Estrogen-related receptor functions via the 20-hydroxyecdysone and IIS/TOR signaling pathways to regulate the development and morphology changes of ant Polyrhachis vicina Roger (Hymenoptera, Formicidae).

Estrogen-related receptor (ERR) is a key regulator of insect growth, development, and metabolic processes in insects; however, the molecular mechanisms underlying its effects are not fully understood. We investigated roles of 20-hydroxyecdysone (20E) and insulin/insulin-like signaling/target of rapamycin (IIS/TOR) signaling pathways in the effects of PvERR on larval development, metamorphosis, and adult growth in ant Polyrhachis vicina Roger. PvFOXO expression levels depended on caste and developmental stage. PvERR RNAi significantly reduced the expression levels of IIS/TOR signaling pathway genes and 20E signaling pathway genes in fourth-instar larvae, pupae, females, and workers and significantly increased the expression levels of IIS/TOR signaling pathway genes PvFOXO and PvAkt in males. PvFOXO RNAi resulted in developmental defects and increased mortality. After PvFOXO RNAi, the expression of PvERR, 20E signaling pathway genes, and IIS/TOR signaling pathway genes decreased significantly in pupae, females, and workers and increased significantly in fourth-instar larvae. Exogenous 20E attenuated expression changes induced by PvFOXO RNAi in a sex- and stage-specific manner. These results indicate that ERR interacts with 20E and IIS/TOR signaling pathways to regulate caste determination, metamorphosis, and male fertility in P. vicina and that correlations between PvERR and PvFOXO are caste- and stage-specific.

Toxicity effect of the edible pigment carmoisine on Polyrhachis vicina Roger (Hymenoptera: Formicidae).

Carmoisine belongs to a water-soluble synthetic dye and is often used as a food additive. Previous research has shown that carmoisine is toxic to rats and zebrafish, but there have been few reports on the effect of carmoisine on soil-dwelling social insects. The present study evaluated carmoisine toxicity in Polyrhachis vicina Roger. We found that the effects of different concentrations of carmoisine on the mortality of workers were dose-dependent. The 10% lethal dose (LD10), 50% lethal dose (LD50) and 90% lethal dose (LD90) of carmoisine to workers at 96 h was calculated to be 0.504, 5.491 and 10.478 g/L, respectivily. LD10 of workers were selected to treat the fourth instar larvae, pupae and adults for 10 days. The results showed that the survival rate of all ants, except for females, was significantly reduced, especially larvae and workers. The body weight of larvae, pupae and males decreased significantly, while weight gain was observed in the females and workers. The appearance of larvae, pupae and workers changed after carmoisine treatment, such as body darkening and epidermis shrinking of larvae and pupae, as well as body segment expansion of workers. Furthermore, carmoisine altered the expression of the estrogen-related receptor, tailless and homothorax of P. vicina (Pv-ERR, Pv-tll and Pv-hth) to varying degrees in larvae and adults. We believe that variations in body weight can lead to a decrease in survival rate and appearance changes in the ants, which may be related to abnormal gene expressions caused by carmoisine treatment. Therefore, we confirm that carmoisine has negative effects on the growth and development of P. vicina.

Expression of 20-hydroxyecdysone-related genes during gonadal development of Teleogryllus emma (Orthoptera: Gryllidae).

Insect gonads develop under endocrine signals. In this study, we assessed the characters of partial complementary DNAs encoding the Teleogryllus emma orthologs of 20-hydroxyecdysone (20E)-related genes (RXR, E75, HR3, Hsc70, and Hsp90) and analyzed their expression patterns in both nymph and adult crickets. 20E treatment suppressed expression of TeEcR, TeRXR, TeE75, TeHR3, TeHsc70, and TeHsp90. Temporal expression analysis demonstrated that TeERR and 20E-related genes were expressed in four stages of gonadal development from the fourth-instar nymph stage to the adult stage. The expression pattern of these genes differed in testicular and ovarian development. TeRXR, HR3, TeHsc70, and TeHsp90 were irregularly expressed in gonads of the same developmental stages, while mRNAs encoding TeERR, TeEcR, and TeE75 accumulated in higher levels in ovaries than in testes. RNA interference (RNAi) of TeEcR expression led to decrease of the expression levels of TeEcR, TeRXR, TeHR3, and TeHsc70, while it enhanced TeE75 and TeHsp90 expressions. These results demonstrate that the TeERR and 20E-related genes help regulate gonadal development, while TeEcR appears to inhibit TeE75 expression, TeE75 inhibits HR3 expression. Hsc70 indirectly regulated the expression of the primary and secondary response genes E74A, E75B, and HR3. Hsp90 regulated Usp expression with no direct regulatory relationship with EcR.

Vitellogenin regulates estrogen-related receptor expression by crosstalk with the JH and IIS-TOR signaling pathway in Polyrhachis vicina Roger (Hymenoptera, Formicidae).

The Estrogen-related receptor (ERR) can regulate the growth and development, metabolism, reproduction, and other physiological activities of insects, but its specific mechanism of action is still unclear. The aim of this study was to explore the relationship between expression of ERR and Vitellogenins (Vg) and the juvenile hormone (JH) and insulin/insulin-like growth factor/target of rapamycin (IIS/TOR) signaling pathways in Polyrhachis vicina Roger. P. vicina was used as the experimental model to clone the PvVg gene, perform double-stranded RNA synthesis and delivery and observe the effects of pharmacological treatments. The full-length PvVg cDNA product is 5586 bp. Higher PvVg mRNA expression was seen in the pupa and adults, and varying levels were seen in the different body parts of three different castes. RNA interference of PvVg expression led to disturbed development, an abnormal phenotype, and high mortality. PvVg RNAi also led to a reduction in mRNA levels of PvERR, ultraspiracle (PvUSP), forkhead box protein O (PvFOXO) and PvTOR genes in fourth instar larval, but a significant increase was seen in pupa and females. No significant change was seen in workers and males. After PvVg knockdown, application of exogenous JHIII reduced the expression of these genes in pupa and females, increased expression in workers, and decreased PvUSP mRNA expression in males. Both protein and mRNA expression levels of PvFOXO were affected by PvVg RNAi. PvERR RNAi increased PvVg expression in pupa and females and Kruppel-homolog 1 (PvKr-h1) and PvFOXO expression in males. The results of this study suggest that there is an interaction between PvERR and PvVg, and that crosstalk with the JH and IIS/TOR signaling pathways can affect development and reproduction. This effect is caste and developmental stage specific. We also speculate that the FOXO/USP complex participates in JH regulation of PvVg in P. vicina.

Molecular Cloning and Expression Analysis of 5-hydroxytryptamine Receptor 7 in Ant Polyrhachis vicina Roger (Hymenoptera: Formicidae).

Serotonin (5-hydroxytryptamine [5-HT]) is a monoamine neurotransmitter that plays an important role in regulating a variety of physiological and behavioral activities. In this study, the 5-HT7 receptor gene was cloned from the ant Polyrhachis vicina Roger (1863). The complete Pv5-HT7 receptor cDNA is 3054 bp, including a 5'-untranslated region (UTR) of 790 bp, a 3'-UTR of 752 bp and an open reading frame of 1512 bp encoding polypeptide of 503 amino acids. Hydrophobic analysis suggests that seven trans-membrane domains are the major sequence characteristic of the Pv5-HT7 receptor. In addition, the Pv5-HT7 receptor has three potential N-glycosylation sites, a palmitoylation site, three protein kinase A phosphorylation sites, and four protein kinase C phosphorylation sites. Phylogenetic analysis revealed that the deduced Pv5-HT7 receptor sequence shared a high homology with 5-HT7 receptor sequences of other species, such as a 78% similarity with the Am5-HT7 receptor (Apis mellifera). Real-time quantitative PCR (qRT-PCR) results showed that the expression level of the Pv5-HT7 receptor was low in the eggs and 1th-4th larval stages, but it was increased in the pupae stage and reached its peak in the adult workers. Western blot results showed that the highest protein expression was in the male body, head, and thorax. These results suggest that the Pv5-HT7 receptor may have specific functions in regulating the development of P. vicina, especially in adult formation and caste differentiation, feeding and caring behaviors of workers in the nest, and in the development of motor organs and mating behaviors in males.

Morphological changes in different caste adult ant specificities of Polyrhachis vicina Roger (Hymenoptera, Formicidae) caused in estrogen-related receptor.

The estrogen-related receptor (ERR) gene is a member of the nuclear receptor subfamily. Previous studies have indicated that ERR plays important roles in regulating insect growth and development. How ERR is associated with ant caste specificities remains unclear. In this study, we attempted to identify the role of ERR in the regulation of different adult caste specificities of Polyrhachis vicina Roger. Significant variations were detected in the ants including PvERR expressions, some physiological indexes and morphological traits including survival rate, body weight, body length, head width and abdominal appearance by different techniques. The results revealed that when PvERR expressions is up-regulated, boundaries of the abdominal segments were indistinct on the ventral side of the abdomen in males. Down-regulation of PvERR expressions caused abdominal swelling in males and a distended ventral abdomen in females and workers. Variation in PvERR expressions led to a remarkable decline in ant survival rates, particularly for males. These results indicated that different caste adults appeared to have different degrees of sensitivity in physiological response and morphological changes caused by variation in PvERR expressions. Thus, our data demonstrate that PvERR plays an important role in regulating the different adult caste specificities of P. vicina.

Relevance of estrogen-related receptor gene and ecdysone receptor gene in adult testis of the cricket Teleogryllus emma (Orthoptera: Gryllidae).

Estrogen-related receptor gene (ERR) and ecdysone receptor gene (EcR) belong to the nuclear receptor gene superfamily, both of which are associated with the regulation of insect reproductive development. However, the relationship between ERR and EcR and whether ERR participates in the 20E signal pathway during male reproduction are unclear. In this paper, adult male crickets Teleogryllus emma Ohmschi & Matsumura were divided into the experimental group, negative group, and control group. Crickets of the experimental group were injected with TeERR or TeEcR-dsRNA, and those in the negative group received EGFP-dsRNA. The efficiency of TeERR and TeEcR-RNAi was detected in the experimental group. Furthermore, the transcription level, morphological characteristics as well as weight were analyzed in the TeERR or TeEcR knocked-down testis. Results showed that the expression level of TeERR or TeEcR was significantly down-regulated (P < 0.05) when treated with 2000 ng TeERR or TeEcR-dsRNA for 48 h. The expression level of TeERR could be down-regulated (P < 0.05) using TeEcR-RNAi and vice versa. TeERR and TeEcR-RNAi caused morphological changes in testes, but they had no obvious effect on weight (P > 0.05). These results indicate that TeERR and TeEcR are intimately related to each other. In addition, TeERR may be involved in the 20E signal pathway and maintain the function of adult cricket testis.

Molecular cloning, characterization, and expression analysis of an ecdysone receptor homolog in Teleogryllus emma (Orthoptera: Gryllidae).

Ecdysteroids are steroid hormones that play important roles in the regulation of Arthropoda animal growth development, larvae ecdysis, and reproduction. The effect of ecdysteroids is mediated by ecdysteroid receptor (EcR). The ecdysone receptor (EcR) belongs to the superfamily of nuclear receptors (NRs) that are ligand-dependent transcription factors. Ecdysone receptor is present only in invertebrates and plays a critical role in regulating the expression of a series of genes during development and reproduction. Here, we isolated and characterized cDNA of the cricket Teleopgryllus emma (Ohmachi & Matsuura) (Orthoptera: Gryllidae) and studied mRNA expression pattern using real time-polymerase chain reaction. The full-length cDNA of T. emma EcR, termed TeEcR, is 2,558 bp and contains a 5'-untranslated region of 555 bp and a 3'-untranslated region of 407 bp. The open reading frame of TeEcR encodes deduced 531-amino acid peptides with a predicted molecular mass of 60.7 kDa. The amino acid sequence of T. emma EcR was similar to that of known EcR especially in the ligand-binding domain of insect EcR. Real-time quantitative reverse transcription-polymerase chain reaction was performed to compare TeEcR mRNA expression level at the whole body and gonad during T. emma development. The data revealed that TeEcR mRNA is differentially expressed during T. emma development, with the highest expression level in late-instar larvae of the body and lowest in third instar. The levels of TeEcR transcripts also vary among gonads development, and levels in ovaries were higher than in testes at every developmental stage. These results suggest that TeEcR may have potential significance to regulate the morphological structure and gonad development of T. emma, due to its expression in different developmental periods.

Effect of aspartame on survival, morphological and molecular levels of Polyrhachis vicina Roger (Hymenoptera, Formicidae).

Aspartame is a food additive that is widely used in the food industry. Previous data have shown that aspartame is toxic to humans and animals. However, there are few reports on the effect of aspartame on social insects living in the soil. The present study was designed to evaluate aspartame toxicity for Polyrhachis vicina Roger. Five dosages of aspartame were fed to the workers and 40 mg/kg (acceptable daily intake, ADI) aspartame was chosen to feed the 4th instar larvae, pupae, females and males in P. vicina. The results showed that the effect of aspartame on the survival rate of workers was in a time and dose dependent manner. The survival rate of 4th instar larvae, pupae and males decreased at 40 mg/kg aspartame. Meanwhile, we found that aspartame toxicity can cause weight and morphological changes. These changes may be related to the abnormal gene expression of Ecdysone receptor, Tailless and Extradenticle of P. vicina (PvEcR, PvTll and PvExd) resulting from aspartame treatment. The present study confirms the effect of aspartame toxicity on P. vicina even at the ADI dosage.

Cloning and expression analysis of a muscarinic cholinergic receptor from the brain of ant, Polyrhachis vicina.

Muscarinic acetylcholine receptors (mAchRs) are the predominant cholinergic receptors in the central and peripheral nervous systems of animals. They also have been found in various insect nervous systems. In this article, a full-length cDNA of a pupative mAchR (PmAchR) was obtained from the brains of ant Polyrhachis vicina by homology cloning in combination with rapid amplification of cDNA ends. PmAchR encodes a 599-amino acid protein that exhibits a high degree of homology with other mAchRs. Real-time quantitative RT-PCR analysis showed that PmAchR is differentially expressed in the brains of workers, males, and females. By in situ hybridization, it is revealed that PmAchR is widely expressed in different soma clusters of the brain, including the mushroom bodies, the antennal lobes, as well as the optic lobes (OL), and the most intensely staining is found in Kenyon cells. Nonetheless, there are more positive nerve fibers in the OL of males' brains than in females' and workers' brains.

Molecular cloning, characterization, and expression analysis of an estrogen receptor-related receptor homologue in the cricket, Teleogryllus emma.

The estrogen receptor-related receptors (ERRs) are a group of nuclear receptors that were originally identified on the basis of sequence similarity to estrogen receptors. The three mammalian ERR genes have been implicated in diverse physiological processes ranging from placental development to maintenance of bone density, but the function and regulation of ERRs in invertebrates are not well understood. A homologue of human ERR was isolated from the cricket Teleogryllus emma (Ohmachi and Matsumura) (Orthoptera: Gryllidae). The full-length cDNA of T. emma ERR, termed TeERR, has 1618 base pair (bp) and contains a 5'-untranslated region of 140 bp and a 3'-untranslated region of 272 bp. The open reading frame of TeERR encodes a deduced 401 amino acid peptide with a predicted molecular mass of 45.75 kilodaltons. The results of sequence alignments indicate that the TeERR protein shares an overall identity of 65%-82% with other known ERR homologues, and is most closely related to that of Nasonia vitripennis (Hymenoptera: Pteromalidae) and Apis mellifera (Apidae). Real-time quantitative reverse transcription-polymerase chain reaction was performed to compare the TeERR mRNA expression level at the whole body and gonad during T. emma development. The data revealed that TeERR mRNA is differentially expressed during T. emma development, with the highest expression level in embryos and the lowest in the body of late-instar larvae. The levels of TeERR transcripts also varied throughout gonad development; interestingly testicles had higher higher expression levels than ovaries at every development stage. These results suggest that TeERR has potential significance in the regulation of development in T. emma, due to its expression during different developmental periods.

RNA-Seq analysis of Polyrhachis vicina Roger and insights into the heat shock protein 90 and 70 families.

The heat shock protein 90 (Hsp90) and heat shock cognate proteins (Hsc70) have been identified as chaperones of the ecdysone receptor (EcR)/ultraspiracle protein (USP) heterocomplex. However, little is known about the status of Hsp90 and Hsc70 in Polyrhachis vicina Roger. Here, we sequenced the transcriptomes of adult ants in P. vicina for the first time. Clean reads in female, male, and worker ants were annotated into 40,147 transcripts, and 37,488, 28,300, and 33,638 unigenes were assembled in female, male, and worker ants, respectively. According to RPKM, the numbers of differentially expressed genes between female and male ants, between female and worker ants, and between male and worker ants and the common differentially expressed genes were 12,657, 21,630, 15,112 and 3704, respectively. These results reveal that caste differentiation, caste specificity formation, and social divisions of P. vicina ants may be due to gene expression differences. Moreover, PvEcR and PvUSP were also detected as differentially expressed genes in the ants; specifically, PvUSP expression was higher than PvEcR expression in all castes. We speculate that PvUSP may have a role similar to that of juvenile hormone receptor. Four identified PvHsp90 family members and 23 identified PvHsp70 family members were found in the ants, and 2 PvHsp90 genes and 8 PvHsp70 genes were analyzed by qRT-PCR. Among those genes, the expression of 2 PvHsp90 genes and 5 PvHsp70 genes coincided with the expression profiles of PvEcR and PvUSP, which suggest that the characterization of PvHsp90 and PvHsc70 may be as EcR/USP molecular chaperones in P. vicina.

A study on the correlation between IL1RAPL1 and human cognitive ability.

This study aimed to investigate the effects of IL1RAPL1 on the human cognitive ability. Four genetic marker sites, i.e., DXS1218, DXS9896, rs6526806 and rs12847959 on IL1RAPL1 were genotyped in 332 Qinba Mountain Area children. Meanwhile, a cognition test with a C-WISC scale was performed to study the relationship of genotype with cognition test scores. Results indicated that genotypes of DXS1218, DXS9896 and rs12847959 were associated with memory/concentration factor intelligence quotient (IQ) (P=0.027, 0.042, 0.029, respectively). DXS1218 also associated with full IQ, verbal IQ, and performance IQ (P=0.006, 0.014, 0.006, respectively). rs12847959 were related to verbal comprehension factor and perceptual organization factor IQ (P=0.021, 0.043, respectively). Further study on rat brain revealed that Il1rapl was mainly expressed in memory/concentration-associated encephalic regions, such as hippocampus, dentate fascia, osmesis perithelium, and piriform cortex. mRNA expression levels of Il1rapl in brains of rats with different learning and memory abilities showed significant difference. Combined data suggested that IL1RAPL1 affected human cognitive ability to some extent, especially the memory and concentration capability.

[Research progress on nonsyndromic X-linked mental retardation related gene PAK3].

Mutations of the gene coding for PAK3 (p21-activated kinase 3) are associated with nonsyndromic X-linked mental retardation (MRX). It may be associated with generalized or specific cognitive ability. Studying the biological and cognitive function of PAK3 is important for MR diagnosis and prevention. This paper reviews the progress on PAK3 including its gene product, biological and cognitive function. The future directions on the study of PAK3 are also discussed.