin. Fifth, experimental treatments led to reduced female body weights, sizes and longevity. Taken together, the point amount to a very strong argument that targeting NlAC9 would exert extremely negative impacts on BPH reproduction and populations.
The reduced accumulations of mRNAs encoding NlAC9 would influence a wide range of physiological functions, possibly all functions that are signaled via the AC/cAMP/PKA pathway. Many of these functions lie beyond the scope of this paper, including various homeostatic functions such as water and salt balance, feeding behaviors, digestion, excretion, learning and memory. The AC/cAMP/PKA pathway also acts in insect pathologies. The Cry1Ab toxin, for example, kills insects by binding to a receptor, which activates a cAMP/PKA that leads to a toxin-induced cell death pathway [24]. The reduced reproductive potentials recorded here may be the outcome of direct effects on the parameters we recorded and the indirect effects of overall impaired organismal functioning. Down-regulating NlAC9 led to increased ecdysone concentration. 20E acts via nuclear receptors, which enhance gene transcription via cAMPdependent activation of PKA [25]. More to the point, cAMP content in fat body is controlled by ecdysone [26].
dsNlAC9 treatments lead to reduced JH concentrations, which help understand the reduced insect sizes reported here because JH is one of the regulators of adult sizes. Experimentally, reducing JH concentrations by ablating the corpora allata led to undersized larvae [27][28][29][30]. The influence of NlAC9 silencing on decreased JH titers may be seen in reduced Vg synthesis and reduced body weight. Similarly, numbers of eggs laid is an index of fecundity. In BPH, JH stimulates fat body Vg biosynthesis and uptake by the developing oocytes [29]. The reduced JH reported here may directly lead to decreased Vg expression and ovarian uptake. NlAC9 influences growth and fecundity in Nilaparvata lugens (Stål) This helps understand how suppressing NlAC9 leads to smaller body sizes and weights, reduced ovarian development and reproduction at the organismal and population levels.
One indirect effect of suppressing NlAC9 is the reduced fat body YLS abundances. YLS act in BPH nutrition [31] and loss of the YLS may interfere with reproduction via malnutrition, particularly with respect to nitrogen metabolism. We infer that the increased 20E signaling can exert negative influences at the organismal level. The idea of induced malnutrition may also explain the decreased body weights and sizes we recorded. The malnutrition may develop from reduced populations of YLS and from reduced feeding behavior. Quantitative nutrition is a problematic field and nearly impossible to analyze in very small insects such as BPH. Nonetheless, one of the broader outcomes of suppressing AC/PKA signaling would be seen in behavioral defects, such as feeding. cAMP/PKA signaling acts in feeding in honeybees and other insects [32,33]. We infer that altered feeding behaviors may account, in part, for the reduced body sizes and weights.
In BPH, reduced egg-laying is usually attended by decreased oviposition periods and elongated pre-oviposition periods. Here, dsNlAC9 treatments led to decreased egg-laying and decreased oviposition periods, without altering the pre-oviposition periods. This is an interesting outcome because the reduced AC/PKA signaling led to changes in one reproductive behavior but not the other. The separate effects indicate to us that some, certainly not all, behavioral aspects of BPH reproduction are influenced by AC/PKA signaling.