Characterization and Developmental Expression Patterns of Four Hexamerin Genes in the Bumble Bee, Bombus terrestris (Hymenoptera: Apidae).

Hexamerins are members of the hemocyanin superfamily and play essential roles in providing amino acids and energy for the nonfeeding stages of insects. In this study, we cloned and analyzed the expression patterns of four hexamerin genes (hex 70a, hex 70b, hex 70c, and hex 110) at different worker development stages and queen diapause statuses in the bumble bee, Bombus terrestris. The results of this study showed that hex 110 has the longest open reading frame (ORF; 3,297 bp) compared to the ORFs of hex 70a (2,034 bp), hex 70b (2,067 bp), and hex 70c (2,055 bp). The putative translation product of Hex 70a, Hex 70b, Hex70c, and Hex 110 has 677, 688, 684, and 1,098aa with predicted molecular mass of 81.13, 79.69, 81.58, and 119 kDa. In the development stages of workers, the expression levels of hex 70a, hex 70b, and hex 70c increased gradually from the larval stage and exhibited high expression levels at the pink eyed and brown eyed pupae stage, whereas hex 110 exhibited the highest expression level at the larval period. Four hexamerin genes were highly expressed at the prediapause status of queen (P < 0.05), and compared to the eclosion queen, the lowest upregulation was 3.7-fold, and the highest upregulation was 1,742-fold. The expression levels of hex 70b, hex 70c, and hex 110 at diapause were significantly higher than those at postdiapause (P < 0.05). In conclusion, hexamerins may play important roles in queen diapause and metamorphosis of larval and pupal stages.

Assessment of the value of ecosystem services in water sources of the South-North Water Diversion Central Project: the case of Dengzhou City, Henan Province.

Dengzhou City is the first ecological water recharge city after the commissioning of the South-North Water Diversion Project. A study on the ecological service value of Dengzhou City and its evolutionary characteristics can provide a theoretical basis for the water supply security of the Central Line Project and regional ecological environmental protection. Based on the ecosystem service value equivalent factor method, GIS, ENVI and GeoSOS-FLUS software were used to monetize the ecosystem service values of Dengzhou City in 2007, 2011, 2014, 2015 and 2020; analyze the spatial and temporal change characteristics under its land use change conditions and predict the regional ecosystem service values of Dengzhou City in 2025. The results show that since the completion and commissioning of the South-North Water Transfer Central Project in 2014, the value of ecosystem services in Dengzhou City has rapidly increased from US$3.792 billion to US$4.612 billion, and in 2025, the value of ecosystem services in Dengzhou City is expected to reach US$4.8 billion.

Evaluation of wetland ecosystem services value of the yellow river delta.

Analyzing and evaluating the ecosystem service value of wetlands can provide technical support and theoretical basis for regional socio-economic development, ecological environmental protection, and other planning measures. Based on the factors of hydrology, meteorology, society, economy, resources, environment, and culture, the wetland ecosystem services value of Yellow River Delta (YRD) was evaluated by the method of functional value and equivalent factor, which has certain reference significance for future research. Two methods were used to calculate the ecosystem services value of YRD in 2016, and the results were $3.28 × 109 per year and $4.53 × 109 per year, respectively, and the regulation services accounted for 76.3% and 16.7%, respectively. Through comparative analysis of ecosystem services value from 2001 to 2016, the average proportion of regulation services was 51%. Regulation service is the leading function of wetland ecosystem of YRD. Equivalent factor method is more suitable for the assessment of wetland ecosystem service value of YRD in 2016. In terms of time, ecosystem services value tended to increase, with an average rate of $2.02 × 109 per year. In terms of space, it gradually increased from inland to shallow sea water. On the premise of rational development and utilization, it is predicted that the value of ecosystem services will continue to grow in the future.

IRP30 promotes worker egg-laying in bumblebee, Bombus terrestris (Hymenoptera: Apidae).

Bumblebees are important pollinators that have evolved between solitary and advanced eusocial insects. Compared with advanced honeybees, workers of social bumblebee species are prone to laying eggs during the competition phase, which leads to the end of the colony. Therefore, worker reproductive behavior has become a popular research topic for exploring various biological phenomena. Here, we demonstrate a novel reproduction-related function of an immune response protein-encoding gene (Immune Responsive Protein 30, IRP30) in Bombus terrestris by employing RNA interference (RNAi) and a transgenic Drosophila melanogaster system. The results show that worker egg-laying was significantly affected by IRP30 expression levels (P < 0.01). Compared with those in the dsGFP-treated groups, the first egg-laying time was delayed by 3.7 d and the egg number was decreased by 41% in the dsIRP30-treated group. In addition, the average size of the largest oocyte and the relative mRNA expression levels of Vg (vitellogenin) were significantly reduced in the dsIRP30-treated group (P < 0.05). Cellular localization by immunofluorescence demonstrated that IRP30 has important functions in the germ cells of workers' ovarioles. Overexpression of IRP30 was confirmed to increase the reproductive capability of the transgenic D. melanogaster. In conclusion, IRP30 regulates worker egg-laying by affecting the expression of Vg, the size of the ovary and the formation of the oocyte. These findings provide essential information for understanding the mechanisms underlying worker reproductive regulation.

Characterization of a Vitellogenin Receptor in the Bumblebee, Bombus lantschouensis (Hymenoptera, Apidae).

The vitellogenin receptor (VgR) belongs to the low-density lipoprotein receptor (LDLR) family, responsible for mediating the endocytosis of vitellogenin (Vg) into the ovaries to promote ovarian growth and oviposition. Here, we cloned and measured VgR gene expression characteristics in the bumblebee Bombus lantschouensis. RNA interference was used to validate VgR function. The results showed that the full length of the BLVgR cDNA was 5519 bp, which included a 5280 bp open reading frame encoding 1759 amino acids (AAs). Sequence alignment revealed that the protein contained 12 LDLa, 5 EGF, 2 EGF-CA and 10 LY domains. Phylogenetic analysis showed that BLVgR and the VgR of Bombus terrestris clustered together and the tree of bumblebees (Bombus) appeared as one clade next to honeybees (Apis). Transcript expression analysis showed that BLVgR was expressed in all tested tissues and showed the highest abundance in the ovaries. BLVgR expression was present in all developmental stages. However, the expression level in larvae was extremely low. In addition, the expression of BLVgR was significantly upregulated after egg laying in both workers and queens. In new emerging workers injected with 5 µg of VgR dsRNA, the expression level of BLVgR was 4-fold lower than that in the GFP dsRNA-injected group after 72 h. Furthermore, BLVgR silencing significantly reduced the number of eggs laid (3.67 ± 1.96 eggs) and delayed the first egg-laying time (16.31 ± 2.07 days) in worker microcolonies when compared to dsGFP (37.31 ± 4.09 eggs, 8.15 ± 0.22 days) and DEPC-treated water injected controls (16.42 ± 2.24 eggs, 10.00 ± 0.37 days). In conclusion, the BLVgR gene and its reproductive function were explored in the bumblebee B. lantschouensis. This gene plays an important role in egg laying time and egg number.

microRNA-126 inhibits tube formation of HUVECs by interacting with EGFL7 and down-regulating PI3K/AKT signaling pathway.

It's critical for tube formation and angiogenesis to repair ischemic myocardium or stroke. This study aimed to investigate role of microRNA-126 (miR-126) in tube formation in human umbilical vein endothelial cells (HUVECs) and associated mechanisms. Primary neural stem cells (NSCs) and HUVECs were cultured and transfected with microRNA-126 mimics and miR-126 inhibitor. Cell counting kit-8 (CCK-8) and cell cycle assay were conducted for evaluating NSCs viability. Transwell assay was conducted to observe invasive ability of HUVECs. Quantitative real-time PCR (qRT-PCR) assay was used to examine epidermal growth factor like domain 7 (EGFL7) and miR-126 mRNA both in vitro and animal models. Tube forming capability was evaluated in HUVECs. Dual luciferase assay was performed to evaluate interaction between miR-126 and EGFL7 gene. Western blot assay was used to determine phosphoinositide-3-kinase/protein kinase-B (PI3K/AKT) signaling molecules and EGFL7. The results indicated that miR-126 significantly decreased cell viability, inhibited invasive ability and modulated cell cycle of NSCs compared to miR-NC group (p < 0.05). miR-126 significantly inhibited tube formation of HUVECs compared to miR-NC group (p < 0.05). miR-126 significantly down-regulated EGFL7 mRNA and protein expression compared to miR-NC (p < 0.05). Atorvastatin significantly increased CD34 and enhanced EGFL7 expression in traumatic brain injury (TBI) rats brain tissues compared to Model group (p < 0.05). miR-126 significantly down-regulated and atorvastatin up-regulated PI3K/AKT signaling pathway (p < 0.05). Atorvastatin significantly increased EGFL7 and down-regulated miR-126 expression in TBI rats brain tissues compared to Model group (p < 0.05). miR-126 interacted with and negatively correlated with EGFL7 gene both in vitro and in TBI models. In conclusion, microRNA-126 inhibited tube formation of HUVECs by interacting with EGFL7 and down-regulating PI3K/AKT signaling pathway.

The complete mitochondrial genome of wild honeybee Apis florea (Hymenoptera: Apidae) in south-western China.

We sequenced almost complete mitochondrial genome of Apis florea (Insecta: Hymenoptera: Apocrita: Apidae) with length of 15,933 bp. The genome has similar codon usage and gene organization to those of mitogenome reported for other Hymenoptera. It includes 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNA genes and a noncoding region with very high AT-base content. Phylogenetic analyses showed that, Apis florea and (Apis cerana+ Apis mellifera) are the sister taxa, and more original, which is widely accepted view.

mRNA expression and DNA methylation in three key genes involved in caste differentiation in female honeybees (Apis mellifera).

In honeybee (Apis mellifera) colonies, queens and workers are alternative forms of the adult female honeybee that develop from genetically identical zygotes but that depend on differential nourishment. Queens and workers display distinct morphologies, anatomies and behavior, better known as caste differentiation. Despite some basic insights, the exact mechanism responsible for this phenomenon, especially at the molecular level, remains unclear although some progress has been achieved. In this study, we examined mRNA levels of the TOR (target of rapamycin) and Dnmt3 (DNA methyltransferase 3) genes, closely related to caste differentiation in honeybees. We also investigated mRNA expression of the S6K (similar to RPS6-p70-protein kinase) gene linked closely to organismal growth and development in queen and worker larvae (1-day and 3-day old). Last, we investigated the methylation status of these three genes in corresponding castes. We found no difference in mRNA expression for the three genes between 1st instar queen and worker larvae; however, 3rd instar queen larvae had a higher level of TOR mRNA than worker larvae. Methylation levels of all three genes were lower in queen larvae than worker larvae but the differences were not statistically significant. These findings provide basic data for broadening our understanding of caste differentiation in female honeybees.

Quantification of major royal jelly protein 1 in fresh royal jelly by indirect enzyme-linked immunosorbent assay.

Rabbit polyclonal antibody produced by a major royal jelly protein 1 (MRJP1) specific peptide reacted only with a MRJP1. Indirect ELISA with the antibody revealed a MRJP1 level of 4.12-4.67 g/100 g in different company's royal jelly, which almost agreed with that of a hexametric form of MRJP1 (apisin) measured by high performance liquid chromatography. These results suggest that MRJP1 exists mainly as apisin in royal jelly.

[Phylogenetic relationships of honey bees (Hymenoptera: Apinae) inferred from mitochondrial and nuclear DNA sequences].

The sequences of mitochondrial ND2, CO2, and 16S rRNA genes and nuclear ITPR gene were obtained from 22 samples of 5 Apis species from China. The characteristics of the sequences and the pairwise distances among species were analyzed. Phylogenetic trees were reconstructed for Apis species using maximum parsimony, neighbor-joining and maximum likelihood methods together with the sequences of the other 4 Apis species downloaded from GenBank. Results supported that Apis species were divided into three major clusters: dwarf bees (A. florea and A. andreniformis), giant bees (A. dorsata and A. laboriosa), and cavity-nesting bees (A. mellifera, A. cerana, A. koschevnikovi, A. nigrocinta, and A. nuluensis). The dwarf honey bees were confirmed as basal lineage. Our study also revealed a high level of genetic divergence between A. dorsata from Hainan Island and China mainland.

Role of procalcitonin in the diagnosis of severe infection in pediatric patients with fever and Neutropenia--a systemic review and meta-analysis.

OBJECTIVE: The aim of this study was to determine the accuracy of the procalcitonin (PCT) test for diagnosis of bacterial sepsis in pediatric cancer patients with febrile neutropenia. METHODS: Three major databases, MEDLINE, EMBASE and the Cochrane Library were searched for studies that evaluated the diagnostic value of PCT alone or compared with other laboratory markers such as C-reactive protein (CRP) to identify bacterial sepsis in children with fever and neutropenia. A bivariate model was used to derive summary sensitivity and specificity of the diagnostic tests. RESULTS: A total of 10 studies looking into PCT tests and 8 studies looking into CRP tests were included in the final analysis. The prevalence of bacterial sepsis was 304 of 1031 (29.5%) in PCT studies and 741 of 1316 (56.3%) in CRP studies. In terms of area under the receiver operating characteristic curve, PCT had comparable discrimination to CRP (area under the curve: 0.75 versus 0.74). PCT was not as sensitive as the CRP test. The pooled sensitivity of PCT was 0.59 (95% confidence interval [CI]: 0.42-0.74) as compared with 0.75 (95% CI: 0.61-0.85) for CRP. PCT was more specific than sensitive whereas CRP was more sensitive than specific in this population. The pooled specificity was 0.76 (95% CI: 0.64-0.85) for PCT and 0.62 (95% CI: 0.49-0.73) for CRP. PCT had greater likelihood ratio positive (2.50; 95% CI: 1.64-3.81), making it the better rule-in test. CONCLUSIONS: Of three markers potentially useful for diagnosing bacterial sepsis in children with fever and neutropenia, PCT had comparable diagnostic accuracy to CRP.

A method of extracting ginsenosides from Panax ginseng by pulsed electric field.

The objective of this study was to develop and optimize a pulsed electric field (PEF) extraction method. Various experimental conditions, including electric field intensity and frequency, were evaluated against extraction methods. The content of six major ginsenosides (Rg(1), Re, Rb(1), Rc, Rb(2), and Rd) were quantified by HPLC. The results indicated that the highest yield of the ginsenoside is 12.69 mg/g by PEF using the conditions of 20 kV/cm electric field intensity, 6000 Hz frequency, 70% ethanol-water solution, and 150 L/h velocity. The yield of the ginsenoside of PEF extraction method is higher than the other five methods, such as microwave-assisted extraction, heat reflux extraction, ultrasonic-assisted extraction, accelerated solvent extraction, and ultrahigh pressure extraction, The whole extraction process of PEF takes less than 1 s, which is much less than the heat reflux extraction method for 6 h and even newly used technique ultrahigh pressure extraction method of 2 min. The high efficiency, shorter extraction times, and lower energy cost of PEF extraction method can be applied in the industrial production of saponins from Panax ginseng. The PEF extraction method is a promising and constructive method to extract ginsenosides.

Gigantism in honeybees: Apis cerana queens reared in mixed-species colonies.

The development of animals depends on both genetic and environmental effects to a varying extent. Their relative influences can be evaluated in the social insects by raising the intracolonial diversity to an extreme in nests consisting of workers from more than one species. In this study, we studied the effects of mixed honeybee colonies of Apis mellifera and Apis cerana on the rearing of grafted queen larvae of A. cerana. A. mellifera sealed worker brood was introduced into A. cerana colonies and on emergence, the adults were accepted. Then, A. cerana larvae were grafted for queen rearing into two of these mixed-species colonies. Similarly, A. cerana larvae and A. mellifera larvae were also grafted conspecifically as controls. The success rate of A. cerana queen rearing in the test colonies was 64.5%, surpassing all previous attempts at interspecific queen rearing. After emergence, all virgin queens obtained from the three groups (N=90) were measured morphometrically. The A. cerana queens from the mixed-species colonies differed significantly in size and pigmentation from the A. cerana control queens and closely approximated the A. mellifera queens. It is inferred that these changes in the A. cerana queens reared in the mixed-species colonies can be attributed to feeding by heterospecific nurse bees and/or chemical differences in royal jelly. Our data show a strong impact of environment on the development of queens. The results further suggest that in honeybees the cues for brood recognition can be learned by heterospecific workers after eclosion, thereby providing a novel analogy to slave making in ants.