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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Mech Dev. Author manuscript; available in PMC Jul 1, 2010.
Published in final edited form as:
Mech Dev. Jul 2009; 126(7): 563–579.
Published online Mar 24, 2009. doi:  10.1016/j.mod.2009.03.005
PMCID: PMC2739235
NIHMSID: NIHMS127365

Juvenile hormone regulation of male accessory gland activity in the red flour beetle, Tribolium castaneum

Abstract

Male accessory gland proteins (Acps) act as key modulators of reproductive success in insects by influencing the female reproductive physiology and behavior. We used custom microarrays and identified 112 genes that were highly expressed in male accessory glands (MAG) in the red flour beetle, Tribolium castaneum. Out of these 112 identified genes, 59 of them contained sequences coding for signal peptide and cleavage site and the remaining 53 contained transmembrane domains. The expression of 14 these genes in the MAG but not in other tissues of male or female was confirmed by quantitative real-time PCR. In virgin males, juvenile hormone (JH) levels increased from second day post adult emergence (PAE), remained high on third day PAE and declined on fourth day PAE. The ecdysteroid titers were high soon after adult emergence but declined to minimal levels from 1-5 days PAE. Feeding of juvenile hormone analog, hydroprene, but not the ecdysteroid analog, RH-2485, showed an increase in size of MAGs, as well as an increase in total RNA and protein content of MAG. Hydroprene treatment also increased the expression Acp genes in the MAG. RNAi-mediated knock-down in the expression of JHAMT gene decreased the size of MAGs and expression of Acps. JH deficiency influenced male reproductive fitness as evidenced by a less vigor in mating behavior, poor sperm transfer, low egg and the progeny production by females mated with the JH deficient males. These data suggest a critical role for JH in the regulation of male reproduction especially through MAG secretions.

Keywords: Acps, microarray, JH, ecdysone, gene expression, RNAi

1. Introduction

The red flour beetle, Tribolium castaneum, is a well studied economically important model organism for sexual selection processes. Adults are highly promiscuous and employ several post-mating mechanisms to bias paternity. The factors influencing the male paternity share of female's progeny have been extensively studied (Schlager, 1960; Nilsson et al. 2003; Fedina and Lewis, 2006). The influence of male accessory gland products in determining the male paternity share remains unknown (Fedina and Lewis, 2008), mainly due to lack of detailed molecular and physiological characterization of these substances.

The secretory proteins from the male accessory glands (MAG), commonly referred to as Acps, are transferred to females along with sperm during copulation, and these substances have been shown to exert profound influences on female reproductive physiology and behavior (Eberhand and Cordero, 1995; Gillott, 2003; Wolfner, 2002). The reproductive tract genes of male Drosophila melanogaster are the best characterized system. So far, 112 Acps have been identified in D. melanogaster; the functional and mutational analyses on a few of them have shed light on Acps functions and their interactions with female physiological processes (Ravi Ram and Wolfner, 2005; 2007; Mueller et al. 2005; 2008). Attempts to identify orthologues of Acps outside D. melanogaster have met with limited success due to the rapid evolutionary change of male reproductive tract genes (Swanson et al. 2001). Therefore, it is necessary to identify Acps for each species individually.

Recent advances in bioinformatics, genomics and proteomics research have greatly helped in identifying Acps of non-drosophilids such as Ceratitis capitata (Davies and Chapman, 2006), honey bees (Collins et al. 2006), some orthopterans (Braswell et al. 2006), Anopheles gambiae (Dottorini et al. 2007), and Aedes aegypti (Sirot et al. 2008). Though the morphology of MAGs has been studied in tenebrionid beetles (Roberts and Grimnes, 1994; Novaczewski and Grimnes, 1996; Chen, 1984), the identification and functional characterization of Acps of beetles have not been reported so far. There are a few reports on secretory proteins from MAG of Tenebrio molitor (Paesen and Happ, 1994; Feng and Happ, 1996; Paesen et al. 1992; 1996; Yaginuma et al. 1996). Some genetic studies in T. castaneum showed influence of male gland substances on female physiology (Attia and Tregenza, 2004; Nilsson et al. 2002).

Insect hormones play key roles in reproduction (Raabe, 1986; Gillott and Gaines, 1992; Gillott, 1996). Ecdysteroids are important in the pupal growth and commitment to the adult pattern of protein synthesis for MAG during the preimaginal stage in most of the insects (Shinbo and Happ, 1989; Sridevi et al. 1988; Gallois, 1989). However, in postecdysial adult, JH accelerates the maturation of MAG in many insects (Chen, 1984; Couche et al. 1985; Davey, 1985; Leopold, 1976; Regis et al. 1985; Gold and Davey, 1989). In vitro studies and application of JH analogs suggested that JH stimulates Acps secretion in MAG (Yamamoto et al. 1988). Mutational studies identified JH binding proteins and the involvement of JH in protein synthesis and male mating behavior in D. melanogaster (Shemshedini et al. 1990; Wilson et al. 2003). The hormonal regulation of MAG secretions in other insects remains unknown.

Here we utilized the availability of T. castaneum genome sequence (Richards et al., 2007), custom microarrays, hormone analog treatments and RNAi in T. castaneum (Tan and Palli, 2008; Konopova and Jindra, 2007; Parthasarathy and Palli, 2008) to identify the Acps and study their hormonal regulation. Microarray analysis identified 112 genes that were highly expressed in MAGs of T. castaneum. The bioinformatics approach classified 59 out of 112 genes as putative secretory Acps. 33 out of the 59 secretory Acps showed sequence and/or protein function similarity with Acps identified in other insects. Expression analyses, hormone analog treatments and RNAi determined that JH, but not ecdysteroid play a major role in male reproduction by influencing the MAGs activity resulting in modulation of mating behavior and reproductive physiology in the male T. castaneum beetles.

2. Results

2.1. Male reproductive tract of T. castaneum

The male reproductive system comprises of a pair of testis, the efferent ducts, and glands (Fig. 1, panel a). A pair of lobed testis (Ts) occupies the major space in the male abdomen. The median tube is the secretory ejaculatory duct (Ed). Testes are connected to ejaculatory duct through the distal thin walled tube, vas deferentia (Vd), and a proximal thick walled tube, seminal vesicle (Sv). There are two pairs of male accessory glands: one pair of coiled tubular accessory glands (TAG) and one pair of rod-shaped accessory glands (RAG). The ducts of RAG, TAG, and the seminal vesicle terminate into ejaculatory duct at grand junction (Gj). The male accessory glands exist as rudimentary structures during the larval stage, proliferate and grow during the pupal stages. At imaginal ecdysis, the MAGs are fully formed. The sizes of MAGs increase during adult stage (Fig. 1, panels b-f); there is a significant increase in size during 2-3 days PAE which might mark the terminal differentiation and maturation of MAGs. For example, the TAG increase in size as evidenced by the increase in size of nuclei during 3-5 days PAE (subpanel d & f), but not in number, when compared to the size and number of nuclei on 1 day PAE (subpanel b).

Fig. 1
Nuclear staining of male reproductive system including accessory glands of T. castaneum by DAPI (a-f). Panel a: whole male reproductive tract; Ts, testis; Sv, seminal vesicle; Vd, vas deferentia; Ed, ejaculatory duct; Gj- grand joint; TAG, tubular accessory ...

2.2. Identification of Acps

Total RNA samples isolated from tissues of whole body or male accessory glands (MAG) dissected on day 5 PAE were labeled and hybridized to T. castaneum custom microarrays. Three biological replicates were included for whole body as well as MAG samples. Out of the 15,208 probe sets screened, hybridization to 11,739 probe sets was detected in at least one of the six RNA samples analyzed. The spot intensity data for these probe sets were statistically analyzed using GeneSpring software (Agilent Technologies). Fold differences in expression (calculated by dividing the mean value of signal intensities in MAG with that in the whole body sample) and the significance of difference (p value from t-test) for 11,739 probe sets are shown as a Volcano plot (Fig. 2). When compared to their expression in the whole body 924, 1410, and 1662 genes were expressed at three fold or more with a p value of <0.0001, 0.001 and <0.01 respectively in MAG (Table 1). Some of these observed differences could occur by chance; therefore, we applied multiple testing corrections to the microarray data. A less stringent Benjamini and Hochberg false discovery rate multiple testing correction applied to the microarray data identified 401, 1234, and 1621 genes that were expressed at three fold or more with a p value of <0.0001, 0.001 and <0.01 respectively in MAG when compared to their expression levels in the whole body. However, more stringent Bonferroni multiple testing corrections applied to the microarray data identified only 1, 4, and 69 genes that were expressed at three fold or more with a p value of <0.0001, 0.001 and <0.01 respectively in MAG when compared to their expression levels in the whole body (Table 1). 1234 genes identified by Benjamini and Hochberg test using the criteria of three-fold or more expression with a p-value of <0.001, were used for further analysis. The predicted protein coding regions of 249 out of these 1234 genes showed signal peptide cleavage sites based on screening by SignalIP 3.0 program. Comparison of expression of these 249 genes (code for proteins that contain potential signal peptide cleavage site) in MAG with their expression in the female whole body, fat body and ovary, identified 91 genes that were expressed at three fold or higher in MAG when compared to their expression in female tissues (data not shown).

Fig. 2
Volcano-plot of differentially expressed genes from microarray analysis. The p-values of t-test were plotted against fold-change in gene expression for all the genes. The horizontal lines in the plot represent the significance of 0.001, 0.0001 and 0.00001 ...
Table 1
Number of genes expressed at 3-fold or higher in MAG when compared to their expression in whole body on 5th day PAE

Comparison of amino acid sequences of 120 Acps found in D. melanogaster, Aedes aegypti, Anopheles gambiae, and Ceratitis capitata with the T. castaneum sequences, identified 48 orthologues with around 30% amino acid sequence identity. Out of these 48, 25 genes were identified in our microarrays as expressed at more than three-fold higher in MAG with p values less than 0.01 when compared to their expression in the whole body (Table 2). Interestingly, the proteins coded by 21 out of these 25 genes contained signal peptide cleavage sequences as predicted by SignalIP 3.0 program. The amino acid sequences coded by these 112 putative Acps (91 identified by microarrays and 21 identified by bioinformatics) were run in an InterProScan program. This program identified signal peptide in all 112 proteins. This program also identified transmembrane domains in 53 out of 112 proteins. Therefore, 59 out of these 112 proteins were probable secretory proteins. 16 out of these 59 putative Acps were orthologues of Acps identified in other insect species (Table 2) and the remaining 43 putative Acps are listed in Table 3. Probable functions of only 35 out of 59 Acps were predicted by annotation programs (Tables 2 & 3). The identified functions of Acps include protein processing including biosynthesis, folding, and metabolism (9); proteolysis (4); protease inhibitors (2); other metabolism (6); peptides/prohormones (4); protein binding (2). Among these 35 Acps, eight of them belong to immune response genes family with proteolysis and protease inhibitor functions. The functions of other 24 Acps are not known.

Table 2
Orthologues of Acps of other insects in T. castaneum
Table 3
Other Putative Acps of Tribolium castaneum

18 out of 59 Acps identified as described above were selected on the basis of different functional role or unknown functions, and analyzed for their expression in MAG and the whole body during 1-5 days PAE by qRT-PCR. The expression of all 18 Acps was detected in MAG. Out of 18 genes, the expressions of three Acps (TC009018, TC010066, TC013598) were expressed in the whole body of female at about 2-3-fold lower than that in MAG at day 5 PAE. TC012278 mRNA was detected in the whole body of male beetles at similar levels as in MAG. As the differences in the expression of these four genes between MAG and whole body male/female were lower than 3-fold observed in microarray analysis, they were eliminated from further analysis. The relative expression levels of the 14 confirmed Acps in the MAG, the whole body of male beetles during 1-5 days PAE, and in the whole body of the female at 5 days PAE are shown in Figure 3A. The expression levels of all the 14 Acps were very low in the whole body when compared to their expression in MAG. Particularly on day 5 PAE, the expressions levels of Acps in the MAG were 2.5-109.5-fold higher than in the whole body of male and female beetles, confirming our microarray data. However, the expression of TC004425 was only 2.5-fold higher in MAG than their expression in the whole body by qRT-PCR analysis, as opposed to their greater than 3-fold expression in MAG when compared to their expression in the whole body by microarray analysis.

Fig 3A
Relative mRNA levels of 14 putative Acp genes in MAG, the whole body of male beetles dissected at 1-5 days PAE, and the whole body of female beetles dissected at 5 day PAE analyzed by qRT-PCR. The expression level of ribosomal protein (rp49) was used ...

To analyze the gland-specific secretion of Acps, the expression of 14 secretory Acps were determined separately in TAG and RAG by qRT-PCR (Fig. 3B). 9 out of 14 Acps tested showed significantly higher expression levels in TAG than in RAG. Out of 9, two Acps (TC005744, TC009364) appeared to be TAG-specific since the expression of these two genes was very low in RAG. Three Acps showed significantly higher expression levels in RAG than TAG, out of which, two Acps (TC000232, TC012466) appeared to be RAG-specific. The expression levels of TC009307 and TC015200 did not differ significantly between TAG and RAG.

Fig 3B
Expression ratio of 14 putative Acps in TAG and RAG. TAGs and RAGs are dissected separately from 5 day-old male beetles. The relative mRNA levels were determined by using the expression level of ribosomal protein (rp49) as internal control. The X-axis ...

2.3. Hormonal titers in adult males

Reverse Phase HPLC/MS/MS Tandem mass spectrometry was used to determine the JH titer in the hemolymph of male beetles during 1-5 days PAE (Fig. 4A). JH III is identified as the major JH in T. castaneum. On day 1 PAE, 0.4 ng JH III/μl of hemolymph was detected. JH III levels gradually increased during 2-3 days PAE. The maximum level of 1.6 ng JH III/μl of hemolymph were detected on day 3 PAE. The titers then declined to 1.0 ng III/μl of hemolymph on day 4 PAE. JH titer decreased significantly on day 5 PAE.

Fig 4A
JH titer and expression patterns of JH biosynthesis genes (JHAMT) and other JH-response genes (jhe, Met, kr-h1, jhips, pepck) in the whole body of adult males determined by HPLC/MS/MS and qRT-PCR respectively. JH titer is expressed as ng of JH III/μl ...

An enzyme immunoassay (EIA) was used to determine the ecdysteroid levels in the male beetles during 0 to 5 days PAE (Fig. 4B). Ecdysteroids at 450 pg/male beetle were present on the first day of adult emergence. The ecdysteroid levels gradually decreased during 1-5 days PAE and reached 7 pg/male beetles by day 5 PAE.

Fig 4B
Ecdysteroid titer and mRNA levels of genes known to be involved in ecdysteroid biosynthesis (phantom and shade) and action (EcR and USP) in the whole body of adult males. Ecdysteroids were estimated by Enzyme immunoassay (EIA) in six independent groups ...

2.4. Expression profiles of JH- and 20E-response genes

The expression profiles of some of the genes known to be involved in biosynthesis and action of JH were determined in the whole body of male beetles from the time of adult emergence at 24 h intervals until 5 days PAE. Expression levels of juvenile hormone acid O-methyltransferase (JHAMT), juvenile hormone esterase (jhe), methoprene-tolerant, Met (known to play key role in JH action), Kruppel homologue 1 (kr-h1), JH inducible protein genes (jhip-07665, jhip-15272, jhip-14914) and pepck were quantified using qRT-PCR. As shown in Figure 4A, JHAMT mRNA levels were low at the beginning of the adult stage and started to increase on day 1 PAE and reached the maximum levels by day 3 PAE. Then the JHAMT mRNA levels declined and lower levels were detected on days 4 & 5 PAE. Low levels of JHE mRNA were detected on the day of adult emergence. Then the mRNA levels decreased and remained low until 3 days PAE. JHE mRNA levels increased again and showed higher levels on days 4 & 5 PAE. Met mRNA levels were higher on day 0 then declined gradually, and reached minimum levels by 4 days PAE. Kr -h1 mRNA levels were low on day 0, increased beginning on day 1, and reached the high levels by day 2 PAE and remained high during the 3 -5 days PAE. JHIP-07665 mRNA levels increased beginning on day 3 and reached the maximum by day 5 PAE. JHIP-15272 mRNA levels increased beginning on day 1 and reached the maximum by day 4 PAE and remained high during day 5 PAE. Higher levels of both JHIP-14914 and pepck mRNA were detected on days 3-5 PAE. These data showed that the titers of JH as well as mRNA levels genes known to be involved in biosynthesis (JHAMT), metabolism (jhe) and action (kr-h1) of JH increased from 0-5 days PAE. The expression profiles of some of the genes known to be involved in biosynthesis and action of ecdysteroids were also determined by qRT-PCR (Fig. 4B). The Phantom mRNA levels were high on the day of ecdysis and declined gradually and reached minimum levels by day 5 PAE, while the Shade mRNA levels fluctuated during 0-5 days PAE. The relative expression level of EcR mRNA did not change during 1-5 days PAE, while the mRNA levels of USP gradually increased to higher levels during 1-5 days PAE.

2.5. Hormonal regulation of gland size and content

To determine the hormonal regulation of gland size, the male beetles were fed on diet containing hydroprene (JH analog) and RH-2485 (ecdysteroid analog) upon emergence, and the length and area of both TAG and RAG dissected on day 5 PAE were determined (Table 4). Hydroprene treatment showed an increase in size of MAGs as evidenced by the significant increase in length and area of both TAG and RAG (Table 4), while RH-2485 treatment showed little effect on the size of MAGs when compared to the growth parameters of MAGs in control beetles treated with acetone. Also, the total RNA and protein content of MAGs dissected on day 5 PAE were determined (Table 5). Hydroprene exposure to adult showed highest total RNA (205.88%) and protein (38.81%) in MAGs when compared to those levels in MAGs of control beetles treated with acetone. RH-2485 treatment showed higher total RNA (64.71%), while the protein contents were lower (14.27%) in these MAGs when compared to those levels in MAGs of control beetles treated with acetone.

Table 4
Effect of hormone analogs and dsRNA injections on the size of MAGs
Table 5
RNA and protein concentrations in MAG by hormone analogs treatments

2.6. Effect of knock-down in the expression of genes involved in biosynthesis and action of JH and 20E

To study the role of individual genes involved in biosynthesis and action of JH and 20E on male reproduction, the expression of JHAMT, Met, kr-h1, Phantom, Shade, EcR, and USP were knocked down by injecting dsRNA into adult males soon after emergence. The expression levels of these genes were knock-down by 94% (JHAMT), 59% (Met), 63% (kr-h1), 98% (phantom), 92% (shade), 61% (EcR), and 96% (USP) when compared to the expression of respective genes in control insects injected with malE as determined by qRT-PCR on 4th day PAE (Fig. 5).

Fig. 5
Knock-down efficiency of genes by RNAi. dsRNAs were injected into males on day 0 PAE. At 4 days after injection, RNA was extracted and the relative expression in comparison to ribosomal protein (rp49) was determined by qRT-PCR. The expression levels of ...

RNAi mediated knock-down in the expression of JHAMT was shown to cause JH deficiency in T. castaneum (Minakuchi et al., 2008). To confirm that the knock-down in the expression of JHAMT results in JH deficiency in adult male beetle, the mRNA levels of JH-response gene, JHE were compared between acetone and hydroprene treated male beetles injected with JHAMT or malE (control) dsRNA. The JHE mRNA levels were reduced by 4.5-fold in JHAMT dsRNA injected insects when compared to the JHE levels in control insects injected with malE dsRNA (Fig. 6). Interestingly, JHE mRNA levels increased by 10-fold in beetles injected with JHAMT dsRNA and treated with hydroprene when compared to the levels of JHE mRNA in acetone treated insects. Also, in beetles injected with JHAMT dsRNA, the JHAMT mRNA levels were reduced by 10-fold when compared to the JHAMT mRNA levels in malE dsRNA injected beetles (Fig. 6). These data suggests that knock-down in expression of JHAMT results in reduced synthesis of JH and this JH deficiency could be compensated by application of JH analog, hydroprene.

Fig. 6
Effect of JHAMT RNAi on the expression of JH-responsive gene, jhe. dsRNA for JHAMT or malE were injected into 2-day old pupa. The adults were staged upon emergence. Acetone and hydroprene (1μg per insect in acetone) was applied topically on 3-day ...

The dsRNA injected males were mated with normal virgin females to determine the effect of RNAi on the egg and progeny production. The progeny production was evaluated as the number of eggs hatched out of the number of eggs laid by each pair in a 7-day period. The number of eggs and progeny produced were quantified and compared with control insects injected with malE dsRNA (Fig. 7A). Egg production was reduced by 1.7-fold and 2.5-fold in females mated with JHAMT and Met RNAi males when compared to the number of eggs produced by females mated with control males. In contrast, females mated with males injected with kr-h1, Phantom, Shade, EcR, and USP dsRNA did not show significant reduction in egg production when compared to the control group. A similar trend was observed with the progeny production. Also, treatment with hydroprene of both control insects injected with malE dsRNA and beetles injected with JHAMT dsRNA showed significant increase in egg production by 35 % and 48% respectively (Fig. 7B). These studies suggest that JH, but not ecdysteroids, is involved in male reproduction.

Fig. 7A
Effect of knock-down in expression of genes involved in biosynthesis and action of JH and 20E in males on the number of eggs and progeny produced by females mated with these males. dsRNAs were injected into males on day 0 PAE. At 4 days after injection, ...
Fig. 7B
Effect of hydroprene treatment in RNAi insects on egg production. dsRNA were injected into 2-day old male pupae. Adults were fed continuously on an acetone or hydroprene diet (1 ppm) soon after emergence. The mattings were performed as described in Fig. ...

To analyze the reasons for less egg/progeny production in JH deficient insects, the mating behavior, sperm production and transfer were compared among the insects injected with JHAMT, Met or malE dsRNA (Table 6). The mating behavior was evaluated by the time to first copulate and the frequency of re-mating within first 30 min. of exposure. The control male beetles injected with malE dsRNA copulated immediately on exposure to a female beetle (2.4 min.), while the time for first copulation of JHAMT and Met RNAi male beetles with female beetle was 6.9 min. and 9.0 min. respectively. malE dsRNA injected male beetle re-mated at high frequency (10 times) in first 30 min. of exposure while JHAMT and Met dsRNA injected male beetles mated 2-3 times only during 30 min. period. The highest number of sperms (1.21×105) per insect was present in the male reproductive tract of control beetles injected with malE dsRNA. Male beetles injected with JHAMT and Met dsRNA produced 40-50% less sperm than the control beetles. Also, the number of sperms transferred to female beetles after mating was 200 and 500% less in JHAMT and Met dsRNA injected male beetles when compared to control beetles injected with malE dsRNA. Hydroprene treatment of malE dsRNA injected beetles did not significantly increase the sperm production or transfer, though 11.5% increase in sperm production and a 27.3% increase in sperm transfer was observed. Hydroprene treatment of malE dsRNA injected beetles did not influence the mating behavior as the time to first copulate and mating frequency were similar to the untreated malE dsRNA injected beetles. However, JHAMT RNAi beetles treated with hydroprene registered 2-fold more number of sperm transferred to the female reproductive tract when compared with untreated JHAMT RNAi beetles. Hydroprene treatment of JHAMT RNAi beetles did not rescue the mating behavior, while 13.8% increase in sperm production was observed in male beetles.

Table 6
Effect of RNAi on mating behavior, sperm quantity and transfer

To confirm the role of JH in Acp production, the expression of JHAMT and Met were knocked down using RNAi technique in male beetles and the gland size and Acps expressions were determined in RNAi insects. Depletion of both JHAMT and Met mRNA levels in male beetles decreased the gland size (Table 4). A reduction of 37% of area of TAG and 21% of area of RAG was observed in JHAMT RNAi insects when compared to the area of MAGs of control beetles injected with malE. Met RNAi insects showed a decrease of 22% in area of TAG and 7% in area of RAG when compared to control beetles. Also, JHAMT and Met RNAi insects showed 20% and 15% reduction in the length of TAG and RAG respectively when compared to the length of MAGs of control beetles. Hydroprene treatment of JHAMT RNAi beetles rescued the size of RAG, where the length and area of RAG significantly increased when compared to that of untreated JHAMT RNAi insects (Table 4). However, hydroprene treated JHAMT RNAi beetles showed only 7% and 18.9% increase in the length and area of TAG that were not significantly different from the measurements of TAG of untreated JHAMT RNAi beetles.

In addition, the expression levels of 14 putative Acps in MAGs were compared among JHAMT, Met, and malE (control) RNAi insects (Fig. 8). The expression levels of 10 Acps were decreased by 40-60% in MAGs of JHAMT RNAi insects when compared to their expression levels in MAGs of control insects, while the expression levels of the remaining four Acps tested did not vary much from their expression in control insects. The mRNA levels of two Acps were reduced around 50% in MAGs of Met RNAi insects when compared to their expression levels in MAGs of control insects. The mRNA levels of 9 Acps were similar to the levels in control; however, the expression levels of three Acps were higher in MAGs of Met RNAi insects when compared to their expression levels in control insects. Out of 10 Acps that showed less mRNA levels in JH deficient insects (JHAMT RNAi insects), the expression levels of 4 Acps in MAGs were increased by 1.2 to 3-fold by hydroprene treatment, of which, 2 Acps (TC004425, TC014505) showed significant increase when compared to control insects treated with acetone (Fig. 9). These data suggest that the expression of some of the genes coding for Acps could be regulated by JH.

Fig. 8
The effect of knock-down in JHAMT and Met mRNA levels on the expression of 14 putative Acps. JHAMT, Met, and malE dsRNA were injected in two-day-old male pupae and MAGs are dissected from four-day-old adults. Total RNA was extracted and converted to cDNA ...
Fig. 9
Effect of hydroprene treatment on mRNA levels of selected secretory Acps in MAGs. The male beetles were fed continuously with either hydroprene (1 ppm) or acetone alone soon after emergence. MAGs were dissected 3 days PAE. The relative expression levels ...

3. Discussion

In this study, we have identified putative Acps of T. castaneum using the genome-wide screening. We used the following stringent criteria to filter the putative Acps from the microarray data: a) Benjamini and Hochberg false discovery rate multiple testing correction, b) p value of <0.001, c) greater than 3-fold expression in MAG than in the male whole body or the female whole body, ovary or fat body tissues, d) greater than 30% amino acid sequence homology to other insect's Acps. Based on these criteria, we have identified 112 genes that are MAG specific. The proteins coded by these genes are identified as secretory based on the presence of the signal sequences (Nielsen et al. 1997). Out of these 112, 53 of them contained transmembrane domains and hence, 59 out of these 112 are designated as putative secretory Acps of T. castaneum. Interestingly, the predicted functions of 35 secretory Acps are similar to the seminal-fluid protein functional classes across the organisms (Mueller et al. 2004; Braswell et al. 2006; Collins et al. 2006; Davies and Chapman, 2006, Ravi Ram and Wolfner, 2007). Besides, 16 out of 59 secretory Acps are orthologues of Acps of other insects. Conservation of sequence in 16 out of 59 secretory Acps is surprising considering that many Acps are rapidly evolving and diverge extensively among related insect species (Clark et al. 1995). 14 out of 59 putative Acps were confirmed to be male-specific by quantitative real-time PCR analysis. Nevertheless, these are the first Acps identified from the beetle genome.

The secretory proteins from TAG and BAG (equivalent to RAG of T. castaneum) of T. molitor have been reported (Happ, 1992). TAG produces a watery proteinaceous fluid that mixes with the sperm, while BAG produces secretions that contribute to the formation of spermatophore. Most of the TAG specific Acps of T. castaneum identified in the current study have conserved functions related to protein processing, metabolism or protease inhibitor activity most likely involved in sperm processing or storage, while most of the RAG specific Acps functions are unknown.

Regarding the hormonal regulation of male reproductive biology, numerous studies indicated the role of ecdysteroids in cell cycle, differentiation and commitment to adult fate of MAGs in several insects including T. molitor during the pre-ecdysial stage (Happ, 1992; Grimes and Happ, 1987; Happ and Happ, 1982; Happ et al. 1985). After adult ecdysis, JH has been reported to accelerate the maturation of accessory glands or is required for the renewal of secretory products after depletion during mating in several insects (Chen, 1984; Davey, 1985; Leopold, 1976). However, the differentiation of MAGs appears to be correlated with differences in reproductive strategies employed by different species of insects. For example, in the oriental silk moth, the glands terminally differentiate by adult ecdysis; in locusts, the process of maturation takes 15 days after adult eclosion, while in D. melanogaster, accumulation of adult specific proteins in MAGs occurred in pharate adults (Shinbo et al. 1987; Gallois, 1989; Chapman and Wolfner, 1988).

The JH and ecdysteroid levels determined in the present study showed high JH III levels during 2-4 days PAE in male beetles, while ecdysteroid levels steadily decline during 0-5 days PAE. The size of both TAG and RAG significantly increased starting from day 2 to day 5 PAE. Hence, in T. castaneum, ecdysteroid may play a role in MAGs differentiation and commitment to adult fate in the pharate adult stage as observed in several other insects; while JH appears to stimulate the growth of MAGs post adult ecdysis as observed in grasshoppers and locusts. We confirmed this effect of JH by oral feeding of hormone analogs. Both hydroprene and RH2485 is effective in male beetles; hydroprene induced JH-responsive genes (JHE, JHIP-07665), and RH-2485 induced 20E-responsive genes (EcR and HR3) in 12 h. after topical application at the dose of 1 μg/insect (data not shown). Hydroprene treatment showed significant increase in length and area of both TAG and RAG. Also, hydroprene treatment resulted in higher levels of total RNA and protein in MAG. Similar results were observed in other insects (Gold and Davey, 1989; Yamamoto et al. 1988). Treatment with ecdysteroid analog, RH-2485 resulted in an increase in total RNA to a lesser extent than with hydroprene treatment. In addition, RH-2485 treatment did not cause an increase in the protein content in the MAGs. It is not surprising because there are reports on the involvement of 20-hydroxyecdysone in the expression of few MAG secretory proteins, and putative EcRE has been identified in the upstream region of some of the Acp genes (Yaginuma and Happ, 1989; Feng and Happ, 1996). Since the ecdysteroid levels are declining after adult emergence, we assume that ecdysteroids do not play a significant role in male reproduction. However, the effect of existing ecdysteroids on MAG secretions could not be ruled out.

Genes involved in JH biosynthesis (JHAMT, Minakuchi et al. 2008a), JH action (Met, Konopova and Jindra, 2007; Parthasarathy et al. 2008; kr-h1, Minakuchi et al. 2008b), pepck (Beckstead et al. 2007), JHIPs (from our microarray data identified as JHIPs from BLAST search with homology to JHIPs of Aedes aegypti, Nene et al. 2007) and ecdysteroid biosynthesis (Phantom and Shade, Warren et al. 2004, Petryk et al. 2003; Niwa et al. 2004) and ecdysteroid action (EcR and USP, Yao et al. 1993) were selected for quantifying their mRNA levels in 0-5 day PAE male beetles. We selected only the terminal enzymes that are involved in the hormone synthesis owing to their importance in the biosynthetic pathway. Expression analysis in male beetles during 0-5 days PAE showed that JHAMT mRNA levels increased from 1-3 days PAE followed by an increase in the mRNA levels of JH-inducible genes during 3-5 days PAE. Interestingly, JHAMT mRNA levels correlated with the JH III levels. JHAMT mRNA levels were low during 0-2 days leading to low levels of JH titer during 1-2 days PAE. JHAMT mRNA levels increase more than 10-fold during 2-3 days PAE resulting in peak of JH titer at 3 days PAE. JH levels drops down with the decrease of JHAMT mRNA levels during 4-5 days PAE. JHE mRNA levels also increased after 3 days PAE following an increase in JH III titer. Previous studies in our laboratory showed that JH III induces the expression of jhe gene in T. castaneum (Parthasarathy et al., 2008). The expression levels of ecdysteroid biosynthesis gene, Phantom correlated well with the ecdysteroid titer. It is interesting to note that EcR mRNA levels did not change during 1-5 days PAE and USP mRNA levels increased gradually 1-5 days PAE indicating that these genes might be involved in the regulation of existing 20E action. The other genes involved in 20E action (e.g. HR3, br, ftz-f1) showed a steady decrease in their mRNA levels during 1-5 days PAE (Tan and Palli, unpublished). Comparison of expression patterns of genes involved in JH biosynthesis, metabolism and action as well as 20E biosynthesis and action during the critical 5 days PAE reveals that JH and its activity, but not the ecdysteroids and their activity, exhibits a major role in regulating reproduction.

This hypothesis was verified by knock-down in the expression of JHAMT, Met, kr-h1, Phantom, Shade, EcR, and USP genes by RNAi and studying their effect on reproduction. Interestingly, knocking down the expression of JHAMT or Met by RNAi decreased the egg production and progeny production by untreated females mated with RNAi males. In contrast, knocking-down the expression of Phantom, Shade, EcR, and USP did not show any effect on reproduction. Though EcR and USP mRNA levels remain high during 1-5 days PAE, knockdown of either of them did not show any effect on egg/progeny production. These studies support a critical role for JH in the regulation of male reproduction especially MAG activity.

Studies on male reproductive fitness showed that JH deficiency or its block of action by JHAMT and Met RNAi resulted in reduction of reproductive vigor of the injected male beetles. These male beetles had low sperm production, less vigorous mating behavior, latency for re-mating and poor sperm transfer when compared to their control counterparts. Similarly, Met27 mutants of Drosophila showed depressed courtship behavior (Wilson et al. 2003). Though sperm production in JHAMT and Met RNAi insects showed around 50% reduction when compared to control RNAi insects, these numbers are not statistically significant. Hence, JH role in spermatogenesis remain unclear. The significant reduction in mating behavior and sperm transfer observed in JH deficient insects might have resulted in the reduction of egg/progeny production. This might be a direct action of JH or the JH action might be mediated through MAG secretions. Interestingly, unlike other insects that show refractoriness to re-mating (Gillot, 2003), both males and females of T. castaneum tend to re-mate several times in a short span of time (Fedina and Lewis, 2008). In the present study, the frequency of re-mating and time to first copulate by JHAMT and Met RNAi male beetles were significantly lower when compared to those in control beetles. Further studies are required to understand the role of JH in these behavioral processes.

JH deficiency has been shown in several studies to result in lowered MAG protein synthesis (Shemshedini et al. 1990; Herndon, et al. 1997) and treatment with methoprene restored protein accumulation. Mutations in Met of D. melanogaster showed lowered protein content in MAG (Wilson et al. 2003). In the present study, depletion of JHAMT and Met mRNA levels by RNAi decreased MAG size as evidenced from the reduction in the length and area of both TAG and RAG. Also, the expression levels of some of the Acps were reduced by 40-60% in JHAMT or Met RNAi insects. Here we provide the direct evidence of JH deficiency caused by JHAMT RNAi by comparing the expression levels of jhe mRNA in malE and JHAMT RNAi insects that were treated with hydroprene after knock-down. It is interesting to note that addition of exogenous JH by hydroprene did not drastically increase the sperm production and transfer or mating behavior or egg production in the control insects injected with malE dsRNA. This could be due to the presence of sufficient quantities of endogenous JH in the system. However some of these suppressed effects (gland size, sperm transfer, and egg production) in JHAMT RNAi insects were partially rescued by hydroprene treatment, indicating that JH is indeed required for successful male reproductive functioning. Also, hydroprene treatment up-regulated the expression of 2 out of 14 Acps genes tested in MAG. Further studies are required to determine the JH role in the regulation of these Acps.

Met RNAi resulted in 1.5-2.5-fold increase of three Acps genes (TC000232, TC001124, TC015200) when compared to the malE RNAi insects. It is not clear whether JH action suppresses the expression of these Acps. It is interesting that Met mRNA levels decrease in male adults from emergence until day 5 PAE; JH III levels and mRNA levels of JH-response genes increased during this time (Fig. 4A). However, egg and progeny numbers reduction by untreated females mated with Met RNAi insects suggests a role for Met in male reproduction. Taken together, these data suggest that Met and other unidentified proteins play critical roles in JH action to regulate male reproduction. Further studies are required to identify various players in JH action that regulate male reproduction especially MAG development, Acp synthesis and secretion. Nevertheless, this study puts forth the possibility of utilizing the male reproductive system as a model system to understand the functional roles of Acps as well as the molecular mechanisms of JH actions in this model insect.

4. Materials and Methods

4.1. Rearing and staging

Strain GA-1 of T. castaneum was reared on organic wheat flour containing 10% yeast at 30°C under standard conditions (Parthasarathy et al. 2008). The pupae were sexed based on the structural differences of genital papillae according to Tribolium rearing protocol (http://bru.gmprc.ksu.edu/proj/tribolium/wrangle). Adult were staged as soon as emergence; the adults with untanned cuticle (teneral adults) were designated as 0 h and staged thereafter. The staged insects were maintained under similar conditions as mentioned above.

4.2. Determination of Juvenile hormone levels

Hexane extraction method was applied for JH extraction as described by Min et al. (2004) and Chen et al. (2007) with slight modification. Briefly, hemolymph samples were added immediately after collection to a glass tube containing 0.5 ml acetonitrile. The samples were kept under -80°C until JH extraction. Ten nanogram methoprene was added to each sample as internal standard. 0.5 ml of 0.9% NaCl was added, and the sample was extracted twice with 1 ml hexane. The tube was vortexed vigorously after adding the hexane and centrifuged at low speed(~5,000g) for 5 min. The hexane (upper) phase was removed and dried completely under Speed-Vac. One hundred microliter hexane was added to each tube for collecting JH at the bottom of the tube and dried completely as above. 15-40 μl MeOH was added, and the samples were stored at -80°C until analysis.

Reverse Phase HPLC/MS/MS Tandem mass spectrometry was carried out as described by Chen et al. (2007) with slight modification using a micro-capillary gradient LC system (1100 series, Agilent Technologies, San Jose, CA) on-line with an Esquire-LC ion-trap (Bruker Instruments, Bellerica, MA). A reverse phase C18 MS column (TARGA C18 3μm, SN 140715, 300 μm × 5 cm, purchased from Higgins Analytical, Inc., Mountain View, CA USA) was used at a flow rate of either 6 μl/min using mobile phase A, water and mobile phase B, methanol. The column was equilibrated at 10% B and the gradient was started at 10% B, increased to 60% B in 5 minutes, followed by an increase to 80% B within 3 minutes, held at 80% B for 2 minutes, increased to 90% B in 3 minutes, followed by an increase to 100% B in 5 minutes, followed by a decrease to 10% B within 5 minutes. A total separation time of 28 minutes was used. The effluent from the HPLC separations was introduced on-line into the orthogonal Esquire-LC electrospray source. The ion spray for most experiments was performed in positive mode at +4000 V; endplate offset was -500V. Ions were scanned from 150 to 1000 m/z; the nebulizer gas was set to 23 psi, the dry gas to 7 L/min, and the drying temperature at the capillary entrance was 250°C. Capillary exit was set to 76.1 V, skimmer 1 at 26.1 V, trap drive to 58; an average of 3 spectra were acquired over a time period of 100 ms. The collision gas was ultra-pure helium, and MS/MS experiments were performed in the auto mode. JH from unknown sample was quantified based on the peak area of internal standard, methoprene.

4.3. Determination of Ecdysteroid levels

The staged insects were washed thoroughly in double distilled water and dried. The insects were homogenized in 250 μl of ice-cold 75% aqueous methanol and centrifuged at 13,000g at 4°C. Supernatants were transferred to 6×50 mm borosilicate glass tubes; precipitates were resuspended in an additional 100 μl of aqueous methanol and kept on ice for 30 min. After centrifugation as above, the precipitates were vacuum dried. An enzyme immunoassay (EIA) was used to estimate ecdysteroid titers as previously described (Kingan and Adams, 2000; Gelman et al. 2002; Margam et al., 2006).

4.4. Hormone analogues treatment

Hydroprene (Ethyl (2E,4E,7S)-3,7,11-trimethyl-2,4-dodecadienoate) was a gift from Wellmark International (Dallas, TX). Technical grade compound was dissolved in acetone and used at a final concentration of 1 ppm in feeding bioassays. RH2485, an ecdysone analog, was used at 1 ppm in feeding bioassays. 1 g of diet containing wheat flour with 10% yeast was used and the compounds were mixed to give a final concentration of 1 ppm. Topical application of hydroprene or RH-2485 on beetles was done with the concentration of 1 μg/insect in acetone solvent. The adults were also treated with equivalent amounts of acetone alone as a control in both cases.

4.5. Microarray analysis

Total RNA was isolated from the whole body and MAGs at 5 days PAE using spin columns (RNeasy, Qiagen). The integrity of RNA was verified using an Agilent 2100 Bioanalyzer (Agilent Technologies). 200 ng of total RNA from three replicates from each treatment was labeled using Agilent Low RNA input fluorescent linear amplification kit following manufacturer's instructions. Labeled cDNAs were purified using RNAse mini purification columns (Qiagen). 15 pmoles of fluorescently labeled cDNAs were used for hybridization.

The 60-mer-oligonucleotides designed based on 15,008 genes selected from the 16,000 genes predicted by T. castaneum genome annotations and 736 control probe sets were printed onto glass slides at Agilent Technologies. The hybridizations were performed according to manufacturer's instruction (Agilent Technologies). The microarray slides were scanned using Typhoon 9410 scanner (GE HealthCare). The images were analyzed using ArrayVision v.8.0 software (Imaging Research Inc.). The normalization and t-test statistical analysis of the data were done. The data were finally subjected to the Bonferroni and Benjamini & Hochberg false discovery rate multiple testing corrections using GeneSpring GX v.9.0.1 software.

4.6. Double-stranded RNA (dsRNA) synthesis and injection

dsRNA were synthesized using the Ambion MEGAscript transcription kit (Ambion, Austin, TX). Cognate primers designed based on the sequences available in the Beetlebase with T7 polymerase promoter sequence at their 5' ends were used for PCR reactions. The resultant PCR products were used for transcription reaction as per the instruction manual. Briefly, dsRNA was injected into the male beetle on day 0 PAE on the ventral side of the first abdominal segment using a aspirator tube assembly (Sigma) fitted with 3.5” glass capillary tube (Drummond) pulled by a needle puller (Model P-2000, Sutter Instruments Co.). Injected adults were reared under standard conditions until use. Control adults were injected with dsRNA for Escherichia coli malE gene. Injection of Phantom, Shade, EcR, and USP dsRNA during the pupal stage causes block in pupal-adult metamorphosis and hence all dsRNA injections were carried out in the adult stage for uniformity, unless otherwise stated.

4.7. cDNA synthesis and Quantitative real-time reverse-transcriptase PCR (qRT-PCR)

Total RNA was extracted from the whole body/fat body/ovaries of staged adults and from insects injected with dsRNA for specific genes using TRI reagent (Molecular Research Center Inc., Cincinnati, OH). cDNA was synthesized using 2 μg of DNAse1 (Ambion, Austin, TX) - treated RNA and iScript cDNA synthesis kit (Biorad Laboratories, Hercules, CA) in a 20 μl reaction volume as per the manufacturer's instructions. Real-time quantitative reverse-transcriptase PCR was performed using MyiQ single color real-time PCR detection system (Biorad Laboratories). PCR reaction components were: 1 μl of cDNA, 1 μl each of forward and reverse sequence specific primers (designed based on the sequences available in the Beetlebase, Table 7), 7 μl of H2O and 10 μl of supermix (Biorad Laboratories). PCR conditions were: 95°C for 3 min followed by 45 cycles of 95°C for 10seconds, 60°C for 20 seconds, 72°C for 30 seconds. Both the PCR efficiency and R2 (correlation coefficient) values were taken into account prior to estimating the relative quantities. Relative expression levels of each gene were quantified using ribosomal protein, rp49 expression levels as an internal control.

Table 7
List of primers used

4.8. Estimation of sperm, egg, progeny counts, and mating assays

JHAMT, Met, and malE (control) dsRNA were injected into freshly emerged male beetles as described above. The sperm counts were determined in male beetles injected with dsRNA and also in female beetles mated with dsRNA injected males as per the methods described (Reichardt and Wheeler, 1995). Briefly, the sperm dispersed in the buffer (1X PBS containing 1% BSA) from the reproductive tracts were either fixed or stained directly with nuclear stain, DAPI. After serial dilutions, 10 μl of sperm suspension were placed on a Neubaeur hemocytometer and analyzed under the microscope. For male beetles, the virgin beetles were reared in isolation and the sperm collected in the seminal vesicle of male reproductive tract at the end of 5 days PAE were dissected out and counted. For female beetles, the virgin female beetles were exposed to dsRNA injected male beetles on a single pair basis after 24 h of pre-mating in the mating arena as described in Pai and Yan (2003). The mating experiments were done 5 days after dsRNA injection. The time to first copulation and the frequency of copulation in the first 30 min. were determined. After 30 min., the sperm transferred to females (mainly seen in the bursa and oviduct of the female reproductive tract) were dissected out and counted as described above.

JHAMT, Met, Kr-h1, Phantom, Shade, EcR, and USP and malE (control) dsRNA were injected into freshly emerged male beetles as described above. After five days of injection, the dsRNA injected male beetles were mated with same age virgin female beetles on a single pair basis. The eggs laid by female beetles were determined after 7 days of exposure. The beetles were removed and the no. of progeny hatching from the eggs was determined after 14 days of exposure.

4.8. Imaging and documentation

For fluorescent images, an Olympus 1×71 Inverted Research Microscope fitted with reflected fluorescence system was used. DAPI was excited using 405 nm laser line. Control of the microscope, as well as image acquisition and exportation as TIFF files, was conducted using MegnaFire software version 1.5. Exposure settings that minimized oversaturated pixels in the final images were used. Figures of all micrographs were assembled using Photoshop 7.0.

Acknowledgement

This work was supported by National Science Foundation (IBN-0421856) and National Institute of Health (GM070559-04). We would like to thank Dr. Nigel Cooper and Ms. Xiaohong Li of University of Louisville for help with microarray analysis. We also would like to thank Dr. Perry of University of Kentucky for microscope facility. This is contribution number 07-08-075 from the Kentucky Agricultural Experimental Station.

Footnotes

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