extfieldsofstudy: Medicine Biology

text:
Screening of immune-related differentially expressed genes from primary lymphatic organs of broilers fed with probiotic bacillus cereus PAS38 based on suppression subtractive hybridization

To explore the molecular mechanism of the effect of Bacillus cereus PAS38 on the immunity of broilers, sixty 7-day-old broilers were divided into two groups with three replicates. The control group was fed with basal diet, and the treatment group was fed with basal diet containing Bacillus cereus PAS38 1×106 CFU/g. Thymus and bursa of fabricius were taken from two groups of broilers at the age of 42 days, total RNA was extracted, differential gene library was constructed by SSH technology, and immune-related differential genes were screened. Then, we used siRNA to interfere with the expression of some differential genes in the original generation lymphocytes of broiler blood to detect the change of cytokines mRNA expression level. A total of 42 immune-related differentially expressed genes were screened, including 22 up-regulated genes and 20 down-regulated genes. When 7 differentially up-regulated genes associated with enhanced immune function were interfered with in lymphocytes, some immune-promoting cytokines were down-regulated. These results showed that Bacillus cereus PAS38 might up-regulate the expression of JCHAIN, PRDX1, CD3E, CDK6 and other genes in immune organs of broilers, thereby affecting the development of immune organs, the expression of various cytokines and the transduction of immune signals, improving the immune capacity of broilers.

Introduction
The most prominent feature of probiotic Bacillus is that it can produce stress-resistant spores under adverse environmental conditions such as high temperature, ultraviolet radiation and chemical reagents. It can also tolerate stomach acid and bile salt in animal digestive tract, thus, it has strong stress resistance and environmental adaptability [1]. Various studies have shown that probiotic Bacillus preparation can not only promote the growth and development of animal, but also can be used as an immune activator, promote the development of immune organs, activate immune-related signaling pathways, and improve the immune capacity of animals, hence, it is widely used in the poultry industry [2,3]. Bacillus cereus is a common soil bacterium. Some of its strains have been proved to be used as probiotics, and have been developed as probiotics and applied in the field of animal husbandry and veterinary [4]. Zhao et al. [5] reported that adding Bacillus cereus EN25 to the diet of sea cucumber could significantly improve the immune function and reduce the cumulative mortality after infection by Vibrio. Scharek et al. [6] found that Bacillus cereus var. Toyoi could increase the number of CD8+ T cells and γδ T cells in jejunal epithelium and Peyer's lymph nodes of sows and piglets, and reduce the frequency of pathogenic Escherichia coli in piglets' feces. Similarly, Roos et al. [7] found that Bacillus cereus var. Toyoi as adjuvant could effectively improve the effect of BoHV-5 vaccine, and increase the expression of cytokines such as IFN-γ, IL-12 and IL-10 in mouse blood. Feng et al. [8] fermented wheat bran with Bacillus cereus which could produce xylanase, and fed it to broilers, found that it could improve the amylase activity of duodenal and the abundance of intestinal flora. It was also found that Bacillus cereus fed Carassius auratus gibelio could significantly ameliorate the immunosuppression, oxidative stress and intestinal flora disorder caused by metal cadmium, and effectively alleviate cadmium poisoning [9,10].
Bacillus cereus PAS38 is a strain isolated from the gut of animal that can produce high levels of cellulase and amylase. Previous studies have shown that the addition of Bacillus cereus PAS38 to the diet of broilers can improve the growth performance of broilers, improve the activity of intestinal digestive enzymes, regulate the intestinal microecological environment, promote the development and maturity of immune organs, increase the content of serum immunoglobulin and the level of cytokines such as IRF1 and IL-1 [11,12]. However, at present, there are few studies on the molecular mechanism of the effect of Bacillus cereus PAS38 on broiler immune system, so it is necessary to examine differential expression genes to understand the probiotic mechanism of the strain.
Suppression subtractive hybridization (SSH) technique combines the advantages of suppression PCR and subtractive hybridization, which can enrich rare transcripts with differences and is very suitable for samples with highly similar genetic backgrounds, so it is often used to screen differentially expressed genes in the field of zoology [13][14][15]. Our group has used SSH to construct the splenic differential gene library of broilers fed Bacillus cerus PAS38, and screened 9 immune-related differential genes including JCHAIN, FTH1, IGF1R, TLR7 and others [16].
In this study, Bacillus cereus PAS38 was fed to white feather broiler. Primary lymphoidorgans (thymus and bursa of fabricius) of the broilers were collected at 42 days of age, and total RNA was extracted to synthesize cDNA. Then the differentially expressed gene library was constructed through SSH technology to screen the differentially expressed genes related to immunity. Absolute qRT-PCR was used to verify the differential genes, and siRNA gene interference technology was used to analyze the function of differential genes. So as to preliminarily explore the key genes regulating immunity of Bacillus cereus PAS38, and lay a foundation for wide application in broilers production.

Ethics statement
All animal experiments were performed in accordance with the guidelines for the care and use of laboratory animals and approved by the Institutional Animal Care and Use Committee of Sichuan Agricultural University (approval number: DYS20174513-1).

Laboratory animals and strains
Sixty one-day-old avian white feather broilers were purchased from Sichuan Zhengda Animal and Poultry Co., Ltd. Bacillus cereus PAS38 strain was provided by the Animal Microecology Research Center of Sichuan Agricultural University. Bacillus cereus PAS38 preparation was obtained by solid fermentation at 37˚C for 48 h. Corn powder was added to regulate the number of viable spores to 10 9 CFU/g.

Lymphocytes
Lymphocytes were isolated and purified from peripheral blood of healthy broilers by Animal Microecology Research Center of Sichuan Agricultural University.

Experimental design
Sixty one-day-old avian white feather broilers were pre-fed with basic diet for 7 days to stabilize the metabolic conditions. The broilers were randomly divided into control group and treatment group, each group consisted of three replicates with 10 chickens per replicate. The control group was fed the basal diet. The treatment group was fed the basal diet supplemented with 0.1% Bacillus cereus PAS38 preparation. The experimental broilers were raised in singlelayer cage with ten broilers in each cage. The broilers of control group and the treatment group were fed in two animal houses respectively to avoid spore interference. Broilers were fed the diet and water ad libitum. At the age of 42 days, two broilers were randomly selected from each replicate of each group. Then the broilers were executed by exsanguination from the carotid artery under anaesthesia with diethylether, and the thymus and bursa of fabricius were taken and immediately stored in liquid nitrogen.

Construction of SSH libraries
The tissues were ground rapidly in liquid nitrogen, and total RNA was extracted by Trizol reagent. 1.0 μg total RNA was taken and the Smarter TM PCR cDNA Synthesis Kit was used to synthesize double-stranded cDNA. During the synthesis of the second-stranded cDNA, from the 18 th cycle to the 33 th cycle, 5.0 μL mixture was taken out every 3 cycles for 1.2% agarose gel electrophoresis to select the optimal number of cycles. The double-stranded cDNA was digested by Rsa I enzyme, and the adaptor was connected as the tester, and the unconnected cDNA as the driver. The cDNA in the treatment group was used as tester, and the cDNA in the control group was used as driver for hybridization, which was regarded as SSH forward library; on the contrary, the reverse library used the cDNA in the control group as tester, and the cDNA in the treatment group as driver for hybridization. Then the Advantage 2 PCR Kit was used to conduct nested PCR on the hybridization products to amplify the differential ESTs. Finally, differential ESTs were connected to pBM16A T vector and transformed into competent Escherichia coli, which were coated on LB solid medium containing ampicillin antibiotics, IPTG and x-gal, and cultured overnight in a constant temperature incubator. The detailed reaction systems and reaction procedures are described by Li et al. [16].

Bacterial liquid PCR verification and sequencing
200 white single colonies were randomly selected from each library, and cultured in 1 mL LB liquid medium containing ampicillin antibiotics in a constant temperature incubator overnight. Then, 1.0 μL bacterial liquid was extracted for bacterial liquid PCR to detect whether the differential gene fragment was successfully connected to the vector, and the size and band unicity of the inserted fragment.
The bacterial liquid with positive PCR results was selected and sequenced by Beijing TsingKe Biological Technology Co., Ltd. After the plasmid sequence and nested PCR primer sequence were removed from the sequencing results, the sequencing results were blasted through the blast function of NCBI website (https://www.ncbi.nlm.nih.gov/), and then the repetitive sequences, vector sequences, non mRNA sequences and the sequences that remained unmatched were removed, so as to count the effective sequences.

Screening and verification of immune-related genes
Through Blast2Go software, GO annotation on Level 2 of SSH library was analyzed, and immune-related genes were screened. Nine genes were selected from the immune-related differential genes of each organ, and absolute qPCR was used to further verify whether they matched the results of SSH. The standard curve was made through the plasmid standard. At the same time, 1 μg single-stranded cDNA was used as template to detect the different genes. The results of qPCR were analyzed by Bio-Rad CFX manager 3.1 software, and the standard curve was obtained to calculate the copies of differential expressed genes. The detailed reaction systems and reaction procedures were described by Li et al. [16]. The primers of differential expressed gene and their optimum annealing temperatures are shown in Table 1.

Functional analysis of differential genes
Some genes were selected from the immune-related differential genes verified by qPCR, and their complete mRNA sequences were obtained according to NCBI website. The corresponding siRNA sequences and negative siRNA sequence were designed and synthesized by Shanghai GenePharma Co., Ltd.
The siRNA interference test of lymphocytes was divided into three groups: the treatment group (Group A) was interfered by the siRNA of different genes, the negative control group (Group B) was interfered by the negative siRNA, and the blank control group (Group C) was added with RNAase-free water as control. Each group was divided into three replicates. The experiment was conducted in 24-well plates, each well contained 500 μL lymphocytes (1×10 6 / mL), and the final concentration of siRNA was 30 nM/L. After 48 h of interference, the total RNA of the cells was extracted and reverse transcribed using the M-MLV 4 First-Strand cDNA kit. Absolute qPCR was used to detect mRNA expression of the interfered gene to verify the interference effect. Finally, the cDNA of lymphocytes with successful interference was selected, and the mRNA expression levels of 6 cytokines: IL-1B, IL-2, IL-6, MYD88, IRF1, TLR4 were detected by absolute qPCR to analyze the effect of differential gene silencing on immune gene expression. The primers of 6 cytokines and their optimal annealing temperatures are shown in Table 2.

Statistical analysis
All the experimental data were analyzed by one-way ANOVA with SPSS 23.0 software and Duncan's method was used for multiple comparisons, with P<0.05 as the statistically significant, P<0.01 as the highly statistically significant.

Synthesis of double-stranded cDNA
Double-stranded cDNA was synthesized by different PCR cycles, and 1.2% agarose gel electrophoresis was shown in Fig 1 (S1 Fig). It can be seen that the brightness and dispersion range of the bands are the best when the cycles are 24. If the number of cycles is more than 24, the brightness of band decreases instead. If the number of cycles is less than 24, and the brightness and range of the bands are significantly lower. In order to better enrich transcripts, especially rare transcriptional information, 24 cycles were selected for synthesis of double-stranded cDNA.

Detection of bacterial liquid PCR
The partial results of bacterial liquid PCR are shown in Fig 2 (S2 Fig). It could be seen that the size of bands is concentrated between 200 bp-1000 bp, and most of the bands are single bright bands, and the bacterial liquid with single bright bands is selected for sequencing.

Sequencing results statistics
The sequencing results were blasted through NCBI, and the valid ESTs were counted, as shown in Table 3. Among them, 128 and 122 differential ESTs were screened separately from the forward and reverse library of the thymus, and 118 and 96 differential ESTs were screened separately from the forward and reverse library of bursa of fabricius. And these ESTs were submitted to NCBI's dbEST database with the GenBank accession numbers JZ982461-JZ982710 (Thymus) and JZ982751-JZ982964 (Bursa of Fabricius).

GO annotation analysis of SSH library
Through Blast2Go software, Go annotation analysis of the differential genes in SSH library was performed, and the results are shown in Fig 3. The ESTs of SSH differential gene library in thymus were involved in the biological process including 12 categories, among which the ESTs in forward library were mainly involved in cellular process (19%), metabolic process (15%), response to stimulus (9%), biological regulation (9%), etc., and 4% of ESTs were involved in the immune system process; the ESTs in reverse library were mainly involved in cellular process (25%), metabolic process (20%), response to stimulus (9%), cellular component organization or biogenesis (8%), and 4% of ESTs were also involved in the immune system process. In the classification of cellular component, differential ESTs in thymus includes 7 categories. The forward library was mainly involved in cell (25%), cell part (25%), organelle (21%), proteincontaining complex (12%), etc.; the reverse library was mainly involved in cell part (24%), cell (24%), organelle (21%), protein-containing complex (10%), etc. Differential ESTs in thymus were involved in 3 categories of molecular function, with forward library involved in binding (63%) and catalytic activity (37%), the reverse library involved in binding (59%), catalytic activity (32%), and transcription regulator activity (9%) (Fig 3A). There were 17 categories which the SSH ESTs of bursa of fabricius involved in, the forward library mainly included cellular process (18%), metabolic process (14%), biological regulation (11%), and regulation of biological process (10%), etc., 2% of the ESTs were involved in immune system process; the reverse library was mainly involved in cellular process (17%), metabolic process (13%), response to stimulus (10%), cellular component organization or biogenesis (8%), etc., and 3% of ESTs were involved in immune system process. In the classification of cellular component, there were 8 categories which the differential ESTs of bursa of fabricius involved in, the forward library was mainly involved in cell (21%), cell part (21%), organelle (16%), organelle part (11%), etc.; the reverse library was mainly involved in cell part (23%), cell (23%), organelle (21%), protein-containing complex (13%), etc. There were 4 categories of molecular function involved in the differential ESTs of bursa of fabricius, including binding (69%) and catalytic activity (31%) in the forward library; and binding (55%), catalytic activity (30%), structural molecular activity (8%), and molecular function regulator (7%) in the reverse library (Fig 3B).

Screening and verification of immune-related differential genes
Through the GO annotation, immune-related differential genes were screened separately from SSH libraries of two organs, as shown in Table 4. There were 26 immune-related genes in thymus, 15 of which were up-regulated and 11 down-regulated. There were 21 immune-related differentially expressed genes in bursa of fabricius, 10 of which were up-regulated and 11 down-regulated.
Absolute qPCR was performed separately on 9 genes selected from the immune-related differentially expressed genes of two organs to verify whether they matched the results of SSH. The copies of differential genes were calculated by the standard curve (S3A-S3M Fig). The results showed that the expression of 9 genes (5 up-regulated genes, 4 down-regulated genes) in thymus had significant difference except for the down-regulated gene BLNK, but the copies of BLNK in the treatment group was also down 57% compared with that in the control group. The expressions of 9 genes in bursa of fabricius (5 up-regulated genes, 4 down-regulated genes) were also significantly different except for the up-regulated gene BTLA, the copies of BTLA in the treatment group was increased by 19% compared with that in the control group. This shows that the detection results of qPCR were basically consistent with screening results of SSH (Fig 4).

Functional analysis of differential genes
Considering the relationship between primary lymphoid organs and secondary lymphoid organs, and the results of our study on spleen differential genes [16], we selected these seven differential genes: JCHAIN, FTH1, IGF1R, PRDX1, CD3E, TLR7 and CDK6 to further analyze their gene functions. Shanghai GenePharma Co., Ltd. was entrusted to design and synthesize corresponding siRNA and negative control siRNA, and the siRNA sequence is shown in Table 5.
The interference results of 7 immune-related differential genes are shown in Table 6. It could be seen that, compared with the negative siRNA control (Group B) and the blank control (Group C), the copies of the 7 differential genes after the corresponding siRNA interference (Group A) were significantly reduced (P<0.01), and there was no significant difference between the negative siRNA control group and the blank control group (P>0.05). Table 4. Screening of immune-related differential genes.

Organ
Forward library (Up-regulate) Reverse Library (Down-regulate) Gene Accession number Gene Accession number The cDNA of lymphocytes after the interference of differential genes was taken, and the mRNA expression levels of 6 cytokines were detected by absolute qPCR (S4A-S4F Fig), as shown in Table 7. The expressions of IL-1B, IL-2, IL-6 and TLR4 were highly significantly down-regulated after interference of JCHAIN and PRDX1 (P<0.01). After FTH1 was interfered, IL-1B, IL-2, IL-6, TLR4 and MYD88 were significantly down-regulated (P<0.01). After the interference of IGF1R, cytokines TLR4, IL-6, MYD88 and IL-1B were significantly downregulated (P<0.01). After TLR7 was interfered in lymphocytes, IL-6 and IL-1B were significantly down-regulated (P<0.01). After interfering with the expression of CDK6 and CD3E, the copies of all six cytokines were significantly reduced (P<0.05).

Discussion
In this study, SSH technology was used to construct the differential gene libraries of thymus and bursa of fabricius in broilers fed with Bacillus cereus PAS38. Through sequencing, sequence alignment and statistics, the SSH library of thymus contains 250 ESTs, 51.2% of https://doi.org/10.1371/journal.pone.0235476.g004 Table 5. siRNA sequence of immune-related differential gene. which were up-regulated and 48.8% were down regulated. There were 214 ESTs in the SSH library of bursa of fabricius, 55.14% of which were up-regulated and 44.86% were down regulated. At the same time, the GO annotation of these libraries showed that most of the genes were mainly involved in biological process or molecular function such as cellular process, metabolic process, catalytic activity, binding, etc., and some genes were also involved in the immune system process. This indicates that the molecular mechanism of Bacillus cereus PAS38 affecting the physiological activities of broilers is comprehensive and complex, which might regulate the growth, development, metabolism and immunity of broilers through multiple signaling pathways. According to the GO annotation of SSH library, 42 immune-related differential expression genes were screened from two organs, 54.76% of them were up-regulated and 45.24% down regulated. Nine immune-related differentially expressed genes were selected from each of the two organs for absolute qPCR verification, and the results showed that the expression level of 88.89% genes was significantly changed compared with the control group. Although the changes in the expression levels of BLNK and BTLA were not significant, they also changed to some extent, which was basically consistent with the screening results of SSH, indicating that SSH technology is generally reliable and can be used to isolate differential genes.

Gene Forward sequence (5'-3') Reverse sequence (5'-3')
Some of these immune-related differential genes were differentially expressed in both thymus and bursa of fabricius. Among them, JCHAIN, PRDX1 and CDK6 were significantly upregulated in both organs, while IPO7 and RPS3 were significantly down-regulated. In our previous study, JCHAIN was also significantly up-regulated in the spleen [16]. JCHAIN protein (Joining chain of multimeric IgA and IgM) is a glycoprotein that can initiate polymerization by linking to homocysteine in the tail of IgM and IgA and bind to poly IgM and IgA stably by isulfide bonds, and JCHAIN plays an important role in the secretion and transport of immunoglobulin, and the activation of complement [17,18]. JCHAIN protein has highly conserved structural characteristics in various vertebrates and invertebrates. It has been found that the amino acid composition of JCHAIN protein in chicken and human is highly similar [19]. Some scholars have studied the expression level of JCHAIN in adult chicken brain, intestine, thymus, spleen, bursa of fabricius and other tissues and organs, and found that the expression level in spleen and rectum was higher, while the expression level in thymus is lower [20]. In addition, some studies have shown that Lactobacillus bulgaricus can activate lymphocytes, synthesize IgA dimer through JCHAIN, and significantly increase the level of sIgA in breast milk and feces of lactating women [21]. In this experiment, the expression of JCHAIN in the immune organs of broilers increased significantly after feeding Bacillus cereus, which may be a positive signal for the immunity of broilers.  catalytic oxidation-reduction. Therefore, PRDX1 may be a good regulator of "H 2 O 2 " signal [22]. Matulova et al. [23] found that PRDX1 was up-regulated in the spleen of broilers infected with Salmonella enteritidis. Lavrič et al. [24] also found that the expression of PRDX1 increased after macrophages of chicken were exposed to Mycoplasma synovium or Escherichia coli. In addition, some studies have shown that the expression of PRDX1 protein in kidney of broilers decreased after infectious bronchitis coronavirus infection [25]. These two different results suggest that chickens may have different defense mechanisms in response to bacterial and viral infection, and PRDX1 may play a role in chicken resistance to bacterial infection. In this study, PRDX1 gene was up-regulated in thymus and bursa of fabricius of broiler chickens fed with probiotics. Thymus and bursa of fabricius are important places for the body to produce lymphocytes. It suggests that Bacillus cereus PAS38 may promote lymphocytes production of PRDX1 in immune organs, so as to improve the ability of organism to deal with bacterial infection. CDK6 (Cyclin-dependent kinases 6) is a cell cycle regulator, and the activity of CDK6 protein depends on its binding to Cyclins. It can combine with Cyclin D to form a complex, thus transform the cell cycle from G1 to S [26,27]. Uras et al. [28] found that the lack of CDK6 would make red blood cells more sensitive to mechanical stress in vitro and lead to a shorter life span, which would lead to anemia in mice. Grison et al. [29] found that CDK6 deficiency could slow the proliferation of basal progenitor cells in the dorsal and ventral forebrain of mice. These studies indicate that CDK6 plays an important role in the development of organism. Meanwhile, Henrique et al. [30] found that the high expression of CDK6 in the cytoplasm of CD8 memory cells was conducive to the rapid cell division, which showed that CDK6 also played a role in the immune process. In this experiment, the expression of CDK6 in thymus and bursa of fabricius increased after broilers were fed with Bacillus cereus. Combined with previous studies, Bacillus cereus can promote the development of immune organs and increase the number of lymphocyte [6,11], indicating that Bacillus cereus PAS38 may achieve these effects by up regulating CDK6 gene.
IPO7 (Importin-7), as a member of the nuclear adaptor protein importin-β superfamily, is a cell transporter, which can mediate proteins, hormones and nucleic acids into the nucleus [31]. At present, the research on IPO7 is mainly focused on its transport mechanism of various proteins and hormones in organism, and there are few studies on immunity [32]. However, some researchers have found that HIV-1 virus can use IPO7 to maximize the nuclear input of its DNA genome [33]. Others have found that the expression level of IPO7 is related to the clinical symptoms of human asthma [34]. After feeding broilers with Bacillus cereus PAS38, the expression of IPO7 in thymus and bursa of fabricius decreased, which might be beneficial to inhibit the proliferation of virus in vivo.
RPS3 (Ribosomal protein S3) belongs to ribosomal protein S3P family and is a part of ribosomal 40s subunit [35]. RPS3 has endonuclease activity and is believed to play a role in repairing damaged DNA and apoptosis [36,37]. RPS3 leads kappa light chain enhancer of NF-κB subunit of activated B cells to specific κB site, which plays an important role in the innate response of bacterial infection. Wu et al. [38] found that E. coli had multiple mechanisms to block RPS3 mediated transcriptional activation, thus interfering with innate immunity. Park et al. [39] found that RPS3 could also act as a binding protein of TLR4 to induce the maturation and activation of dendritic cells, which has a potential auxiliary role in dendritic cell tumor vaccine. In this experiment, after the broilers were fed with Bacillus cereus PAS38, the RPS3 expression in thymus and bursa of fabricius was down-regulated in the treatment group, which might be related to the reduction of anaphylaxis. Of course, its specific mechanism of action needs further study.
In this experiment, 6 cytokines were selected to detect the expression changes after the interference of differential genes. Among them, IL-1B, IL-2 and IL-6 are all common interleukins, they play an important role in lymphocytes activation, production of cytokines, antibodies and complement [40][41][42]. And MYD88, IRF1 and TLR4, play key roles in pathogen recognition, activation and conduction of immune signals [43][44][45].
The expression of IL-1B, IL-2, IL-6 and TLR4 in the lymphocyte decreased significantly after JCHAIN was interfered. These three kinds of interleukin and TLR4 are mainly produced by mature macrophages and T cells. In previous studies, JCHAIN was mainly related to the secretion of immunoglobulin [18]. This experiment shows that in broilers, JCHAIN may also have influence on the generation of certain interleukins and toll-like receptors, and participate in the process of immune activation. Similarly, interference with PRDX1 in lymphocytes also resulted in significant down-regulation of IL-1B, IL-2, IL-6, and TLR4. PRDX1 is an important signaling protein that can regulate intracellular H 2 O 2 level. Combined with the results of this experiment, it is suggested that PRDX1 may play a role in the resistance of chickens to bacterial infection through the activation of immune signals.
FTH1 (Ferritin heavy chain 1) is the main subunit of ferritin, responsible for the oxidation and integration of ferrous ions to maintain the balance of ferrous ions in the body [46]. Some studies have shown that FTH1 is related to the egg-laying traits of chickens [47], and some studies have also proven that expression of FTH1 changed significantly after poultry were infected with some pathogenic bacteria and viruses [23,48]. After the interference of FTH1, the five cytokines: IL-1B, IL-2, IL-6, TLR4 and MYD88 were all significantly down-regulated. This suggests that in broilers, FTH1 may be involved in the defense mechanism against viruses and bacteria by influencing the production of some cytokines.
IGF1R (Insulin Like Growth Factor 1 Receptor) can combine with insulin like growth factor (IGFs) specifically to mediate IGFs signaling and promote the development of various tissues and organs [49]. Previous studies have reported that IGF1R played a positive role in weight gain and bone growth and development in broilers [50], and selenium deficiency in broilers could inactivate the IGF-1R/PI3K/Akt/mTOR pathway, reduce the growth rate of the spleen and the number of splenic lymphocytes [51]. This study found that, after the interference of IGF1R, the expression of TLR4, IL-6, MYD88 and IL-1B were significantly down-regulated, which suggests that IGF1R may regulate the expression of certain cytokines by affecting the development of immune organs and the number of lymphocytes in broilers, play a positive regulatory role in the development of the immune system.
When TLR7 (Toll-like receptor 7) was down-regulated by interference in lymphocytes, the copies of IL-6 and IL-1B were significantly reduced. TLR7 belongs to the toll-like receptor family and is mainly expressed in the lungs and spleen, secreted by macrophages, B lymphocytes and mast cells [52]. It can be activated by single strand RNA, and then activates NF-κB pathway through MyD88 and TRAF6, it can lead to activation, leading to cytokines secretion and inflammatory response [53]. It has been found that the levels of IFN-β, IFN-γ, IL-1β and IL-4 in peripheral blood mononuclear cells of broilers were significantly up-regulated after TLR7 agonist Resiquimod was used [54]. It has also been found that the highly virulent NDV strain can block the expression of TLR7 in chicken macrophages and enhance replication [55]. In this study, interference of TLR7 down-regulated two kinds of interleukins, suggesting that TLR7 may be involved in the defense against viruses by promoting the expression of immune factors in broilers.
After interfering with the expression of CDK6 and CD3E, the copies of all six cytokines were significantly reduced. CDK6 is a cell cycle regulator that plays a role in the development and the immune system of organism [26][27][28][29][30]. CD3E (Cluster of differentiation 3e) is a oneway type I membrane glycoprotein located on the surface of T cells [56]. Mainly as one of the subunits of T cell receptor CD3 complex, it plays an important role in antigen recognition, various intracellular signal transduction and T cell development, and also initiates the assembly of CD3 complex [57]. At present, the research on CDK6 and CD3E in broiler immunity has not been reported. This experiment should be the first time to explore its immune function in broilers. It was found that the interference of these two genes led to the down-regulation of six cytokines. It is speculated that CDK6 may participate in the proliferation process of lymphocytes, and CD3E may participate in the development of T cells and intracellular signal transduction, thus affecting the expression of many cytokines.
Through interference tests on these 7 differential genes, it was preliminarily analyzed that the mechanism of Bacillus cereus PAS38 to improve broiler immunity might be as follows: (1) By up-regulating the expression of some growth factors, such as IGF1R and CDK6, it can promote the development of immune organs and the proliferation of lymphocytes. (2) By promoting the expression of JCHAIN, PRDX1 and FTH1, it is beneficial to the production of immunoglobulin, and interleukin, toll like receptor and other cytokines. (3) By up-regulating genes such as TLR7 and CD3E that play important functions in antigen recognition and intracellular signal transduction, which can affect the expression of various cytokines and also promote the development of immune cells, so as to enable the body to cope with the invasion of bacteria and viruses more efficiently.

Conclusion
The expression of many genes in thymus and bursa of fabricius were significantly affected after broilers were fed with Bacillus cereus PAS38, including 42 immune-related genes. When some of the immune-related differential genes were interfered in the original generation lymphocytes of broiler, some cytokines were significantly down-regulated. It is suggested that Bacillus cereus PAS38 may promote the development of immune organs and the proliferation of lymphocytes by up-regulating some genes of immune organs such as JCHAIN, PRDX1, CDK6, and CD3E, etc., which is conducive to the activation and transmission of immune signals, as well as the synthesis of cytokines and immunoglobulins, thus play a regulatory role in the immune system of broilers.


provenance: AgriculturalAndFoodSciences,2019-2020-Agricultural And Food Sciences-2020.jsonl.gz.gz

text:
Signiﬁcance of Apoidea as Main Pollinators. Ecological and Economic Impact and Implications for Human Nutrition

: Wild and managed bees provide pollination services to crops and wild plants, as well as a variety of other services beneﬁcial to humans. Honey bees are the most economically valuable pollinator worldwide. It has been calculated that 9.5% of the total economic value of agricultural production comes from insect pollination, thus amounting to just under USD 200 billion globally. More than 100 important crops depend on pollination by honey bees. The latter pollinate not only a wide number of commercial crops but also many wild plants, some of which are threatened by extinction and constitute a valuable genetic resource. Moreover, as pollinators, honey bees play a signiﬁcant role in every aspect of the ecosystem by facilitating the growth of trees, ﬂowers, and other plants that serve as food and shelter for many large and small creatures. In this paper, we describe how the reduction in honey bee populations a ﬀ ects various economic sectors, as well as human health.

Introduction
The honey bee plays a significant role as a pollinator of a wide range of agricultural and wild plant species [1,2]. Furthermore, wasps, flies, butterflies, moths, and beetles also contribute to the pollination of a variety of plants [3]. Since the beginning of the 21st century, there has been a growing trend of decreases in the diversity of both managed and non-managed animal pollinators-some birds (hummingbirds, sunbirds, spider hunters, etc.) and mammals (bats, rodents, etc.) [4]. Numerous studies have documented a significant decline in the diversity and range of non-managed wild pollinators over the past three decades worldwide [5]. Additionally, many animal pollinators (birds and mammals) are going extinct and have been narrowing their habitats globally over the past 25 years [6].
This review provides data about the vital role of the honey bee as the main pollinator. It also surveys data regarding the essential role of honey bees in ecosystem services and human nutrition, as well as the medicinal significance of honey bee products.

Some Factors Leading to Honey Bee Colony Decline
Colony collapse disorder (CCD) is an abnormal phenomenon occurring when the majority of worker honey bees in a colony disappear, whereas the queen is left behind with plenty of food and a few nurse honey bees to care for the remaining immature honey bees [7]. It was described for the first time in the late 2006, when 30-40% of the managed honey bee colonies in the United States declined [8].
Additional pollinator taxa, besides honey bees, have been the subject of monitoring concerns: several local and national level butterflies (Lepidoptera) have been recorded, mainly in the USA. Based on the intensive long-term monitoring of individual butterfly species, rigorous quantitative estimates of declines have been established. It should be noted that the eastern North American monarch has declined by over 85% [33] and the western North American monarch by over 95% [34] over the last two decades. Butterfly populations in Europe have also decreased drastically in recent decades. In England and in the Netherlands, respectively, 42% and 24% of the resident breeding species became extinct in the late 19th and the 20th centuries [35]. There have been reports of similar declines in Japan, and in hotspots of butterfly endemism such as Brazil, South Africa, and Australia [36][37][38][39]. The intensification of human land use, for the purposes of agriculture, in particular, remains the major cause of butterfly declines and of the changes in community composition [40].
There has been strong evidence of declines in mammalian and bird pollinators as well. The latter include larger and more visible species, and thus are more often included in monitoring schemes. At least 45 species of bats, 36 species of non-flying mammals, 26 species of hummingbirds, seven species of sunbirds, and 70 species of passerine birds-all of which are known pollinators-are of global conservation concern [41].

Negative Impact of Monocultures on Beehives
Poor honey bee management is associated with specific peculiarities in beekeeping practices and directly adds to the combination of stressors that can contribute to honey bee population decline. These stressors include artificial substances and unilateral feeding, antibiotics, acaricides, and insecticides applied to the hive, exposure to adverse temperatures and temperature fluctuations, infections and parasites, the overexploitation of honey bee products, and unreliable sources of honey bees and honey bee queens [16,42]. The one-sided selection of honey bees results in genetic erosion in the species population and a lack of resistance to infectious diseases, mites, acaricides applied by beekeepers in hives, etc. [17,18].
Studies on the arrival of honey bees to pollinate almond flowers in California orchards-primarily in five counties between the San Francisco Bay Area and Los Angeles-have reported the start of a brief frenzy of activity. According to researchers, this is the world's largest pollination event. Depending on where a farm is located, certain pollination strategies may be more appropriate than others, because wild bees are more often found in orchards near natural habitats [5,25]. In such areas, it is important to maintain the natural habitat. Growers whose orchards are far from the habitat can diversify their pollination strategies by using alternatively managed honey bees, such as the blue orchard bee (Osmia lignaria), in addition to honey bees, and by adding flowering resources to support these managed honey bees and to attract wild species. The feeding of honey bees is often insufficient, be it due to the overcrowding of hives or irregular grazing, and in the conditions of prolonged cold and rainy weather, it is lacking [43,44]. Feeding is considered to be deficient in areas with intensive agricultural production where the so-called stress from a monotonous or "monocultural" diet is evidenced [6]. The latter refers to the continuous grazing of honey bees on crops in mass flower, and which are grown over large areas, such as sunflower or rapeseed, as well as acacia, where the purpose is to produce honey, or just to pollinate the plants.
Some investigations have revealed that the monocultural diet may be increasing pollinators' visits to defended plants [45]. Such is the case with the presence of cyanogenic glycoside amygdalin in almond pollen. The authors of the relevant study established that the amygdalin levels in the nectar and in the honey were below the lethal threshold for honey bees, as no unusual levels of bee mortality were detected. Additionally, the authors suggested that amygdalin deters honey bees, and that these concentrations are high enough to account for the preference for white mustard flowers over almond in almond orchards. It was hypothesized that the presence of secondary compounds in nectar and/or pollen may enhance plant fitness by attracting more specialized pollinators.
Similarly, Stephenson [46] found that the iridoid glycosides of Catalpa speciosa increase floral constancy by inhibiting nectar "thieves" but not legitimate pollinators. These data suggest that the presence of cyanogenic glycosides in the nectar and pollen of almond flowers might inhibit inefficient pollinators or nectar "thieves". Honey bees, as the most effective pollinators of almonds, can probably tolerate the toxicity of amygdalin, up to a certain level.
The African continent is characterized by a significant number of managed honey bee subspecies, which are exposed to a plethora of factors attributing to colony losses in other biogeographic realms. Data about Africa show that, with the exception of the bacterial American foulbrood epidemic in the Western Cape, there has been an insignificant decline in honey bee populations [47]. There are some preconditions for this picture: (1) Honey bee populations in some countries in Africa (South Africa) may have a natural immunity to pathogens and diseases coming from other continents [48]; (2) as mentioned earlier, African populations are more genetically diverse than their European counterparts (due to a large wild population), and are less commercialized [49,50]; (3) the African honey bees are known to exhibit specific behavior in response to unfavorable climatic conditions and the presence of various pests or predators [51]. All of the listed factors may have contributed to the reduced impact of stressors on honey bee health in Africa. Thus, the honey bee colony losses observed in Africa have been lower than those on other continents [47,48].
As far as Europe is concerned, honey bee population declines vary in different parts of the continent. In central Europe, the observed losses since 1985 have amounted to a quarter of the honey bee colonies, with a 54% loss in the United Kingdom [5]. Since 1998, weakening and mortality in colonies have been recorded in Europe, especially in France, Belgium, Switzerland, Germany, the UK, the Netherlands, Italy, and Spain. Lethality is the highest at the end of the winter and at the beginning of the spring [23]. During the last several winters, honey bee colony mortality in Europe averaged about 20% (with a wide range of 1.8% to 53% among European countries). Over the winter of September 2008, honey bee losses in Europe ranged between 7% and 22%, and over the winter of October 2009, between 7% and 30% [37]. In addition to the decline in managed bee colonies, a global decrease in native wild pollinators has been reported as well [5,52]. Typical examples in this regard are the UK and the Netherlands [53].
All these data have led to suggestions that honey bee declines are localized in specific regions, mostly in Europe and North America, whereas in other parts of the world, honey bee population declines seem to be of lower values. Thus, the declines seem to be compensated by increases in the major honey-producing countries (Argentina, China, and Spain) [17].

Impacts of Pollinator Declines on Crop Production
Despite the considerable number of existing pollinators, the honey bee plays a significant role for many agricultural plants. It has been suggested that 75% of all crops that are used for human consumption depend on insect pollination [54]. Plenty of the world's most important crop plants are wind pollinated (wheat, rice, corn, rye, barley, and oats) [55]. Moreover, fruits constitute a substantial part of human diet, as they provide high amounts of essential nutrients such as vitamins, antioxidants, and fiber [56,57]. The most significant pollinator-dependent crops are vegetables and fruits, representing about €50 billion each, followed by edible oil crops, stimulants (e.g., coffee, cocoa, etc.), nuts, and spices [58]. The authors calculated the world value for the contribution of pollinators to the production of crops used directly for human food to be as much as €153 billion, which is about 9.5% of the total value of the production of human food worldwide. In case of the total loss of pollinator services, the average global fruit supplies could drop by 22.9%, vegetables by 16.3%, and nuts and seeds by 22.1% [59]. Such a decline in these valuable resources may have a radical impact on global human health [60].
The expansion of pollinator-dependent crops in certain regions has been analyzed in the paper of Aizen et al. [61]. The authors conclude that if pollinator-dependent agriculture continues to grow, there will be an increasing global demand for pollination services and a risk of pollination shortfall caused by reduced biodiversity in both managed and wild pollinators. The consequences of this trend may vary at regional and country levels. It is necessary to identify which countries and regions are particularly vulnerable, because the steep increase in their agricultural pollinator dependence involves a potentially high environmental cost and may not be compensated by the economic and social benefits associated with more diverse agricultural practices.
It is well known that urban areas are growing globally and this has consequently been affecting biodiversity and ecosystem services, such as pollination [62]. The influence of urbanization on pollinators depends on the urban sprawl, and positive responses were observed with moderate levels of urbanization of rural, mostly agricultural, land of below 50% impervious surface. Additionally, urbanization generally reduces pollinator diversity in comparison with natural or semi-natural areas, but enhances it in comparison with intensified agricultural landscapes. As a whole, it seems that urban pollinator communities still provide enough pollination services to crops and wild vegetation.
Overall, pollinator diversity depends on the amount of urban green spaces at the landscape scale. Urbanization also increases the competition between managed and wild pollinator with regard to certain flowering sources [63]. It has been established that the pollinating activity of large solitary bees, bumblebees, and beetles is negatively related to the density of honey bee colonies in the surrounding landscape.
Agriculture and land-use intensification also have a negative effect on bee nutrition and health [64]. For example, it is well known that pollen quality has numerous positive effects on honey bee health. Due to primary human activity, most landscapes are characterized by a mix of different habitat types with varying proportions of natural, semi-natural, agricultural, and urban areas. An important consequence of this process is the provision of additional resources, such as pollen and nectar, for pollinators [65]. The influence of pollen diet quality and diversity on the physiology of young nurse honey bees has been investigated [64]. The authors found that pollen quality affected both nurse bee physiology and the tolerance to the parasite. Pollen diet diversity had no impact on the physiology of nurse bees and the survival of healthy honey bees. However, when parasitized, honey bees fed with the polyfloral blend survived longer than honey bees fed with monofloral pollens, with the exception of the most protein-rich monofloral pollen. These results have come to support the perception that both the quality and diversity of pollen can influence honey bee physiology and might help to better understand the impacts of agriculture and land-use intensification on honey bee nutrition and health.
Although the complete disappearance of all pollinators is impossible, it is of great importance to calculate the relative importance of insect pollination as an important agricultural input [66,67]. It should be noted that this calculation takes into consideration the fact that most crops only partially decline in production in the absence of insect pollinators, and this fact is utilized to calculate the economic value [48]. Different crop varieties may have different yield responses to major changes in animal pollination; however, only a little data about these differences are available. With this connection, it is necessary to calculate the global economic value of pollination in accordance with each variety, as well as the area cultivated per variety.

Ecosystem Service Benefits
Estimates of flowering plant dependence on animal pollination vary between 78% and 94% in temperate and tropical ecosystems, respectively [68]. As can be expected, it has been proven that plants which require pollination by animals decline in tandem with their pollinators, although further studies are needed in order to elucidate a more complete picture [53,[69][70][71]. Ashman et al. [70] suggest that pollen limitation may have profound effects on certain species (like obligate outcrossing animal-pollinated annual plants), and this can, in turn, cause additional profound changes to the whole ecosystem. It is not unreasonable to extrapolate the effects of the reduced production of crops caused by pollinator decline to such a decline in wild plants. Allen-Wardell et al. [41] list several cases of decline in certain plant populations or even extinction related to the reduction in pollinator abundance, also highlighting the possibility of an extinction chain within an ecosystem if key species are affected.
Since it has been proven that honey bee health is related to floral diversity, and, correspondingly, honey bee health and growth improve with a diverse diet, it may therefore be expected that plant declines driven by honey bee declines may exacerbate the problem by forming a positive feedback loop [72]. Although plant-pollinator networks are generally characterized by a certain robustness and ability to compensate both for the decline and extinction of a pollinator or a pollinated species, we can reasonably presume that these networks have a limited compensation reserve and may reach a tipping point, leading to an ecosystem collapse and profound species composition reshift [73].
As we mentioned above, honey bees are a significant pollinator not only for many crops, but for many other plants in their natural habitats. What are the benefits of this pollination? Directly or indirectly, pollinators provide ecosystem services in addition to pollination. These contributions include (but are not limited to): carbon sequestration, the water cycle and water purification, biodiversity preservation, organic matter degradation, and others. The first significant benefit is associated with carbon sequestration: no less than 80% of all flowering plants need pollination in order to reproduce [68]. Flowering plants use the carbon dioxide from the atmosphere for growing through the process of photosynthesis, during which they release oxygen as a waste product of their metabolism [74]. A second, very important, issue is related to the water cycle. The latter is a combination of physical processes, one of which is transpiration, through which plants return moisture from the soil into the atmosphere [75]. Plant transpiration contributes to 10% of the total moisture content in the atmosphere, while the other 90% comes from evaporation from water surfaces [76]. A fully grown tree can transpire as much as 760 liters (200 gallons) of water annually. Third, pollination by honey bees plays a crucial role for sustaining many endangered plant species (e.g., in the USA these include American ginseng, Hawaiian sandalwood, rosy periwinkle, and wild yam) [77]. These species serve as valuable genetic resources in many countries, and their disappearance will have an adverse impact on the biodiversity in the respective countries. Furthermore, the loss of endemic plant species will negatively affect the ecosystem equilibrium within the geographical area [78].
Thus, the global decline in honey bee populations, as well as the fact that managing honey bees is more difficult than it used to be, will negatively influence the reproduction and population dynamics of plants in natural areas [79], and, consequently, the ecosystem services provided by such plants, e.g., carbon sequestration and soil retention. Different populations of A. mellifera have recently been introduced to overcome the problem in many regions [80]. However, this activity may have certain negative consequences, such as: competition for floral resources with native pollinators; competition for nest sites; the co-introduction of natural enemies, especially pathogens, or compromised plant reproductive success [81]. These phenomena are of major ecological, evolutionary, and conservation significance, but until now there have been limited data about the importance of honey bees as a pollinator in their native natural ecosystems or as an introduced species in some regions.

Honey Bee Products and Their Medicinal Value
Honey bee products, such as honey, pollen, royal jelly, propolis, bee venom, wax, and bee bread, are important sources for human nutrition and the production of pharmaceuticals and food additives. This makes honey and pollen suitable for the treatment of a plethora of diseases and infections [82].
Honey, the definitive honey bee product, is a natural substance produced by honey bees, mainly from the nectar of blossoming plants. It can also be derived from the secretions of living parts of plants or the excretions of plant sucking insects (e.g., aphids) on the living parts of plants, which honey bees collect, transform, and combine with specific substances of their own metabolism, then store and leave in the honey comb to ripen and mature [83].
The usage of honey as a medicine has been well known since ancient times [84]. It has been found that honey has numerous properties, e.g., antimicrobial and anti-inflammatory. Its wound-healing property is due to the fact that it maintains the moisture of the wound while at the same time its high viscosity provides a protective barrier to prevent bacterial infection [85,86]. Honey is also proven to have an antibacterial property due to the enzymatic production of free radicals and other reactive oxygen species, including hydrogen peroxide [87,88].
In the light of the rising issue of antibiotic resistance, which poses a very serious threat to public health, different antibacterial agents are essential in the fight against many infectious diseases. Therefore, more and more attention is being paid to alternative antimicrobial strategies, and honey fully meets these requirements [84,89,90]. Thus, as well as a nutrient, honey may be used successfully as an alternative medicine therapy, thus replacing antibiotic treatment [91].
Another typical honey bee product with a possible medicinal application is pollen. Pollen pellets are stored in comb cells and are combined with honey, nectar, or glandular secretions. These conditions are favorable for the pollen bolus to undergo a lactic acid fermentation and become what is called "bee bread" (composed of proteins, monosaccarides, organic acids, minerals, fatty acids, tocopherols, and polyphenols), which also has antioxidant and antimicrobial properties [92][93][94].
Propolis is probably the most popular honey bee product with a primarily medicinal application, having its own antimicrobial, antifungal, antivirus, anti-inflammatory, and anesthetic activities [95]. Propolis is a resinous substance and is used for honey bees' protection, disinfection, and isolation of beehives [96,97]. Based on analysis, it has been established that there are at least 149 compounds and 22 minerals in propolis [82].
Royal jelly is known as a valuable source of energy nutrients and essential amino acids, as well as vitamins, minerals, and hormone-like substances [98,99]. Thus, royal jelly improves general wellbeing and brain function, increases appetite, stimulates and controls endocrine function, etc. [100]. Since it can mitigate unfavorable conditions and inconveniences related to ageing, which, among other things, include a deficient diet, royal jelly has proven to be an excellent dietary supplement, particularly for the elderly.
Bee venom is also used as an alternative therapy, most importantly for the treatment of many human diseases [101]. Bee venom contains several active biomolecules, such as peptides and enzymes, which are potentially beneficial in treating inflammation and central nervous system diseases like Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis [102,103]. Furthermore, it has been shown that bee venom has promising benefits against different types of cancer, as well as anti-viral activity, even against the highly challenging human immunodeficiency virus (HIV) [104,105].
Bee bread is the result of the lactic fermentation of pollen collected by honey bees and mixed by their digestive enzymes, then carried into the hive and kept with a thin layer of honey and beeswax. Bee bread is the main food in the hive, particularly for larvae and young honey bees producing royal jelly [106]. Research on the chemical composition of bee bread has shown that it usually consists of water, proteins, free amino acids, carbohydrates, fatty acids, and other bioactive molecules [107,108]. It is known that bee bread definitely has antioxidant activities and effectiveness against all bacteria and fungi [109,110].

The Beneficial Role of Honey Bee Products on Individual Honey Bees' Health and Their Impact on Social Immunity
Honey bees suffer from various, often interrelated, unfavorable factors, such as parasites, pathogens, pesticides, and poor nutrition and management [111]. In this regard, it is particularly important to understand the effects of individual stressors and the interactions among stressors in order to provide solutions to improve colony health and survival. Moreover, it is important to understand how honey bees' natural immune responses (individual immunity) and collective behavioral defenses (social immunity) can increase and maintain honey bee health and counteract stressors without human intervention. One form of social immunity in honey bee colonies is the formation of a propolis envelope within the nest, which acts as an important antimicrobial layer. It is believed that certain types of social immunity behavior, such as the collection of plant resins and their deposition in the nest as an antimicrobial propolis envelope, have evolved to compensate for deficiencies in innate or physiological immunity [112]. These facts have been supported by evidence that propolis may reduce the effects of mycotoxins produced by fungi [113]. There are multiple honey bee products, such as nectar and pollen, that contribute to social immunity and have a direct influence on colony health [114]. In addition, collecting the antimicrobial secondary metabolites of plants can also be beneficial to honey bees [115]. This defense from microbes is conferred to individual honey bees when they ingest the secondary metabolites, as is true for all pollinators that do so. Furthermore, pollinators can counteract pathogens by collecting and storing non-edible plant materials, such as resin. Honey bees also store these antimicrobial molecules within the edible and non-edible substances that they create from these plant materials, thus preserving the antimicrobial properties in the products they make [115].

Impact of Pollination on Human Nutrition
Apart from its impact on crop production, the loss of pollination may also affect not only the macronutrient aspect of human nutrition, but also the micronutrient one. Pollinator-dependent crops are the main sources of a number of micronutrients, such as vitamins A and C, calcium, fluoride, and folic acid [116]. Where the deficiency of such vitamins is observed depends mainly on the geographical region. For example, according to Chaplin-Kramer et al. [117], the production of vitamin A, which is highly pollinator-dependent, reaches a 50% dependence on pollination in North-Central and Southeastern India, Thailand, Western Iran, Romania, Eastern and Southwestern Australia, Argentina, Mexico, and parts of the USA. Vitamin A is important for human growth and development, and folic acid, which is essential for body functions, cannot be synthesized by humans. Thus, the loss of pollinators can further threaten the health of human populations.
Changes in food and nutrient intake due to the reduction in animal pollinators and pollination have been linked to the risk of three groups of diseases: non-communicable (non-infectious, chronic diseases such as cancer, diabetes, and heart disease), communicable (transmissible diseases such as TB and influenza) and malnutrition-related diseases (e.g., vitamin deficiencies such as rickets) [118]. If all pollinators were eliminated, 71 million people in low-income countries could develop a deficiency of vitamin A and another 2.2 billion, whose consumption is already below the average, would experience further declines in supply [119]. As for folic acid, 173 million people may become newly deficient in the vitamin and another 1.23 billion, who are already deficient, would experience further declines [120]. According to published data, about 74% of all lipids produced worldwide are present in oils from plants that are promoted by animal pollination, and these plants are also primary sources of the fat-soluble vitamins [57]. Regarding the water-soluble vitamins, it should be noted that 98% of the available vitamin C comes from animal-pollinated plants, primarily citrus plants, as well as other fruits and vegetables [121]. Plants are also valuable sources of the B vitamins, particularly folic acid. The human body's requirements for folic acid are higher during pregnancy, which is related to the prevention of neural tube defects in infants [122]. Nowadays, another considerable human health concern on a global scale is vitamin A deficiency. Insect-pollinated crops provide about 70% of this vitamin, and pollination increases the yields of these crops by about 43% [57]. The loss of pollinators and the related services could thus create problems for human nutrition, although the magnitudes of the problems might often depend on the geographical location and the degree of social development. Thus, the consequences related to human health generally affect to a greater extent the developing countries where people are poor and often more dependent on their local insect-pollinated crops [57]. The influence of pollinator losses on human health may be less notable in more developed countries, yet it still has the potential to adversely affect the quality of human diets or deepen the dependence on synthetic micronutrients, such as vitamin supplements. An interesting study has been carried out, using a bioeconomic approach, to calculate a world value for the contribution of pollinators to the production of crops that are used directly for human food. This value has been estimated to be $166 billion, which is about 9.5% of the total value of the production of human food worldwide [58].
The authors have established the most vulnerable crop categories-namely, stimulant crops, nuts, and fruits, as well as the categories that stand for the largest part of the economic vulnerability-fruits, vegetables, and edible oil crops. Furthermore, crop vulnerability varies in different geographical regions. The most affected region turns out to be East Asia (15%), followed by Central Asia (14%), South Asia (12%), and non-EU countries (12%). On a global scale, the vulnerability of the northern countries appears higher than that of the southern ones. This suggests that the decrease in insect pollinators may significantly affect North-South agro-food production [58].

Conclusions
Today, there is growing evidence related to significant pollinator losses in many regions of the world. Further studies are needed in order to map the ubiquity of the phenomenon. Integrating the existing national and local monitoring schemes and the establishment of a global program may provide much needed data in order to help and improve direct policy decisions on pollinators. Not only are pollution threats diverse, but they can also interact. Currently, the main challenge is to better determine the relative importance of a number of drivers, and their synergistic effects in particular. Due to the persistent pressure from well-known drivers, such as habitat loss and pathogens, combined with the environmental and economic risks that have been clearly associated with pollinator loss, there is still a continuing need to improve our understanding of the nature, causes, and consequences of the declines in pollinator services locally, nationally, and globally.


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Influence of Zinc Oxide Nanoparticles on Soil Physicochemical Properties and Arachis hypogea Rhizosphere Microbial Community

We evaluated the effect of 4000 mg zinc oxide (ZnO, 99%, 30 nm) nanoparticle on the physicochemical and microbiological properties of organic manure amended ultisol and loam soil cultivated with Arachis hypogaea using standard methods. The results indicate varying effects on the physicochemical properties in relation to the soil type. The pH of the control ultisol at 7.85 ± 0.17 and 8.3 ± 0.12 in the amended ultisol whereas, the control loam was 7.15 ± 0.17 and 7.41 ± 0.11 in the amended soil indicating 1.06and 1.04-times higher difference than the controls respectively. Phosphorus concentration at 57.82 ± 0.54%, 50.81 ± 0.22% and 55.97 ± 0.04%, 59.97 ± 0.02% was 1.14 times lower in the ZnO amended ultisol and 1.07 times higher in amended loam soil compared to the respective controls. The organic matter content in the control and amended ultisol was 2.28 ± 0.32% and 0.91 ± 0.02%, 3.68 ± 0.36% and 0.36 ± 0.02% in the control and amended loam soil. The concentration of nitrate in the control ultisol was 0.05 ± 0.01% and 0.03 ± 0.01% in the amended soil. The nitrate in the control loam soil was 0.08 ± 0.01% relative to Original Research Article Oghenerume et al.; IJPSS, 32(8): 88-100, 2020; Article no.IJPSS.59974 89 0.02 ± 0.01% in the treated soil and these differences were significant at p = 0.05. The concentration of nutritive salts was reduced and in contrast iron, copper, exchangeable acids, exchange capacity, clay and silt increased in the amended soils. Further to this, heterotrophic ammonia and nitrate-oxidizing bacterial population were inhibited in the amended soils and denitrifying organisms were stimulated. The organisms were members of the genera Pseudomonas, Xanthobacter, Enterobacter, Bacillus, Lactobacillus, Citrobacter, Nitrosomonas, Agromyces and Rhizobium. ZnO nanoparticles altered the soil physicochemical properties which exacerbated the negative effect on microbial abundance and varied with the soil type.

INTRODUCTION
The ecological role of the soil ranges from providing habitat for plants and animals, to site for biogeochemical processes mediated by microorganisms and influenced by changes in the soil physicochemical properties. These changes include alteration in the presence and composition of organic matter, environmental conditions and soil organisms [1] and contamination from anthropogenic and emerging xenobiotic substances such as engineered nanoparticles (ENPs). ENPs have a range of sizes less than 100 nm with novel physical, thermal, optical and biological capabilities and are incorporated into a number of consumer products. Most of these products including personal care products, release ENPs into the soil at the end of their life cycle and disposal [2]. Thus, the soil becomes the largest receptor and sink for aged nanoparticles.
ENPs undergo different forms of interactions with soil properties and organisms which in turn alter the ecological function of the soil [3]. The soil properties that makeup up quality of the soil include: organic matter, pH, nitrogen content, cations, cation exchange capacity and minerals among others. Soil-ENPs interactions include aggregates formed with colloids, absorption, change in oxidation state, precipitation or formation of complexes with ligands [4,5]. The effect on soil properties can vary, for example, the soil pH is influenced by the accumulation of different types of nanoparticles [6]. Several studies [7,8,9,10] have reported increased pH of soils on exposure to different nanoparticles. Soil pH determines the function of a soil and is closely related to presence of, and absorption of nutrients, microbial activity and plant growth [8].
The ability of the soil to maintain the presence of positively charged ions (cation exchange capacity) is an important soil property that reflects on the structure, nutrient utilization and pH of soil [11].
ENPs can also indirectly affect soil properties through the effect on soil microbial activities. For instance, copper oxide nanoparticles increased iron (II) oxide (Fe 2+ ) in paddy soils exposed to CuO nanoparticles [8]. Increased Fe 2+ was attributed to the nanoparticles exerting a stimulatory effect on iron reducing bacterial community in the soil. In addition, heavy metals in the soil contribute to plant growth although high concentrations exert harmful effects [12]. Iron in soils is reduced by microbial respiration and involves electron transfer from organic matter to Fe 3+ which serves as the electron acceptor. Reduction of iron in soils affects the carbon, nitrogen and sulphur cycles [13].
However, several studies indicate the presence of ENPs in the soil environment may exert no negative effect on soil properties. For instance, the porosity of soil was not affected by bimetallic (Fe/Pd) nanoparticles [14], copper oxide and magnetite (Fe 3 O 4 ) effect on clogging was minimal [6]. Also, copper oxide and magnetite nanoparticles exerted no effect on sorption capacity and organic matter although there was a change in dissolved organic matter attributed to short exposure time [15]. Here, we evaluated the effect of zinc oxide nanoparticles on the physicochemical properties of tropical ultisol and loam soils and bacterial abundance in groundnut (Arachis hypogaea) rhizosphere under greenhouse conditions. zinc oxide nanoparticles as previously reported [16]. Briefly, 14 kg of soil and 6 kg of organic manure (poultry dropping) were placed into seven wooden troughs (30 cm x 30 cm) lined with polyethene in duplicate. The organic manure amended soils were allowed to stand for 21 days and seeded with Arachis hypogea under greenhouse conditions. The plants were harvested after 10 weeks of growth and the soil sampled for analysis. The zinc oxide (ZnO, 99%, 30 nm) nanoparticles were purchased from Nanostructured & Amorphous Materials Inc. (Texas, USA), the properties were provided by the manufacturer and used without further characterization. Choice of ZnO was based on the wide application in a variety of consumer products.

The greenhouse
The greenhouse was constructed over a land space of 3.6 x 4m and 2.7 m in height to accommodate the troughs with adequate space for walkway. Transparent roofing sheets resting on galvanized pipes as support were used and the perimeter covered with wire net to prevent rodents and trespassers. The whole structure was reinforced with transparent polyethene for enhanced illumination during the day and maintain optimum temperature during the rainy and cold conditions. The ambient temperature of the soil and greenhouse was monitored throughout the period of planting and growth of the plant using a mercury thermometer.

Soil Physicochemical Analysis
The control ultisol and loam soil and the 4000 mg kg⁻¹ ZnO nanoparticles amended soils were selected to represent a pristine soil condition and a worst-case scenario of ZnO nanoparticles input into the garden soil ecosystem through fertilizer application. The bulk ultisol and loam soils were collected from the control and 4000 mg ZnO nanoparticles amendments and analyzed for the following physical and chemical properties. Physicochemical analysis of the soil was carried out according to standard procedures [17,18]. The samples were digested and analysed for hydrogen ion, exchangeable cation and basic salt, potassium, sodium, magnesium, calcium, iron, copper, exchangeable acids, cation exchange capacity, organic matter, available nitrate and phosphate. The Bouyoucos (Hydrometer) method was employed for particle size distribution analysis. The conductivity meter was used to measure the electrical conductivity and the pH was determined by the electrometric method in 1:1 soil/water ratio.

Enumeration, Characterization and Identification of Bacteria
The enumeration, characterization and identification of heterotrophic bacterial abundance were carried out as previously reported [16]. Briefly, matured plants were uprooted to obtain the rhizosphere soil and bulk samples collected, serially diluted, inoculated by pour-plate onto Nutrient Agar (Oxoid) and incubated at 28 ± 2°C for 24 hours and discrete colonies enumerated. The bacterial isolates were characterized based on their cultural and morphological attributes and response to standard biochemical tests [19] and compared with those of known taxa using the Bergey's Manual of Determinative Bacteriology [20].

Statistical Analysis
The data were subjected to analysis of variance (ANOVA) and Kruskal Wallis test on logtransformed values using Statistical Package for the Social Science (SPSS version 20.0, IBM Corp, USA). Results are presented as mean ± standard deviation with levels of significance maintained at 95% for each test.

Greenhouse Condition
Temperature is an important environmental requirement for enzyme activity and microbial growth and different soil microorganisms and plants have varying temperature regime for optimum performance. The average temperature of the greenhouse throughout the period of plant growth was 26.18 ± 0.85°C, 30.36 ± 2.36°C and 28.09 ± 1.66°C in the morning, afternoon and evening respectively. In the ultisol, the average temperature was 26.73 ± 0.63°C, 30.82 ± 1.89°C and 29.09 ± 1.11°C, whereas in the loam soil, 26.73 ± 0.63°C, 31 ± 1.9°C and 29.09 ± 1.11°C were recorded in the morning, afternoon and evening respectively.

Effect of ZnO Nanoparticles on Soil pH, Hydrogen Ion, Exchangeable Cation and Basic Salt
The soil pH determines the function of a soil and relates to presence of and absorption of nutrients, microbial activity and plant growth [8]. ZnO nanoparticles induced a higher pH in the treated ultisol and loam soils in contrast to the control. The pH of the control ultisol at 7.85 ± 0.17 was lower than 8.3 ± 0.12 of the amended ultisol indicating 1.06 times higher value than the control. The pH of the control loam was 7.15 ± 0.17 and 7.41 ± 0.11 in the ZnO treated soil and suggests 1.04 times higher difference than that of the control. The differences between the pH of the treated soils and the controls were significant at p = 0.05. The results indicate that the ZnO nanoparticles induced changes that increased the pH of the ultisol and loam soils. Several studies indicate that ENPs such as copper oxide nanoparticles [8], zero-valent iron nanoparticles [10] and ZnO nanoparticles [7,9] induced changes in soil pH during acute and chronic exposures. These results are consistent with the present study in which ZnO nanoparticles raised the pH of the ultisol and loam soils.
The mean hydrogen ions in the control ultisol was 1.3 ± 0.09 compared to 3.89 ± 0.12 Cmol kg⁻¹ in the control loam soil indicating 2.99 times higher content than the ultisol (Fig. 1b). The hydrogen ions in control ultisol was 1.3 ± 0.09 relative to 0.12 ± 0.01 Cmol kg⁻¹ in the ultisol amended with ZnO nanoparticles suggests 10.83 times higher concentration in the control ultisol than the ZnO amended ultisol. The control loam soil with 3.89 ± 0.12 and 3.49 ± 0.01 Cmol kg⁻¹ in the ZnO amended loam soil indicates 1.11 times higher concentration than the treated loam soil. The difference in the control and amended ultisol and loam soils were significant at p = 0.05. The empirical evidence suggests reduced hydrogen ions in the treated ultisol and loam soils, and subsequent increase in pH compared to the control soils. Thus, ZnO induced a higher pH in the amended soils. The probable reason is that the ZnO nanoparticles consumed H + in the soil solution with the production of Zn 2+ [21].
The mean exchangeable cations content of the control ultisol was 16.09 ± 0.12 relative to 11.52 ± 0.12 Cmol kg⁻¹ in the amended soil ( Fig. 1 c) indicating 1.4 times higher content than the treated soil. In the control loam soil, the mean exchangeable cations content was 19.18 ± 0.19 Cmol kg⁻¹ in relation to 12.08 ± 0.03 Cmol kg⁻¹ in amended soil. The exchangeable cation content in the control soil was 1.59 times higher than the treated soil. These differences were significant at p = 0.05. In the ultisol and loam soils exposed to ZnO nanoparticles, exchangeable cations content was reduced compared to the controls which caused an increase in the potassium concentration. The mean basic salt content of the control ultisol was 96.08 ± 0.03 in relation to 98.74 ± 0.30 Cmol kg⁻¹ in the treated soil ( Fig. 1d) which was 1.03 times higher than the control. In loam soil, the mean basic salt content of the control was 90.92 ± 0.09 compared to 85.29 ± 0.34 Cmol kg⁻¹ in the treated soil and indicates 1.07 times higher than the treated soil. In the control soils, the mean basic salt content of the ultisol was 1.13 times higher than the loam soil and these differences were significant at p = 0.05. The salinity of the control ultisol was higher than the control loam soil, however, salinity increased on amendment with ZnO nanoparticles indicating that the plants and microorganisms in the ultisol were exposed to higher salt stress caused by the ZnO nanoparticles. In the loam soil, the salinity was however reduced on amendment with ZnO nanoparticles (Fig. 1d). Salinity stress usually leads to reduced soil osmotic potential, nutritional imbalance and negative impact on biochemical and physiological processes in the soil [22]. Nevertheless, nanoparticles have been associated with increased plant growth despite salinity stress [23].

Effect of Zinc Oxide Nanoparticles on Soil Potassium, Sodium, Magnesium and Calcium Content
The mean potassium content of the control and ZnO amended ultisol was 0.09 ± 0.01 Cmol kg⁻¹ and 0.05 ± 0.01 Cmol kg⁻¹ respectively (Fig. 2a). The result indicates that the potassium content in the control soil was 1.8 times higher than the treated soil and the difference was significant at p = 0.05. In loam soil, the mean potassium content of the control was 0.08 ± 0.01 Cmol kg⁻¹ compared to 0.08 ± 0.01 Cmol kg⁻¹ in the treated soil. There was no difference between the potassium content in the treated soil and the control.
The mean sodium content of the control ultisol at 0.18 ± 0.01 Cmol kg⁻¹ was higher than 0.12 ± 0.01 Cmol kg⁻¹ in the ZnO amended soil (Fig.  2b). The result shows that the sodium content in the control soil was 1.5 times higher than the ZnO amended soil. The difference was low but significant at p = 0.05. In loam soil, the mean sodium content of the control was 0.21 ± 0.01 Cmol kg⁻¹ in relation to 0.22 ± 0.01 Cmol kg⁻¹ in the ZnO nanoparticle treated soil indicating that the sodium content in the treated soil was 1.05 times higher than the control. The difference between the sodium content in control and the amended soil was however not significant (p = 0.05). In the present study, calcium, potassium and sodium ions concentration in the ZnO amended ultisol were low relative to the control ultisol. The calcium ions were lower in the amended loam soils compared to the control, whereas sodium and potassium ions were stable despite the amendment with ZnO nanoparticles. The reduced cations content indicates that ZnO nanoparticles induced a rapid uptake by soil biota as a response to oxidative stress [24]. The mean magnesium content of the control ultisol was 3.95 ± 0.06 Cmol kg⁻¹ and 2.80 ± 0.12 Cmol kg⁻¹ in the treated soil (Fig. 2c) which was 1.41 times higher than ZnO nanoparticles amended soil. The content in the control loam soil was 3.35 ± 0.01 Cmol kg⁻¹ relative to 2.20 ± 0.23 Cmol kg⁻¹ in the ZnO nanoparticles amended soil suggesting 1.52 times higher value than the amended soil. The differences between the magnesium content in the control and amended soils were significant at p = .05. Magnesium serves as a cofactor for most enzymes, maintain membranes and nucleic acid formation, stabilize ribosome, cell growth and division [25]. Transport of magnesium ion (Mg 2+ ) into cells is usually based on requirements and regulated in response to changing environmental conditions. The concentration of Mg 2+ in the control ultisol and loam soil was higher than the treated soils (Fig. 2c) and indicates that ZnO nanoparticles induced the uptake of Mg 2+ by both microorganisms and Arachis hypogae. ENPs such as TiO 2 nanoparticles induced the activities of Mg 2+ -ATPase of spinach chloroplast and in turn, lead to increased uptake Mg 2+ [26]. An increase in Mg 2+ intracellularly is associated with susceptibility of plants to pathogens, inhibition of cell division and ribosomal activities [27], whereas influx of Mg 2+ is a coping mechanism of microorganisms to stress [28]. Thus, the results suggest that the ZnO nanoparticles was a stressor for the plant and soil microorganisms and led to increased uptake of Mg 2+ .
The mean calcium content of the control ultisol was 11.7 ± 0.12 Cmol kg⁻¹ and 6.4 ± 0.16 Cmol kg⁻¹ in the treated soil (Fig. 2d) which was 1.83 times higher than zinc oxide nanoparticles amended soil. In the loam soil, the mean calcium content of the control was 13.75 ± 0.17 Cmol kg⁻¹ compared to 8.25 ± 0.29 Cmol kg⁻¹ in the ZnO nanoparticles amended soil and indicates 1.67 times higher value than the amended soil. The differences in the calcium content in both soils were significant at p = 0.05. Calcium is an important element in soil which helps in plant growth. Magnesium and calcium ions interact competitively in cells in relation to enzyme activity [25]. Calcium uptake and translocation by plants are influenced by presence or absence of cations, nitrates, exchange capacity, root temperature and metabolic inhibitors in the soil [29]. Here, the control ultisol and loam soils recorded higher calcium ions compared to the soils amended with ZnO nanoparticles (Fig. 2d) and suggest induced use of calcium by the soil biota.

Effect of ZnO Nanoparticles on Iron, Copper, Exchangeable Acids and Exchange Capacity
The iron content of the amended ultisol soil at 93.21 ± 0.23 mg kg⁻¹ was 8.63 times higher than the control with 10.80 ± 0.24 mg kg⁻¹ (Fig. 3a). In loam soil, the mean iron content of the amended soil was 90.9 ± 0.16 mg kg⁻¹ compared to the control at 9.79 ± 0.5 mg kg and indicated 9.29 times higher value than the control. In the control ultisol, the mean copper content was 0.032 ± 0.001 and 0.064 ± 0.002 mg kg⁻¹ in the ZnO nanoparticles treated soil (Fig. 3b) indicating that copper content in the treated soil was 2 times higher than the control. In contrast, the control loam soil was 0.042 ± 0.001 mg kg⁻¹ and 0.071 ± 0.001 mg kg⁻¹ in the treated soil ( Figure) which was 1.69 times higher than the control. The differences between the iron and copper contents in the control and treated soils were significant at p = 0.05.
Iron and copper are both important elements in soils and contribute to plant growth although high concentrations exert harmful effects [12]. Iron in soils is reduced by microbial respiration and involves electron transfer from organic matter to Fe 3+ which serves as the electron acceptor. Reduction of iron in soils affects the carbon, nitrogen and sulphur cycles [13]. The results show an increase in iron (Fe 2+ ) content in the ultisol and loam soil amended with ZnO nanoparticles (Fig. 3a) and indicate negative effect of ZnO nanoparticles on the activity of iron reducing bacteria. Similar findings which Fe 2+ rapidly increased in paddy soils exposed to copper oxide nanoparticles have been reported [8]. The increase in Fe 2+ content is attributed to the nanoparticles exerting a stimulatory effect on dissimilatory iron reduction in soil.
Copper in soils assist the enzymes responsible for photosynthesis, respiration and formation of lignin and deficiencies inhibit nodulation and nitrogen-fixation, delay flowering and plant yield [30]. Copper content in the ZnO nanoparticle amended ultisol and loam soils were higher than the control soils (Fig. 3b) and signify induced accumulation of copper ions. High copper content in soils exacerbates the toxic effect of xenobiotics evidenced in delayed germination and reduced shoot vigor [30]. The germination and growth response of A. hypogaea in the ultisol and loam soils were negatively impacted [16] and accumulation of copper ions, in part, probably contributed to the poor plant performance.
The mean exchangeable acid content of the control ultisol was 0.62 ± 0.03 Cmol kg⁻¹ and 0.6 ± 0.07 Cmol kg⁻¹ in the amended soil (Fig. 3c) and indicates 1.03 times higher value than the treated soil. In loam soil, 1.77 ± 0.02 Cmol kg⁻¹ mean exchangeable acid content of the control was similar to 1.77 ± 0.05 Cmol kg⁻¹ of the amended soil. The differences between the exchangeable acid content were not significant at p = 0.05. The result indicates that the ZnO nanoparticles did not alter the exchangeable acids present in both soils and the reasons are unclear at the moment.
The mean exchange capacity for the control ultisol at 0.15 ± 0.06 d s⁻¹ and 0.23 ± 0.01 d s⁻¹ in the soil amended with zinc oxide nanoparticle (Fig. 3d) indicates 1.53 times higher value in the amended soil than the control. The mean exchange capacity for the control loam soil at 0.35 ± 0.03 d s⁻¹ was lower than 0.53 ± 0.03 d s⁻¹ in the ZnO nanoparticles amended soil with 1.51 times higher value than the control. The differences between the exchange capacity of the ZnO nanoparticles amended and the control soils were significant at p = 0.05. The presence and maintenance of positively charged ions is an essential property with influence on the structure, nutrient utilization and pH of soil [11]. The cation exchange capacity of the ultisol and loam soil exposed to ZnO nanoparticles was higher than the control soils and in agreement with related studies. For instance, volcanic ash nanoparticles increased the cation exchange capacity of Andisol soil which increased at high decomposition rate [31]. Here, there was increased decomposition rates indicated by the reduced organic matter content, in addition, to the enhanced exchange capacity in utisol and loam soils amended with ZnO nanoparticles.

Effect of ZnO Nanoparticles on Particle Fraction, Organic Matter and Available Nitrate
The particle size of the control and ZnO amended ultisol was 91.91 ± 1.06 %, 2.98 ± 0.27%, 4.47 ± 0.26% and 83.04 ± 0.32, 8.08 ± 0.91%, 8.58 ± 0.23% for sand, silt and clay fractions respectively (Fig. 4a). The sand content in the control ultisol was 1.11 times higher than the treatment, whereas the silt and clay content in the treated soil was 2.71 and 1.91 times higher than the control respectively. The particle size of the control and ZnO amended loam soil was 90.83 ± 0.94%, 2.99 ± 0.01%, 4.45 ± 0.41%, and 87.86 ± 0.76, 4.65 ± 0.52% and 6.19 ± 0.22% for sand, silt and clay fractions respectively (Fig. 4a). The sand content in the control was 1.03 times higher than the treated soil, whereas the silt and clay content in the ZnO amended soil was 1.56 and 1.39 times higher than the control. The results indicate changes in the particle fractions of the two soil types because of ZnO nanoparticles interaction, and the differences were significant at p = 0.05. Soil particles confers a structure that cause variations of oxygen availability over a small distance, leading to a presence of both aerobic and anaerobic conditions [32] and ZnO nanoparticle interaction with the soil particles increased this effect.
The organic matter content of the control ultisol was 2.28 ± 0.32% and 0.91 ± 0.02% in the treated soil (Fig. 4b) indicating 2.51 times higher value than the amended soil. The organic matter content of the control loam soil was 3.68 ± 0.36% and 0.36 ± 0.02% in the amended loam soil which was 10.22 times higher than the soil exposed to ZnO nanoparticles. These differences were significant at p = 0.05.
Soil organic matter synthesized primarily from plant remains degraded by microorganisms plays important roles in how the soil is formed, aggregates stabilized and soil fertility maintained [33,34]. The organic matter content in the control ultisol and loam soils were relatively higher than the soils amended with ZnO nanoparticles (Fig.  4b). The ZnO nanoparticles induced an increase in decomposition of organic matter in both the ultisol and loam soils probably because of the catalytic properties of the nanoparticles. This is consistent with other studies in which ENPs are reported to be highly specific and reactive because of their nanoscale size and catalytic properties [35]. Here, the ZnO nanoparticles exerted catalytic effect in the ultisol and loam soils and increased decomposition of the organic matter. Other ENPs such as iron oxide nanoparticles have been associated with increased decomposition of organic matter and soil enzyme activities [36]. The results suggest that the ZnO nanoparticles, in part, stimulated microbial activities that resulted in breakdown and use of the organic matter and, in part, induced catalytic degradation in the soil.

Fig. 4. Effect of ZnO nanoparticles on soil (a) particle fraction, (b) organic matter (c) available nitrate and (d) phosphorus in the ultisol and loam soil
The concentration of nitrate in the control ultisol was 0.05 ± 0.01% compared to 0.03 ± 0.01% in the treated soil (Fig. 4c) and indicates 1.7 times higher than the soil exposed to zinc oxide nanoparticles. However, the differences between the nitrate content in both soils was not significant at p = 0.05. Nitrates in the control loam soil were 0.08 ± 0.01% relative to 0.02 ± 0.01% in the treated soil. The concentration of nitrate in the control loam soil was 4 times higher than the ZnO nanoparticles amended soil and this difference was significant at p = 0.05. Soil microorganisms immobilize and convert nitrate ions to organic forms used by plants for growth and generally contributes to soil fertility.
The nitrate concentration in the control ultisol and loam soils were higher than the ZnO amended soils (Fig. 4c). The result suggests in part, that the ammonia-and nitrate-oxidizing organisms were inhibited in the amended soil, and in part, that ZnO nanoparticles induced nitrate reduction by stimulating the growth of denitrifying organisms in both soils. The reduced nitrate content in both soils treated with ZnO nanoparticles is consistent with other studies, for instance, there was a decrease in denitrifying enzyme activity on exposure of five different soils to copper oxide nanoparticles [37]. Furthermore, nanoparticles act as catalyst for nitrate reducing bacteria such as gamma-Proteobacteria or Acidobacteria, [38] to stimulate growth evidenced in the increased use of nitrate through denitrification and/or dissimilatory nitrate reduction.

Influence on Available Phosphate and Phosphate-solubilizing Bacterial Community
The mean available phosphorus of the control ultisol was 57.82 ± 0.54% in relation to 50.81 ± 0.22% of the treated soil (Fig. 4d) which was 1.14 times higher the zinc oxide nanoparticles amended soil. In loam soil, the mean available phosphorus of the control was 55.97 ± 0.04% in contrast to 59.97 ± 0.02% in the treated soil. The available phosphorus content in the ZnO nanoparticle treated soil was 1.07 times higher than the control and this difference was significant at p = 0.05. Phosphorus plays different roles in photosynthesis, energy transfer and metabolism of carbohydrates, either solubilized or mineralized from insoluble soil phosphate by microorganisms and/or addition of phosphate fertilizers. Examples of some phosphate-solubilizing microorganisms include members of the genera Pseudomonas, Agrobacterium, Aspergillus, Pythium, Azotobacter and Bacillus [39]. Depletion of phosphorus in agricultural soil causes deficiencies in plant growth. Here, the available phosphorus content in the control ultisol was higher than the treated soil indicating inhibition of phosphate-solubilizing bacteria by the ZnO nanoparticles. In contrast, phosphate-solubilizing bacterial activity was stimulated in the loam soil by the ZnO nanoparticles leading to increase in available phosphorus content compared to the control loam soil (Fig. 4d).
Different soil stressors such as increase in sodium chloride and pH influences phosphorus solubilization [40]. Despite the negative effect of ZnO nanoparticles on phosphate-solubilizing bacteria which directly correlated with reduced phosphate content in the amended ultisol, Pseudomonas aeruginosa and P. alcaligenes were isolated from the ultisol and loam soils. ENPs such as ZnO are known to exert selective and varying inhibitory effects on microbial activity. For example, ZnO nanoparticles inhibited biofilm formation, production of pyocyanin, Pseudomonas quinolone potential and hemolytic activity of Pseudomonas aeruginosa with no effect on the cell growth [41]. Toxicity of nanoparticles was likely influenced by soil properties [42,43] which is consistent with the different effect exerted by the ZnO nanoparticles in the ultisol and loam soils. It is safe to assume that the soil component interacted with the nanoparticles for an additive, synergistic or potentiative effect on the microbes in the ultisol, whereas the effect was mitigated in the loam soil.

Influence of ZnO Nanoparticles on
Total Heterotrophic Bacterial Abundance in

A. hypogaea rhizosphere and Bulk Soils
The counts of total heterotrophic bacteria (THB) in the bulk ultisol (control) was 7.69 ± 0.54 relative to 7.30 ± 0.49 Log 10 CFU g -1 in the 4000 mg kg⁻¹ ZnO nanoparticles amended ultisol (Fig.  5a). The results indicate that the THB counts in the control was 1.05 times higher than the ultisol amended with 4000 mg kg⁻¹ ZnO nanoparticles. The mean THB counts in the control loam soil at 7.41 ± 0.51 compared to 7.15 ± 0.4 8 Log 10 CFU g -1 in the 4000 mg kg⁻¹ zinc oxide nanoparticles amended loam soil (Fig. 5a) and was 1.04 times higher than the amended soil. The differences between the THB counts for in the control and ZnO nanoparticle amended soils were significant at p = 0.05.
The ecological role of microorganisms in promoting plant growth and nutrient cycling are essential and influenced by microbial abundance, diversity, intrinsic and extrinsic factors in the soil [44]. Here, the impact of ZnO nanoparticles on the bulk soil indicates that ZnO nanoparticles reduced the bacterial community abundance in both the ultisol and loam soil at 4000 mg kg⁻¹ (Fig. 5a). The result is consistent with other studies where ZnO nanoparticles caused in a decrease in abundance of soil bacterial community [45]. ZnO nanoparticles exerted inhibitory effect on soil bacterial population by the release of zinc ions (Zn 2+ ) to induce oxidative stress, cell membrane damage and cytoplasmic leakage [46].

Fig. 5. Effects of ZnO nanoparticles on microbial abundance in the bulk ultisol and loam soil and A. hypogaea rhizosphere
The THB counts in the A. hypogaea rhizosphere from control ultisol was 7.00 ± 0.55 and 7.28 ± 0.30 Log 10 CFU g -1 in the 4000 mg kg⁻¹ ZnO amended ultisol. The results indicate that the counts in the A. hypogaea rhizosphere from ZnO amended ultisol was higher than the control with a difference of 1.04. The THB abundance in the A. hypogaea rhizosphere for control loam soil was 6.80 ± 0.58 and 6.59 ± 0.48 Log 10 CFU g -1 in the 4000 mg kg⁻¹ ZnO amended loam soil. (Fig.  5b) and indicates a 1.03 times higher counts in the control than the 4000 mg kg⁻¹ amended soil. The differences between the THB counts in the amended and control soils was significant at p = 0.05. Indeed, the decomposition of organic matter had effect on bacterial composition [34] and correlated with the differences in the composition of A. hypogaea rhizosphere bacterial biomass and abundance in the amended and the controls ultisol and loam soil [16]. Further to this, nanoparticles are implicated in enhanced plant growth in environments with high salt concentration [23] which plausibly accounted for some of the positive impacts on the bacterial and A. hypogaea growth response [16] in the ultisol and loam soil despite the salt stress.
The effect of ZnO nanoparticles on rhizosphere microbial abundance was however soil type dependent. For instance, in the ultisol, ZnO nanoparticles stimulated growth and increased bacterial abundance whereas in the loam soil, the ZnO nanoparticles reduced the abundance (Fig. 5b). Soil properties such as pH, texture, particle size distribution, structure, and organic matter content play a key role in the effect of nanoparticles on microorganisms [47]. The coating of soil organic matter on the surface nanoparticles reduced the direct contact between the microbial cells and nanoparticles [8] which probably accounted for the different effect of the nanoparticles on microorganisms in both soils.

CONCLUSION
The outcome of the interaction between the soil physicochemical properties, biomass and ZnO nanoparticles was inconsistent and varied according to soil type. ZnO nanoparticles exerted differential reduction and oxidation effects to alter the soil physicochemical properties. In addition, microbial mediated processes were either stimulated or inhibited based on these alterations. The reduced nitrate concentration in both soils and phosphorus in ultisol reflects the inhibitory effects on phosphate-solubilizing and nitrate-fixing bacteria. ZnO nanoparticles induced an increased pH, exchangeable acids and exchange capacity, iron and copper, whereas the concentration of nutritive salts was reduced and thus, modified the soil health and ecosystem function.