extfieldsofstudy: Biology Medicine

text:
Isolation and Identification of Algicidal Compound from Streptomyces and Algicidal Mechanism to Microcystis aeruginosa

The biological control of cyanobacterial harmful algal blooms (cyanoHABs) is important to promote human health, environmental protection, and economic growth. Active algicidal compounds and algicidal mechanisms should be identified and investigated to control cyanoHABs. In this study, the algicidal actinobacterium Streptomyces sp. L74 was isolated from the soil of a nearby pond which located in the center lake of Guanghzou Higher Education Mega Center. Results showed that the algicidal activities of cyanoHABs are mainly achieved via an indirect attack by producing algicidal compounds. All active algicidal compounds are hydrophilic substances that are heat and pH stable. In the present study, an active compound (B3) was isolated and purified by high-performance liquid chromatography and identified as a type of triterpenoid saponin (2-hydroxy-12-oleanene-3, 28-O-D-glucopyranosyl) with a molecular formula of C42H70O13 as determined by infrared spectrometry, electrospray ionization mass spectrometry, and nuclear magnetic resonance. Active algicidal compounds from Streptomyces sp. L74 were shown to disrupt the antioxidant systems of Microcystis aeruginosa cells.

Introduction
The growth of cyanobacterial harmful algal blooms (cyanoHABs) has become a global concern as they threaten the environment, economy, and human health and require treatment to control pollution. Chemical, physical, and biological treatments have been applied to control cyanoHABs in aquatic environments [1][2][3]. However, chemical and physical methods entail high costs and cause secondary pollution; hence, rapid and highly active biological methods are considered as important tools to control cyanoHABs [4].
Previous studies have shown that bacteria, viruses, fungi, and actinobacteria exhibit algicidal activities [16][17][18]. However, the high specificity of viruses to hosts and the parasitism of fungus to cyanobacteria have limited the application of these two types of microorganisms [19]. Actinobacteria are distributed mainly in soil and produce several active substances, including antibiotics, enzymes, organic acids, amino acids, and peptides. In 1962, Safferman and Morris found that 90% of 213 actinobacteria strains exhibit algicidal activities [17]. Specifically, actinobacteria species such as Streptomyces achromogenes, S. exfoliatus, S. neyagawaensis, and S. phaeofaciens have been shown to exhibit algicidal abilities yet, similar to algicidal bacteria, few compounds have been purified and isolated [20][21][22][23].
In this study, an algicidal actinobacteria was isolated from the soil boarding a fresh water pond. This strain was identified as Streptomyces sp. L74 and exhibits algicidal activities that are harmful to cyanobacteria, Microcystis aeruginosa, Anabaena flos-aquae, Oscillatoria animalis, and Aphanizomenon flos-aquae. An algicidal compound from Streptomyces sp. L74 cultures was isolated, purified, and identified. The mechanism of algicidal activity of Streptomyces sp. L74 was also studied.

Ethics Statement
No specific permits were required for the described field studies in the center lake of Guanghzou Higher Education Mega Center (http://en.wikipedia.org/wiki/Guangzhou Higher Education Mega Center). The research sites are not privatelyowned or protected in any way and field studies did not involve endangered or protected species.

Isolation of Algicidal Actinobacteria
Soil samples were collected from the topsoil near the center lake of Guanghzou Higher Education Mega Center, where cyanobacterial blooms of M. aeruginosa usually form. The soil samples were air dried at room temperature, ground, and sieved. Soil powder (2 g) was suspended in phosphate buffer solution (PBS, pH 7.0) and diluted to 10 −2 , 10 −3 , 10 −4 , and 10 −5 . Approximately 0.1 mL of dilutions was spread on Gause's synthetic agar medium plates [24]. Potassium dichromate (75 µg/L) was added in the medium as a growth inhibitor of actinobacteria as well as other bacteria and fungi [25]. The colonies were grown on plates at 28 °C for 7 d and those with different morphologies were selected and streaked onto new agar plates. The colonies were re-streaked several times to obtain purified isolates.
A modified double-layer agar plate method was used to isolate algicidal actinobacteria according to Yang et al. [26]. Double-layer agar plates contained 20 mL of basal agar BG-11 medium (2% agar) and over-layered soft agar medium. Softagar medium was made of 2 mL of cyanobacterial cell suspension at the exponential growth phase and 3 mL of BG-11 medium with 1% agar. After the cyanobacterial cells were cultivated in double-layer agar plates at 25 °C at a light intensity of 2000 lux for 5 d, Oxford cups containing the isolated actinobacteria colonies were placed on the surface of the agar plates. The double-layer agar plates were cultivated for another 5 d at 25 °C at a light intensity of 2000 lux. A clear zone around the Oxford cups on the double-layer agar plates indicated the algicidal activity of the isolate.
Positive strains were inoculated in fresh fluid of Gause's synthetic medium and incubated for 2 d to determine the algicidal activity. Approximately 5 mL of the strain culture was added to 50 mL of cyanobacterial culture cultivated at 25 °C at a light intensity of 2000 lux and a flask shaking speed of 150 rpm. The concentration of cyanobacterial cells in the culture and the algicidal activity were determined based on optical density at 650 nm (OD 650 ).

Morphology and 16S rRNA Gene Identification of Algicidal Actinobacteria Isolate
The morphology of the isolate was observed under a light microscope (Leica DM2500M, Germany). The spores were fixed and stained with 1% osmic acid, plated with gold film (BAL-TEC SCD-500, Germany), and observed by scanning electron microscopy (SEM, Philips XL-30, Holland).
The nucleotide-nucleotide BLAST (BLASTn) database (http:// www.ncbi.nlm.nih.gov/BLAST), Seqmatch program, and CHIMERA_CHECK program of the Ribosomal Database Project (http://rdp.cme.msu.edu/) were used to analyze the 16S rRNA gene sequence of the isolates. The sequence and the closest matches retrieved from the database were aligned. Phylogenetic trees were constructed using the neighbor-joining method in MEGA 3 software [28].

Determination of Chlorophyll a
Chlorophyll a (Chl a) content in the cyanobacterial culture was determined according to Wintermans et al. [29]. The cyanobacterial culture (V 1 ) was centrifuged at 4,000 rpm for 10 min. The cell pellet was collected, resuspended in 95% (v/v) ethanol, and stored at 4 °C for 24 h. The supernatant (V 2 ) was centrifuged at 4,000 rpm for 10 min and collected. A spectrophotometer (Unico UV-2802S, Shanghai, China) was used to determine light absorbance at 665 nm (Chl a absorption), 649 nm (Chl b absorption), and 750 nm (turbidity correction). Chl a concentration was determined based on Eq. (1)

Extraction, Isolation, and Identification of Algicidal Compounds from Streptomyces sp. L74 Culture
Streptomyces sp. L74 was incubated by shaking in 250 mL of Gause's synthetic liquid medium at 28 °C for 7 d. The bacterial culture was centrifuged at 5,000 rpm for 10 min. The culture supernatant was recovered by filtering through a 0.22 µm microporous membrane. A fourfold volume of ethanol was added slowly to the culture supernatant; the solution was mixed for 30 min at room temperature and stored at 4 °C for 2 h. The supernatant was centrifuged at 5,000 rpm for 10 min and collected. After ethanol evaporated in a rotary vacuum evaporator (Rikakikai NE1001, Japan) at 40 °C, the supernatant was extracted continually thrice by using an organic solvent at equal volume. After the solvent evaporated under reduced pressure at 40 °C, the residue was fractionated by silica-gel flash column chromatography (Bio-Rad DuoFlow, America) and eluted with chloroform in CH 3 OH (15:4, v/v). The fraction exhibiting algicidal activity was subjected to size-gel exclusion chromatography (16 mm ID × 40 cm, Sephadex G15) by using ultrapure water as an eluent. An active fraction was placed in a reversed-phase C 18 column (250 mm × 4.6 mm, Agilent) connected to a high-performance liquid chromatography (HPLC) system (Waters 1525, Waters) and monitored at 210 nm. Elution was performed with 90% (v/v) CH 3 OH aqueous solution at a flow rate of 0.5 mL/min for 20 min. The algicidal activities of the fractionated compounds were identified by determining the concentration of Chl a in M. aeruginosa in BG-11 medium.
The active algicidal compound was dried in vacuum (Christ Alphal-2, Germany) and scanned by SEM to identify the morphology of the compound. The active algicidal compound was filled with potassium bromide at 20 MPa, and the infrared spectrum (IR) of the active algicidal compound was analyzed in Vector 33 (Bruker, Germany). Electrospray ionization-mass spectrum (ESI-MS) was obtained using a Waters 1525 HPLC coupled with an Esquire HCT (Bruker, Germany). The purified active algicidal compound was dissolved in deuterium oxide (CIL, America) and placed in a nuclear magnetic resonance (NMR) spectrometer (Bruker AV400D, Germany) operating at 400 MHz for 1 H NMR and 100 MHz for 13 C NMR.

Determination of Superoxide Dismutase, Catalase, and Peroxidase Activities as well as Malonaldehyde in M. aeruginosa
Approximately 1 mL of compound B3 water solution from HPLC was added to 99 mL of M. aeruginosa culture at the exponential growth phase. The cyanobacterial cells from different co-culture times were collected by centrifugation at 8,000 rpm and 4 °C for 10 min. The cyanobacterial cells were suspended with phosphate buffer saline (PBS, pH 7.8) and ultrasonicated at 400 W for 5 min. The supernatant was collected by centrifugation at 10,000 rpm and 4 °C for 20 min and then stored at -70 °C. This process was performed until enzyme activity was determined. Superoxide dismutase (SOD) activity was determined by nitro blue tetrazolium photoreduction method [30]. Catalase (CAT) activity was determined by ultraviolet spectrophotometry [31]. Peroxidase (POD) activity was determined by Guaiacol method [32].

Nucleotide Sequence Accession Number
The sequence reported in this study was deposited in the GenBank database with accession number JX983654.

Screening and Identification of Algicidal Actinobacteria
A total of 117 actinobacteria isolates were obtained from the soil. Among these isolates, six isolates, namely, L3, L10, L17, L39, L69, and L74, exhibited algicidal activity against M. aeruginosa. Isolate L39 in double-layer agar plate exhibited the largest algicidal zone. In the fluid containing M. aeruginosa, isolate L74 exhibited the highest algicidal activity (Figure 1). After repeat screening, isolate L74 was chosen as the targeted algicidal strain and applied for the next experiments.
Strain L74 exhibited the following morphological characteristics on the agar plate: substrate; aerial; and spore hyphae. The spore hyphae were faint pink straight or winding chains under a light microscope (Figure 2a and 2b). The spores were oval, approximately 1 µm × 0.5 µm to 1 µm in size, and grew separately (Figure 2c and 2d). The microscopic results suggested that strain L74 possibly belongs to the genus Streptomyces.
After sequencing, the 16S rRNA gene of strain L74 (comprising 1393 bp nucleotides) was determined. BLASTn and RDP databases revealed that the sequence of strain L74 is closely related to the genus Streptomyces and exhibited 100% sequence similarity to S. fradiae or S. rubrolavendulae. Considering strain similarity and phylogenetic analysis, we found that strain L74 was Streptomyces sp. L74. A phylogenetic tree was constructed using the 16S rRNA gene of the strain and its closely related sequences in the NCBI database ( Figure 3).

Algicidal Characteristics of Streptomyces sp. L74
The Chl a concentration of cyanobacterial cultures indicated that the growth of M. aeruginosa was affected by Streptomyces sp. L74 cultures with an algicidal rate of 55.23%, while Streptomyces sp. L74 cell-free cultures and washed Streptomyces sp. L74 cells exhibited algicidal rates of 41% and 23.4% to M. aeruginosa, respectively (Figure 4). These results suggested that strain L74 exhibits an algicidal activity mainly by an indirect attack of secreted active compounds and partially by a direct attack of bacterial cells.
Strain L74 exhibited an algicidal activity against M. aeruginosa, A. flos-aquae, O. animalis, and Aph. flos-aquae (Table 1). Strain L74 showed a more rapid algicidal effect on M. aeruginosa at 2 d when the algicidal rate was three times to six times higher than the other tested cyanobacteria. The algicidal activities against M. aeruginosa, O. animalis, and Aph. flos-aquae are mainly facilitated by an indirect attack of algicidal compounds of strain L74 (80% of filter liquor in the cultures). By contrast, algicidal activity against A. flos-aquae is mainly facilitated by a direct attack of strain L74 cells (40% of filter liquor in the cultures).

Physical and Chemical Characteristics of Algicidal Compounds
The algicidal compounds considered as hydrophilic substances were extracted by mineral ether, acetic ether, or butyl alcohol and subsequently dissolved in the water phase. The algicidal compounds were dialyzed out of a 3 kDa bag filter, indicating that these substances have molecular weights of <3 kDa. The algicidal compounds were stable at 70 °C with pH ranging from 3 to 12 ( Figure 5).
The stability of the algicidal compounds to heat and pH indicated that these compounds were not proteins or peptides. Table 2 shows that the specific chemical color reactions of algicidal compounds with reducing sugars, polysaccharides, glycosides, and triterpenes were positive. These results suggested that the algicidal compounds may contain radical groups of reducing sugars, polysaccharides, glycosides, and parent nuclei of triterpenes.

HPLC Purification and Identification of Algicidal Compounds
HPLC results suggested that the three compounds B1, B2, and B3, which eluted at the following retention times: 4.5 min to 5 min, 5 min to 6 min, and 8 min to 10 min, respectively, were present in the active fraction separated by size-gel exclusion chromatography ( Figure 6). B3 compound exhibited an algicidal activity against M. aeruginosa and was applied for the next identification of algicidal mechanism.
SEM revealed that the morphology of algicidal B3 compound exhibited indefinite forms. B3 compound was successfully obtained at high purity (Figure 7). Figure 8 shows that the IR absorption spectra of the residual groups of B3 were at 3420, 2930, 2878, 1634, 1455, 1388, 1311, 1260, 1202, 1076, 1040, 928, and 894 cm −1 . The IR absorption at 3420 cm −1 indicated the presence of several -OH groups. In addition, a larger peak area corresponds to a higher number of -OH groups. The IR absorption at 1634 cm −1 indicated the presence of unsaturated -C=C-. IR absorption at 1040 cm −1 indicates the presence of glycosidic bonds. The active algicidal B3 compound could be a type of saponin based on IR spectrum analysis and comparison with saponins in the database. ESI-MS was recorded in a positive mode and yielded a deprotonated [M+H] + ion showed that the molecular mass of B3 compound could be determined at m/z 782.7. The molecular distinction between m/z 782.7 and 602.7 and between m/z 602.7 and 422.7 was 180. These distinctions are consistent with the IR result, indicating the presence of glycosidic groups in B3 compound. Two glucosyl groups are also present in the B3 compound structure. Similarly, the specific chemical color reactions and IR spectrum analysis revealed a nucleus of terpenoid with a molecular weight of 422.7 in the B3 compound structure. NMR spectral data analysis showed that the molecular formula of B3 compound is C 42 H 70 O 13 . 13 C NMR spectrum data indicated the presence of two alkenyl carbons (124.6 and 132.8 ppm, C12 and C13, respectively) and at least 12 C-O carbons (40 ppm to 90 ppm) in the B3 compound (Table 3). IR spectrum and ESI-MS analyses suggested that one ethylenic bond and two glucosyl groups are present in the compound. The absence of carbonyl groups (150 ppm to 200 ppm) in the 13 C NMR spectrum data suggested that keto-, carboxy-, and ester groups were present in the molecular structure of B3. The 1 H NMR spectrum further suggested that seven methyl groups (C23, C24, C25, C26, C27, C29, and C30) and one alkenyl group (5.15 ppm, C12) were present. These results are consistent with IR results. Among the 42 carbons, 12 and 30 were assigned to the sugar units comprising 2 hexoses and the triterpene moieties, respectively. The triterpene moieties were correlated with those in previous studies. The δ values of C3 at 84.5 ppm and C28 at 69.6 ppm as well as the ESI-MS spectrum (two glucosyls with m/z 180) suggested that the triterpenoid saponin is a bisdesmosidic glycoside with a hexose attached to C3 and C28 positions. These data showed that the structure of compound B3 is 2-hydroxy-12-oleanene-3,28-O-D-glucopyranosyl ( Figure  9).   (Figure 10 B). After the cells were co-cultured with B3 compound, the MDA concentration of M. aeruginosa increased from 3.93 µmol/L to 6.21 µmol/L at 5 d. This concentration was maintained for the next cultural time (Figure 10 C). The antioxidant systems of the cyanobacterial cells were activated immediately after such cells reacted with B3 compound. These cells were damaged after they reached their limits. MDA is a product of lipid oxidation. The MDA concentration in M. aeruginosa indicates the oxidation status of the lipid membrane, maximum MDA limit, and severity of cell damage. Streptomyces sp. L74 released the active compound, thereby exhibiting an algicidal activity against M. aeruginosa by destroying the antioxidant systems of the cyanobacterial cells.

Discussion
S. achromogenes, S. exfoliatus, S. neyagawaensis, and S. phaeofaciens are actinobacterial species exhibiting algicidal abilities [20][21][22]. This study is the first to report about an actinobacterial strain closely related to S. fradiae and S. rubrolavendulae with algicidal abilities. This study is also the first to identify an algicidal compound as a type of a triterpenoid saponin from the strain culture. The algicidal mechanism of this active algicidal compound was also determined based on the toxicity to antioxidant systems of cyanobacterial cells.
Streptomyces sp. L74 exhibited algicidal activity against M. aeruginosa, A. flos-aquae, O. animalis, and Aph. flos-aquae. Streptomyces sp. L74 indirectly attacks M. aeruginosa, O. animalis, and Aph. flos-aquae by using the active substances. Streptomyces sp. L74 directly attacks A. flos-aquae by cell-tocell contact. Cytophaga, Myxobacter, and Saprospira from aquatic systems exhibit algicidal abilities by direct attack; by contrast, Bacillus, Pseudomonas, Alteromonas, and Pseudoalteromonas exhibit algicidal activities by indirect attack [14]. Streptomyces sp. L74 shows two patterns of algicidal activities. This species also exhibits high algicidal activities against harmful types of cyanobacteria. Therefore, this study provids basic information for a wide application of cynaoHAB control in various environments.
The active algicidal compound isolated from Streptomyces sp. L74 cultures was identified as a triterpenoid saponin with a molecular weight of 782. Several heat tolerant and pH stable algicidal compounds with molecular weights of <2 kDa have a wide range of algicidal activities. However, the active component in these compounds remains unidentified [13,23].
Yamamoto et al. [22] isolated L-lysine from the algicidal actinobacteria S. phaeofaciens culture and identified this microorganism as one of the causes of M. aeruginosa lysis. Triterpenoid saponin is a type of saponin found in many plants. These compounds exhibit antimicrobial, antibacterial, antifungal, antiviral, antiyeast, and cytotoxic activities [34][35][36][37]. Triterpenoid saponins are seldom produced by microorganisms. Shigematsu et al. [38] reported a tetracyclic   [40,41]. Diterpene glucosides are parts of terpene glucoside compounds and produced by a fungus [42][43][44]. In contrast to the structures of known triterpenoid saponins, triterpenoid saponin found in this study may be a new type of saponin from actinobacteria. Algicidal microorganisms kill cyanobacteria by indirect attack, in addition to direct cell-to-cell attack, via proteins, peptides, amino acids, antibiotics, nitrogen compounds, alkaloids, and other materials [8][9][10][11][12][13][14]. Various agents produced by algicidal microorganisms exhibit algicidal activities. However, few algicidal mechanisms have been determined. For instance, Yamamoto et al. [22] revealed that L-lysine from S. phaeofaciens culture exhibits algicidal activity by destroying the cyanobacterial cell wall. Lee et al. [8] identified a serine protease from the supernatant of Pseudoalteromonas sp. and suggested that the algicidal effect occurs via the enzymolysis of cyanobacterial protein by this protease. In the current study, the algicidal activity of Streptomyces sp. L74 against M. aeruginosa is caused by the destruction of the antioxidant systems of the cyanobacterial cells by the active terpenoid saponin. SOD, CAT, and POD are three important antioxidases in cells that protect organisms against damages caused by oxygen-free radicals. In 1969, Mccord and Fridovich [45]   proposed a theory of superoxide radical damage to organisms. This theory has been widely applied in toxicology.
In conclusion, the algicidal activity of Streptomyces sp. L74 against harmful bloom-forming cyanobacteria is achieved by an indirect attack. Algicidal compounds are types of hydrophilic substances that are heat tolerant and pH stable. The purified active algicidal B3 compound was identified as a type of triterpenoid saponin (2-hydroxy-12-oleanene-3, 28-O-Dglucopyranosyl) with a molecular formula of C 42 H 70 O 13 . The algicidal mechanism of Streptomyces sp. L74 against M. aeruginosa is suggested to occur by disrupting the antioxidant systems of cyanobacterial cells by the active algicidal compound. Active algicidal compounds should be isolated and identified from Streptomyces sp. L74 to promote the application of this species in the control and treatment of cyanoHABs in various environments. The algicidal mechanism of these active algicidal compounds should also be further investigated. Further studies should be conducted to confirm the structure of


extfieldsofstudy: Medicine Biology

s2fieldsofstudy: Biology Medicine

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Screening and identification of novel B cell epitopes of Toxoplasma gondii SAG1

Background The identification of protein epitopes is useful for diagnostic purposes and for the development of peptide vaccines. In this study, the epitopes of Toxoplasma gondii SAG1 were identified using synthetic peptide techniques with the aid of bioinformatics. Findings Eleven peptides derived from T. gondii SAG1 were assessed by ELISA using pig sera from different time points after infection. Four (PS4, PS6, PS10 and PS11), out of the eleven peptides tested were recognized by all sera. Then, shorter peptides that were derived from PS4, PS6, PS10 and PS11 were predicted using bioinformatics and tested by experimentation. Four out of nine shorter peptides were identified successfully (amino acids 106–120, 166–180, 289–300 and 313–332). Conclusions We have precisely located the epitopes of T. gondii SAG1 using pig sera collected at different time points after infection. The identified epitopes may be useful for the further study of epitope-based vaccines and diagnostic reagents.

Findings
Toxoplasma gondii is an obligate intracellular parasite that infects a variety of mammals and birds, causing toxoplasmosis [1,2]. T. gondii is an important food-borne parasite, and the primary route of transmission from animals to humans is through the consumption of infected meat [3,4]. In some countries, pork is the most common meat consumed, and some ethnic groups consume raw pork; thus, pigs are considered to be the primary source of human infection with T. gondii [5]. In addition, toxoplasmosis is a source of significant economic losses for swine farmers [6]. Therefore, the development of effective diagnostic reagents or vaccines for controlling this infection is required. Attempts to develop a peptide-based vaccine for T. gondii have focused on SAG1 and shown encouraging results [7]. Furthermore, the multiepitope antigen is one of the most promising antigens for the serodiagnosis of toxoplasmosis. Thus, it is very important to determine the precise sequences against which effective immune responses are directed. SAG1 epitopes have been studied by different research groups [8][9][10]. However, it is still unclear which SAG1 peptides are recognized by antibodies from pigs infected with T. gondii. Therefore, B cell epitopes of SAG1 were analyzed using a synthetic peptide technique in combination with software-based prediction.

Serum samples
A total of 51 T. gondii-positive sera, which had been previously collected from experimentally infected pigs by our lab, were investigated. Twelve pig serum samples were collected at the time of presentation of clinical symptoms (G1); 18 follow-up specimens were taken on days 14 to 35 after the onset of symptoms (G2), and 21 further serum samples were taken on days 60 to 120 after the onset of symptoms (G3). Toxoplasma IgM and IgG antibodies were confirmed by T. gondii lysate antigen-ELISA. The serum samples in G1 and G2 were positive for IgM and IgG against T. gondii. The serum samples in G3 were only positive for IgG against T. gondii. Ten sera that were negative for T. gondii IgM and IgG were used as controls. The experimental protocol was approved by the Ethical Committee of the Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

Synthetic peptides
Based on the sequence of T. gondii SAG1 (Genbank Accession No. FJ455529), 20 non-overlapping or overlapping 12-36 mer peptides were synthesized by GL Biochem Ltd (Shanghai, China). Peptide sequences are shown in Table 1.

ELISA analysis
ELISA for each peptide were performed as described by Cardona et al. [10], except that microplates were coated with 10 μg/ml peptide and peroxidase-conjugated anti-pig IgG (1:4000) was used as the secondary antibody. PS1 was negative to all sera; PS2, PS3, PS5, PS7, PS8 and PS9 were recognized by few or partial sera (PS2: 5/51, PS3: 13/51, PS5: 24/51, PS7: 32/51, PS8: 6/51 and PS9: 25/51), and only Four peptides (PS4, PS6, PS10 and PS11) were recognized by all sera (Figure 1). For each of the 4 peptides, no significant differences were observed between the mean absorbances of the three groups, as determined by an ANOVA statistical test. The mean absorbance ± standard deviation of one peptide (amino acids 271-300) was significantly higher (0.337 ± 0.053) than that of the other peptides (PS4: 0.235 ± 0.025, p = 0.000; PS6: 0.237 ± 0.0028, p = 0.000; PS11: 0.266 ± 0.039, p = 0.001). Five peptides (amino acids 61-80, 181-200, 241-260 and 301-320) derived from SAG1 were determined to be B cell epitopes, and one peptide (amino acids 301-320) was the peptide that was most strongly recognized by sera from patients with human ocular toxoplasmosis [10]. A robust immunological response to the SAG1 is associated with chronic Toxoplasma infection in humans [11]. However, we found that peptides derived from SAG1 were capable of being recognized by pig sera from different time points after infection, which is different from previous reports. The discrepancy could be explained by differences in parasite strains, by using different animal models as well as by the different MHC-types between human and pig.

Precise definition of the epitopes
To further determine the epitopes of SAG1 and decrease the number of laboratory experiments, bioinformatics was used to predict the epitopes. The secondary structure and the surface properties of the SAG1 were analyzed as described by Zhang [12]. The results are shown in Figure 2. Based on these results, 9 shorter peptides that were derived   from PS4, PS6, PS10 and PS11 were chosen for further investigation (Table 1). These peptides were tested using pig sera as described above. Four out of 9 peptides (PS4-2, PS6-3, PS10-3 and PS11-2) were recognized by all sera. The results are shown in Figure 3.

Conclusion
Conformational epitope selection needs to determine the tertiary structure of antigen in order to identify antibody interacting residues in antigen. Experimental techniques such as crystallography are expensive and time consuming. Thus, our work focuses on linear epitope selection. In this study, we have located the epitopes of T. gondii SAG1 to a shorter sequence than had been identified previously. The identified epitopes will be useful in vaccine and diagnostic reagent design.


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Characterization of temperature and light effects on the defense response phenotypes associated with the maize Rp1-D21 autoactive resistance gene

Background Rp1 is a complex locus of maize, which carries a set of genes controlling race-specific resistance to the common rust fungus, Puccinia sorghi. The resistance response includes the “Hypersensitive response” (HR), a rapid response triggered by a pathogen recognition event that includes localized cell death at the point of pathogen penetration and the induction of pathogenesis associated genes. The Rp1-D21gene is an autoactive allelic variant at the Rp1 locus, causing spontaneous activation of the HR response, in the absence of pathogenesis. Previously we have shown that the severity of the phenotype conferred by Rp1-D21 is highly dependent on genetic background. Results In this study we show that the phenotype conferred by Rp1-D21 is highly dependent on temperature, with lower temperatures favoring the expression of the HR lesion phenotype. This temperature effect was observed in all the 14 genetic backgrounds tested. Significant interactions between the temperature effects and genetic background were observed. When plants were grown at temperatures above 30°C, the spontaneous HR phenotype conferred by Rp1-D21 was entirely suppressed. Furthermore, this phenotype could be restored or suppressed by alternately reducing and increasing the temperature appropriately. Light was also required for the expression of this phenotype. By examining the expression of genes associated with the defense response we showed that, at temperatures above 30°C, the Rp1-D21 phenotype was suppressed at both the phenotypic and molecular level. Conclusions We have shown that the lesion phenotype conferred by maize autoactive resistance gene Rp1-D21 is temperature sensitive in a reversible manner, that the temperature-sensitivity phenotype interacts with genetic background and that the phenotype is light sensitive. This is the first detailed demonstration of this phenomenon in monocots and also the first demonstration of the interaction of this effect with genetic background. The use of temperature shifts to induce a massive and synchronous HR in plants carrying the Rp1-D21 genes will be valuable in identifying components of the defense response pathway.

Background
The Rp1 locus of maize carries variable numbers of tandemly-repeated genes belonging to the nucleotide binding site, leucine-rich repeat (NB-LRR) class of major disease resistance genes (also known as R-genes; [1]) that confer race-specific resistance to the common rust fungus, Puccinia sorghi [2]. The resistance response conferred by these genes is generally activated by a race-specific direct or indirect recognition of pathogen-derived molecules [1]. It includes the "hypersensitive response" (HR) which is a rapid localized cell death at the point of pathogen penetration, the induction of pathogenesis associated genes and a variety of other responses [3].
The Rp1 locus is complex; the well characterized HRp1-D haplotype is composed of nine tandemlyrepeated Rp1 paralogs [4] while the Rp1 haplotype of the B73 line carries four Rp1-homologous sequences [5] and more than 50 Rp1-homologous sequences have been identified in other lines [6]. This complexity results in the locus being meiotically unstable [7,8], with a high frequency of unequal crossover events [9]. Intragenic recombination events resulting in the generation of chimeric genes derived from two different Rp1 paralogs have been observed several times. In some cases these lead to the generation of novel resistance specificities [10]. In other cases it has led to the creation of dominant mutant genes that confer a "spontaneous necrotic" or "lesion" phenotype [10][11][12][13].
Rp1-D21 is one such gene [13]. It confers a spontaneous lesion phenotype with lesions forming in apparently random locations without the requirement for the presence of a pathogen. Previously we showed that the Rp1-D21 phenotype is influenced by genetic background; in some backgrounds it is very severe, killing the plant during the juvenile phase. In others it is suppressed to the extent that the mutant plants can set seed in a more-orless normal fashion [3,14,15].
The signal transduction pathway leading to the HR response and the mechanism by which the plant is able to restrict the HR to cells in the immediate area surrounding an infection is poorly understood in plants. We are using a series of genetic crosses with diverse lines and with members of segregating populations to identify loci and genes that interact with Rp1-D21 either to suppress or enhance its expression. The expectation is that many of these loci are also important in the regulation of the 'normal' HR induced by wild type R-genes in response to pathogen detection [3,14,15]. This approach, in which a mutant conferring an extreme phenotype in a trait of interest is used to uncover previously inaccessible genotype-dependent variation has been termed Mutant-Assisted Gene Identification and Characterization (MAGIC) [16,17].
Temperature has long been known to play a role in the modulation of plant defense responses in dicots [18], including the HR [19][20][21][22]. In some cases it appears that the temperature-sensitive component of the defense response resides in the NB-LRR genes themselves [23]. Light sensitivity of resistance responses, including HR, is likewise a well known phenomenon [24,25]. In the course of our studies we noted that phenotypic expression of Rp1-D21 seemed to be affected by temperature. This effect had been briefly noted previously [11]. In light of our ongoing work on the genetic dissection of HR using Rp1-D21 in a MAGIC approach, and especially because these experiments were taking place in multiple locations under varying environmental conditions, it was important to characterize this temperature-dependent effect further and to ascertain whether there was an interaction with genotype. We show here that while temperature effects on the Rp1-D21 phenotype were observed in all 14 genetic backgrounds tested, the temperature effects were significantly affected by genotype. The phenotype could be completely suppressed in a reversible way by manipulating temperature or light levels.

Results and discussion
Rp1-D21-associated lesion phenotypes are temperature dependent The Rp1-D21-H95 line has the background of the common maize inbred H95 into which the Rp1-D21 allele has been introgressed. It is 97% genetically identical to H95 [14]. Rp1-D21-H95 plants are heterozygous for the Rp1-D21 mutant allele and therefore the F 1 progeny of any cross between Rp1-D21-H95 and another line segregate in a 1:1 ratio for F 1 progeny carrying an Rp1-D21 gene (i.e. mutant F 1 progeny) and F 1 progeny carrying the wild-type (i.e. non-autoactive) H95 allele at the Rp1 locus (i.e. wild type F 1 progeny). Importantly the wild type and mutant F 1 progeny were almost entirely isogenic outside the Rp1 locus [14].
The Rp1-D21-H95 line and crosses between the Rp1-D21-H95 line and 13 diverse lines were used for this study ( Table 1). All of these lines are among the 26 parents of the maize nested association mapping (NAM) population [26]. Importantly for these purposes, these lines represent a diversity of phenotypic effects on the Rp1-D21 phenotype. Table 2 is adapted from Chintamanai et al. [3] and shows the response of these crosses in the field in two environments, Clayton NC and West Lafayette IN, that we observed in our previous study. Included in this experiment are crosses in which the Rp1-D21 phenotype is substantially suppressed (B97 × Rp1-D21-H95, Mo18W × Rp1-D21-H95, Oh7B × Rp1-D21-H95) and several in which it is substantially enhanced (Tx303 × Rp1-D21-H95, M37W x Rp1-D21-H95, M162W × Rp1-D21-H95 ) as well as several crosses with intermediate phenotypes.
These F 1 crosses, together with the Rp1-D21-H95 line were grown in the greenhouse at three different temperature regimes: 22/18°C, 26/22°C, and 30/26°C higher/ lower for 12 hours each. For simplicity, these regimes are referred to here as, respectively, LowGH, MidGH, and HiGH (see Methods). Two traits associated with the severity of the Rp1-D21 phenotype were scored on all the plants, mean lesion score (LES), and mean mutant to wild type height ratio (HTR). LES is simply a score of the lesion severity (Additional file 1: Figure S1) while HTR determines how much the growth of the plant has been inhibited by the expression of Rp1-D21 with respect to a near-isogenic sibling lacking the Rp1-D21 gene. The mean LES and HTR scores for all crosses at each temperature are summarized in Table 1. The plants grown at the highest temperatures, 30/26°C, had the most suppressed Rp1-D21 phenotypes (lowest LES score, highest HTR) in every case regardless of genotype. However, the ranking of these two traits at 18/22°C and 22/ 26°C depended on the genetic background although in general the phenotype was most extreme at the lower temperature ( Figure 1). The results observed in this study are largely consistent with our previous work [3] with the Tx303 × Rp1-D21-H95, M37W × Rp1-D21-H95, M162W × Rp1-D21-H95 F 1 crosses showing the most severe phenotypes and the B97 × Rp1-D21-H95, Mo18w × Rp1-D21-H95, Oh7B × Rp1-D21-H95 F 1 crosses among the most suppressed.
Analysis of variance (ANOVA) showed that the most significant sources of variation for both LES and HTR  were environment (meaning the two different occasions on which the experiment was performed in 2009 and 2010), temperature and genotype. There were also highly significant (p ≤ 0.001) interactions between genotype and environment, and genotype and temperature for both traits (Table 3). Since LES and HTR measure aspects of the same phenotype, it was not surprising that they were highly correlated. Combined data from Fall 2009 and Fall 2010 showed strong negative phenotypic correlation between LES and HTR (Pearson correlation coefficient = −0.77, p < 0.001). At each temperature range the correlation was strong (Pearson correlation coefficient =−0.79, -0.78, and −0.44 at 18/22°C, 22/26°C, and 26/30°C, respectively). The correlation showed a trend of weakening association between LES and HTR as temperature increased. This was probably because higher temperature suppressed the phenotypic expression of the Rp1-D21 phenotype.
The effect of temperature reduction on the Rp1-D21associated lesion phenotype To investigate the effect of changing the external temperature on the expression of the Rp1-D21 phenotype, we moved some Tx303 × Rp1-D21-H95 mutant plants from the HiGH to the LowGH. Within 48 hours a dramatic phenotypic change was noticed; leaves which had displayed sparse small lesions developed much more numerous and larger lesions within 48 hours of the transfer to lower temperatures ( Figure 2A). Genetically identical plants that remained in HiGH conditions did not display this dramatic change (data not shown).
We subsequently conducted an experiment in which eight F 1 seed from each of four families from the crosses of Rp1-D21-H95 to M37W, Tx303, M162W and B97, which represent a diversity of Rp1-D21supressive and enhancing backgrounds, were planted in growth chambers at 30/34°C (12 hours/12 hours)-i.e. 4°C higher than the HiGH. After three weeks growth at 30/34°C (~four leaf stage) no lesions were visible on any of the plants and it was impossible to discern wild type from mutant segregants. In one growth chamber the temperature was then dropped to 18/22°C. After 48 hours, half the plants (i.e. presumably the plants carrying the Rp1-D21 gene) from the M37W, Tx303, M162W F 1 families developed copious lesions (an example is shown in Figure 2B). The plants from the B97 F 1 family took longer to develop lesions but after seven days some lesions were visible on about half of the plants from this family too. In the growth chamber which had remained at 30/ 34°C all the plants still were indistinguishable from wild type and no lesions were visible on any leaves.
The HR-phenotype associated with Rp1-D21 can be activated/inactivated reversibly using temperature shifts At the four-week time point and using the same plants that had been shifted from 30/34°C to 18/22°C, the location at which the emerging leaves joined the main stem was marked on the mutant plants and the temperature of the growth chamber was increased from 18/22°C back to 30/34°C. After seven days, all the newly-emerged leaves and the newly-emerged portions of the leaves that  had been partially emerged at four weeks showed no, or in just a few cases, a very few lesions ( Figure 2C, D). In a separate experiment (Additional file 2: Figure S2) we grew at least 3 mutant F 1 plants from each of 13 different genotypes for four weeks at 22 /18°C and then shifted to 34/30°C. At this point the 5th leaves were partially emerged from the whorl. In every case a clear transition from a lesioned to a non-lesioned phenotype was apparent on the 5th leaf (Additional file 2: Figure S2).
This result implied two things: Firstly that the Rp1-D21 lesion phenotype could be turned off, then on, then off again simply by increasing the temperature above about 30°C and decreasing it to about 20°C by turns. In other words the activation/inactivation of the HR phenotype was reversible. Secondly, since no lesions were visible on portions of the plant that emerged immediately after the temperature was increased to 30/34°C, it implied that the Rp1-D21-associated lesions did not form before leaf emergence from the whorl but that something associated with leaf emergence, perhaps exposure to light, was required for the lesion phenotype.
An objectively measurable quantitative assay for Rp1-D21 temperature-sensitive phenotype To examine the detailed kinetics of Rp1-D21 lesion development following transfer to lower temperatures, four different genotypic combinations were selected: F 1 progenies of Rp1-D21-H95 crossed to A632, B73, Mo17 and Tx303. The phenotype underlying Rp1-D21 is highly suppressed in a hybrid combination of H95 with A632, but highly enhanced in a hybrid combination with Tx303 while F 1 hybrids of Rp1-D21-H95 with B73 and Mo17 exhibit intermediate phenotypes ( Table 2).
None of the mutants F 1 s developed Rp1-D21-associated lesions in any genetic background when grown at 30°C. Eight days after planting, the temperature was lowered to 26°C at which point HR lesions started to become apparent. The speed at which the HR lesions started manifesting was dependent on genotype of the host (Figure 3). The genetic backgrounds that had previously been associated with a more severe Rp1-D21 phenotype (Table 2), Rp1-D21-H95 × Tx303 and Rp1-D21-H95 × Mo17, displayed lesions within two and three days respectively. Of the backgrounds associated with more suppressed phenotypes, B73 took seven days for half the plants to display lesions while lesions had not formed on Rp1-D21-H95 × A632 mutants at 26°C plants nine days after the temperature shift. These results demonstrate how the temperature-dependent phenotype of Rp1-D21 can be converted to an objectively measurable quantitative trait. Loci responsible for mediating this temperature sensitivity could therefore by mapped using an approach similar to that which enabled us to map loci associated with Rp1-D21-mediated lesion severity in the field [3,14].

Light requirement for Rp1-D21 lesion manifestation
Since it appeared that lesions formed only after emergence from the whorl (see above), we wanted to determine whether exposure to light was important for lesion formation. In a separate experiment, eight seedlings of Tx303 × Rp1-D21-H95 and Rp1-D21-H95 were grown in 34/30°C, with 12 h light/12 h dark for 10 days. The middle sections of the 4th leaves were wrapped in aluminum foil to avoid exposure to light and the temperature was changed to 22/18°C. As before, lesions appeared within two or three days on the mutant segregants. After four days the foil was removed. The tissue that had been covered by the foil had virtually no lesions but after two further days at the lower temperature (with continued exposure to light) lesions started developing in these regions also ( Figure 4). This experiment was repeated a second time with essentially similar results. It should be noted that wrapping a portion of the leaf in aluminum foil as well as blocking out light, may have the effect of locally increasing the humidity and possibly also the temperature of the wrapped part of the leaf. So, conceivably, altered temperature and humidity might underlie the failure to form lesions. However, we have observed in other experiments that Rp1-D21 plants grown in bottles at~100% humidity display characteristic lesions (data not shown). Therefore it seems that both light and a permissive temperature are required for Rp1-D21-associated lesions to form.

Temperature sensitivity of the defense response associated with Rp1-D21
Apart from the obvious lesion formation phenotype, we have shown previously that Rp1-D21 expression is associated with other characteristics of the classical HR response including the generation of H 2 O 2 and superoxide as well as the induction of expression of defense-related genes such as PR1, PR5, PRms and WIP1 [3]. In order to determine whether the Rp1-D21 phenotype could also be suppressed at the molecular (as opposed to the phenotypic) level at elevated temperatures, the following experiment was performed: Twelve seeds from a Tx303 × Rp1-D21-H95 F1 family were germinated and grown in a growth chamber for three weeks at 30°C. At this point all 12 plants appeared as wild type and leaf samples were taken from all of them. The temperature was then reduced to 18°C and within 48 hours, as expected, lesions were visible on the entire area of six of the plants, these were the segregants carrying the Rp1-D21 gene while the plants without lesions were their wild type, near-isogenic, siblings. Leaf samples from two wild type and three mutant plants were taken at the four week time-point (i.e. a week after reducing the temperature). Semi-quantitative RT-PCR analysis was performed on RNA extracted from the leaves of these five plants sampled at both the three-and four-week timepoints (i.e. before and after the temperature drop). The results indicated that, after three weeks of continuous growth at 30°C, PR1 and PRms were not detectably expressed while WIP1 was expressed at relatively low levels in both wild type and mutant segregants. After a week at 18°C, all three of these genes were highly induced relative to their levels at 30°C in mutant but not in wild type plants ( Figure 5). This therefore indicates that the defense response (at least as reflected by defense-related gene expression of these specific genes) associated with Rp1-D21 lesion phenotype is affected by temperature in the same way as the visible HR response.
While this is, to our knowledge the first detailed demonstration of this phenomenon in monocots, examples of temperature sensitive plant disease resistance genes have been long been known, with high temperatures often suppressing function [22]. The tobacco N gene, which confers resistance to TMV and the Arabidopsis RPW8 gene, which confers resistance to powdery mildew are, like Rp1-D21, not effective above 30°C [27,28]. Recently the temperature responses of two NB-LRR genes, the N gene and the Arabidopsis SNC1 gene, another NB-LRR gene associated with the defense response [23], were investigated. It was shown that the temperature sensitivity of the defense response they mediated was likely resided in the NB-LRR gene products themselves and not in some component of the downstream signal transduction pathway. Our work here further suggests that while the NBS-LRR gene product may be the primary cause of temperature-sensitivity the interaction of other loci and alleles can modulate this effect. Alcázar and Parker [18] reviewed the interaction of temperature and immune responsiveness in Arabidopsis (the system in which most of this work has been done). They suggest that temperature sensitivity might be a characteristic associated with the molecular assembly of which the NB-LRR gene is a part, but which also includes other proteins such as HSP90. They also suggest that, given the prevalence of this type of relationship, temperature sensitivity may have evolved as an adaptive strategy to 'tune' the defense response to the local environmental conditions to balance the advantages of a robust defense response (disease resistance) with the disadvantages such as reduced growth and reproductive potential [29,30].
The basal defense response in Arabidopsis has been shown to be regulated in a circadian manner [31]. The expression levels of several genes important for resistance to Hyaloperonospora arabidopsidis and for basal resistance were shown to peak around dawn, the time of peak spore dissemination. In this way, it was hypothesized, the plant was able to synchronize its maximal defense responsiveness to the period of maximal need. Of course dawn is also the coldest period of the day and so perhaps the temperature sensitivity of NB-LRR resistance complexes is another way of fine tuning this system. Similarly, light dependence of the HR mediated by other R-genes has been observed [24,32]. In some cases, this may reflect a relationship between HR, photosynthesis and the oxidative balance in the cell [33,34].
We have been using Rp1-D21 as a reporter for the HR in order to map loci important for controlling natural variation in the maize defense response [3,14,15]. This work is being performed largely in field experiments in North Carolina and Indiana. The results reported here suggest that environmental temperature will be an important factor in determining how severe the Rp1-D21 phenotype is in the field and, more importantly, that different temperatures may differentially affect different genotypes. This is not necessarily surprising. Genotype by environment interactions are observed in the vast majority of quantitative trait mapping studies, emphasizing the need for conducting studies over multiple environments.

Conclusions
We have demonstrated here that: 1. The Rp1-D21 associated lesion phenotype is temperature-sensitive at both a morphological and a molecular level. 2. Rp1-D21 temperature-sensitivity is observed in all genetic backgrounds studied, including backgrounds that both strongly enhance and strongly suppress the phenotype. 3. However, there is a significant interaction between temperature and genotype. In other words, the Figure 5 The expression of three maize genes associated with the defense response PR1, PRms and WIP1 was measured using semi-quantitative PCR in three mutant two wild type seedlings derived from the cross Tx303 × Rp1-D21-H95. GAPDH and EF1α were included as reference genes. RNA was extracted from the plants grown at a constant 30°C for three weeks, the temperature was then dropped to a constant 18°C for seven days and RNA was extracted from leaves of the same plants.
response of the Rp1-D21 phenotype to changes in temperature is, in part, dependent on genetic background. 4. The effect of genotype on temperature sensitivity can be objectively quantified by examining the kinetics of lesion formation after temperature change. 5. The Rp1-D21 phenotype can be suppressed at temperatures above 30°C in all genetic backgrounds studied at both the gross phenotypic and the molecular level. 6. The Rp1-D21 phenotype can be suppressed and activated in a reversible manner by altering the external temperature. 7. Light exposure is also required for the Rp1-D21 lesion phenotype.
As noted above, temperature effects on the R-gene mediated defense response may be a general phenomenon in plants [18,23,35]. However this is the first time that the interaction of this effect with genetic background has been demonstrated. This implies that other genetic factors other than the R-gene itself are responsible for mediating the effect of temperature. Using the Rp1-D21 system and the MAGIC [14,16,17] technique we will now be able to examine the loci and genes underlying this response. Furthermore, the precise ability to turn the defense response on and off in a massive and synchronous way provides new resources with which to characterize the molecular and physiological events associated the maize defense response.

Plant materials
The Rp1-D21-H95 line was generated as described previously [3]. Briefly, the Rp1-D21 variant was crossed to the maize inbred line H95, and subsequently backcrossed to the H95 parent four times, while selecting for the HR phenotype indicated by the spontaneous formation of cell death lesions. Since Rp1-D21 homozygotes in the H95 background are often unable to set seed, this stock is maintained in heterozygous condition by repeatedly crossing it as a male to the H95 inbred. A previous study showed that the Rp1-D21-H95 line is 97% identical genetically to H95 with 121.5 Mbp of the genome heterozygous for the recurrent and donor parent alleles out of a total genome size of 2045 Mbp [14]. The Rp1-D21-B73 line was produced in the same way as Rp1-D21-H95, just substituting B73 for H95.
The Rp1-D21-H95 line was crossed to 13 diverse lines. All of these lines and B73 are among the 26 parents of the maize Nested Association Mapping population [26]. F 1 progenies segregated 1:1 mutant:wild type. Apart from the segregation of the Rp1-D21 gene, these progenies were essentially isogenic.

Growth conditions
Plants were grown in the phytotron greenhouse facility at North Carolina State University with fixed temperatures of 22/18°C, 26/22°C, and 30/26°C higher/ lower for 12 hours each, respectively. For simplicity these greenhouses (GH) were referred to as LowGH, MidGH, and HiGH, respectively. In each year the experiment was laid out in randomized complete block design with two replicates per year per temperature condition. Within each replicate three wild type and three mutant plants were measured per genotype in 2009 and two plants per genotype in 2010. Each replicate was arranged as a randomized complete block. The experiment was performed in fall 2009 and fall 2010. No artificial lighting was used.
In each GH seeds were planted on a plastic germination tray. As soon as it was possible to distinguish wild types from mutant counterparts, seedlings were transplanted to a six inch pot with standard soil mix. Throughout the study period seedlings were watered two times a day on regular basis. Changes in growth and lesion development were photographed and documented.
For all the growth chamber experiments light level were maintained at approximately 325 micromoles/m/s using cool white fluorescent lights. Growth chamber experiments were performed to determine whether constant temperatures above 30°C could abrogate the expression of Rp1-D21 entirely and to determine whether the phenotype was reversible. Four F 1 families: Tx303 × Rp1-D21-H95, M162W × Rp1-D21-H95, and M37W × Rp1-D21-H95, B97 × Rp1-D21-H95 were used for this experiment. In the first three of these F 1 families, the Rp1-D21 phenotype is relatively enhanced; in the last it is relatively suppressed. The Rp1-D21-H95 and Rp1-D21-B73 lines were also included as standard checks for comparison. Eight plants were grown for each of the crosses in two growth chambers. Both growth chambers were initially calibrated at low temperature of 30°C and high temperature of 34°C. Illumation was fixed at 12 h dark and 12 h light and seedlings were regularly watered twice a day. In one growth chamber, after three weeks (corresponding to the four leaf stage), the temperature was changed to a low temperature of 18°C and high temperature of 22°C. After four weeks the temperature in this growth chamber was shifted back to 30/34°C. Temperature shifts took about 20 minutes to complete in every case. Light duration remained unchanged.
For the experiment to measure the kinetics of Rp1-D21-associated lesion formation, F1 crosses of Rp1-D21-H95 crossed to A632, B73, Mo17 and Tx303 were used. Twenty plants from each of the four F 1 families cross were grown in pots (2 plants per pot) in a growth chamber under a daily photoperiod of 12 h. Initially the temperature of the growth chamber was maintained at a constant temperature of 30°C, but after 8-days of growth (V2 stage) the temperature was dropped to 26°C. Plants expressing HR lesions were counted twice every day for the next 9 days.

Phenotypic data collection
Plants were evaluated for a lesion severity phenotype on a 0-10 scale with 0 being no symptoms and 10 being dead. Wild type to mutant height ratio was also measured. This was simply the average height of the mutant plants divided by the average height of the wild type plants [3,14]. Lesion severity was scored twice in both fall 2009 and fall 2010.

Data analysis
ANOVA was performed using SAS version 9.2 (SAS Institute, 2002-2008). SAS Version 9.2 PROC CORR (SAS Institute, 2002-2008) was used to estimate the Pearson correlation coefficients. Heritability estimates were calculated using PROC MIXED procedure of SAS, as described previously [36].

Semi-quantitative RT-PCR
Total RNA was extracted from maize leaf tissue using Trizol (Life Technologies, Carlsbad, CA) according to manufacturer's instructions. RNA concentration, quality and integrity were monitored by the NanoDrop and agarose gel electrophoresis. For cDNA synthesis, 1μg of total RNA was reverse transcribed using M-MLV (Life Technologies, Carlsbad, CA) following standard protocols. Briefly, total RNA was mixed with 1μl of Oligo (dT)20 primers (Life Technologies, Carlsbad, CA), heat denatured at 65°C for 5 min and chilled on ice for 2 min. To each reaction 4 μl of 5× First Strand buffer, 2 μl of 0.1 M DTT, and 1μl RNaseOUT (Life Technologies, Carlsbad, CA) was added. The reaction was incubated at 37°C for 2 min and then 1 μl of M-MLV was added, to a final volume of 20 μl. cDNA synthesis was performed at 37°C for 2 hours, followed by a 15 min incubation at 75°C for enzyme inactivation. The cDNA reaction was then diluted 5× using water.

Additional files
Additional file 1: Figure S1. The scoring scale used for visual scoring of the lesions. Leaves scored from 1-8 are shown. An entirely dead leaf would score a "10" whereas a "9" would be a leaf with just a few patches, about 5%, of living tissue.
Additional file 2: Figure S2. Plants in 13 different backgrounds were grown for four weeks at 22/18°C, 12 hr light/dark and then shifted to 34/ 30°C 12 hr light/dark. At this point at which the 5th leaves were partially emerged from the whorl. Pictures were taken from 5th leaves five days after temperature shift. In every case the genotype of the plant is an F 1 cross between the line indicated and Rp1-D21-H95. The red line indicates the point up to which the 5th leaf had emerged at the time of the temperature shift. While only one plant is pictured for each background, at least 3 mutant F 1 plants were observed in each background and the results were essentially similar in each case.


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Acroframosome-Dependent KIFC1 Facilitates Acrosome Formation during Spermatogenesis in the Caridean Shrimp Exopalaemon modestus

Background Acrosome formation and nuclear shaping are the main events in spermatogenesis. During spermiogenesis in Exopalaemon modestus, a unique microtubular structure called the acroframosome (AFS) forms in spermatids. The AFS links to a temporary organelle called the lamellar complex (LCx) leading to the formation of an everted umbrella-shaped acrosome and a dish-shaped nucleus in the mature sperm. These morphological changes require complex cell motility in which the C-terminal kinesin motor protein called KIFC1 is involved. In this study, we demonstrate that KIFC1 moves along the AFS and plays an important role in acrosome formation and nuclear shaping during spermatogenesis in E. modestus. Methodology/Principal Findings We cloned a 3125 bp complete cDNA of kifc1 from the testis of E. modestus by PCR. The predicted secondary and tertiary structures of E. modestus KIFC1 contain three domains: a) the C-terminus, b) the stalk region, and the c) N-terminusl. Semi-quantitative RT-PCR detected the expression of kifc1 mRNA in different tissues of E. modestus. In situ hybridization demonstrated the temporal and spatial expression profile of kifc1 during spermiogenesis. Western blot identified the expression of KIFC1 in different tissues of E. modestus, including the testis. Immunofluorescence localized KIFC1, tubulin, GM130, and mitochondria in order to elucidate their role during spermiogenesis in E. modestus. Conclusion/Significance Our results indicate that KIFC1 transports the Golgi complex, mitochondria, and other cellular components that results in acrosome formation and nuclear shaping in E. modestus. The KIFC1 transport function depends upon the microtubular structure called the acroframosome (AFS). This study describes some of the molecular mechanisms involved in the acrosome formation and nuclear shaping in E. modestus. In addition, this study may provide a model for studying the molecular mechanisms involved in spermatogenesis in other crustacean species and lead to a better understanding of the fertilization process in crustaceans.

Introduction
There are three types of motor proteins that convert chemical energy into mechanical energy and show cytoskeleton dependency. Kinesin and dynein show microtubule dependency and participate in morphological changes and organelle/vesicle transport [1][2][3][4]. Myosin shows microfilament dependency and participates in various processes like muscle contraction. Kinesin motor proteins use energy released by ATP hydrolysis for organelle/vesicle transport [5]. The molecular mechanisms used by various kinesin motor proteins in morphological changes and organelle/vesicle transport have been elucidated [1,3].
In addition, KIF1C and kinesin II associate with the Golgi complex and transport Golgi vesicles to the endoplasmic reticulum in a retrograde fashion [11,12]. The kinesin II motor protein interacts with the intra-flagellar transport complex and functions in mammalian intra-flagellar transport, including the testis [13]. Kinesin transports a unique oligomeric tubulin complex during giant squid neuron development [14,15]. The KIF5 motor protein associates with a peripheral membrane protein called Gadkin and the clathrin adaptor AP-1 protein and transports endosomal vesicles [16].
Although there are numerous kinesin motor proteins involved in spermatogenesis [17][18][19][20][21][22][23][24], we focused on the C-terminal KIFC1 motor protein which belongs to the kinesin-14 subfamily with a highly conserved motor domain [25]. KIFC1 associated with the nuclear membrane participates in acrosome formation via vesicle transport along the outer face of the machete [25,26]. KIFC1 also transports other cellular components during spermatogenesis in various species [27][28][29][30][31]. For example, the KIFC1 motor protein participates in acrosome formation in Eriocheir sinensis and cell morphological changes in Octopus tankahkeei [27][28][29]. The KIFC1 motor protein also drives acrosome formation and cell morphological changes by interacting with the AFS and LCx during spermatogenesis in Macrobrachium nipponense [30]. However, the relationship between the KIFC1 motor protein and the AFS during spermatogenesis in caridean shrimp remains unclear. In order to explore this relationship between KIFC1 and the AFS, we chose the caridean shrimp Exopalaemon modestus as our animal model.
There are three events in mammalian spermatogenesis which include: a) acrosome formation, b) nuclear shaping, and c) flagellum formation. However, there are only two events in decapod crustacean spermatogenesis which include only acrosome formation and nuclear shaping because mature crustacean sperm lack flagella [32][33][34]. The two events in decapod crustacean spermatogenesis generally result in the formation of a unique shaped acrosome and nucleus. For example, an intricately-shaped acrosome and a cup-shaped nucleus form in Eriocheir sinensis mature sperm [32,33]. An everted umbrella-shaped acrosome and a dish-shaped nucleus form in the caridean shrimp M. nipponense mature sperm [34][35][36]. The morphological changes of caridean shrimp spermatids have been reported in our previous publication [34][35][36][37]. Briefly, at the early stage, the nucleus firstly is changed from spheroidicity to oblate and the cytoplasmic components began to accumulate at one side of the spermatid [34]. At the middle stage, the main cytoplasmic component is a temporary specific organelle LCx, which primarily contained Golgi apparatus, endoplasmic reticulum, mitochondria, lysosome and centriole [35,36]. Simultaneously, centriole drives the formation of the microtubular structure AFS, which extends around continuously and provides motor proteins as a support of movement [37]. In particularly, the center of AFS will form a protuberance which is perpendicular to the nucleus and the protuberance grows as a long spike, and the LCx is condensed, compacted and form the acrosome eventually.
Even though the morphological changes that occur during spermatogenesis in caridean shrimp have been reported, the mechanism of acrosome formation in caridean shrimp remains largely unclear at this time [34]. With regards to acrosome formation in caridean shrimp, the LCx and AFS are two important structures that deserve investigation. The temporary organelle called the LCx was first discovered in M. nipponensis. The LCx components consist of the Golgi complex, endoplasmic reticulum, mitochondria, lysosomes, and centriole [36]. All of these organelles pull and pack together until an everted umbrellashaped acrosome forms during the middle stage of spermatid formation. The microtubular structure called the AFS may participate in the pulling and packing together of the abovementioned organelles that form the LCx. The AFS may originate from the centriole and provide the framework for acrosome formation [37]. The AFS may also provide the framework that holds the LCx together, participate in the accumulation of acrosomal hydrolytic enzymes, maintain the acrosome structure, and facilitate the entry of the sperm nucleus into the ovum during fertilization [30].
Caridean shrimp of different genera share similar processes of spermiogenesis [30,[34][35][36][37]. We previously reported that the morphological changes during spermatogenesis in E. modestus are similar to those in M. nipponensis [35,36]. Therefore, we hypothesize that the AFS may play a similar function during spermiogenesis in E. modestus as it does in M. nipponensis. This hypothesis raises some interesting questions. Is the AFS a ubiquitous microtubular structure in all caridean shrimp? Do other motor proteins ''walk'' along the AFS and contribute to the formation of the everted umbrella-shaped acrosome? In this study, we report that the KIFC1 motor protein carries and transports the LCx components along the microtubules of AFS which thereby facilitates acrosome formation during spermiogenesis in E. modestus.

Animals and Sampling
The wild adult E. modestus shrimp were purchased from Taihu Lake at Huzhou city in Zhejiang Province (China). We detached the zoetic individuals and collected the tissues such as testis, heart, muscle, hepatopancreas, and gill. The detached tissues were put into liquid nitrogen immediately for RNA and protein extraction. In addition, the testis was fixed with 4% paraformaldehyde in phosphate buffered saline (PBS; pH 7.4) for in situ hybridization and immunofluorescence.
No approval for experimentation on caridean shrimp is needed in China.

RNA extraction and reverse transcription
The Phase Lock Gel TM Heavy with Trizol A + reagent (Tiangen Biotech, Beijing, China) was used to extract total RNA from the   s; 72uC for 10 min for the final extension. The PCR products then underwent extraction, purification, and sequencing of the target fragments as described above.

Sequence analysis and phylogenetic analysis
BLAST online provided by the National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih.gov/) was used to analyze the similarity between the kifc1 cDNA/protein sequences and the known sequences. The Vector NT110 (Invitrogen, California, USA) software was used to align multiple sequences of KIFC1 proteins for analysis of conserved sequences. The Mega4 (version 5.0) software was used to construct the phylogenetic tree by the neighbor-joining method. The KIFC1 homologues in different species were used for comparison and

Semi-quantitative RT-PCR analysis of kifc1 mRNA expression in different tissues
The PrimeScriptH RT reagent Kit (Takara, Dalian, China) was used for the reverse transcription assay of different tissues. The Primer Premier 5 software was used to design two specific primers (Table 1) for the analysis of kifc1 mRNA expression in different tissues. Two specific primers of actin (Table 1) were used to amplify a b-actin cDNA fragment for positive control. PCR was run as follows: 94uC for 4 min; 35 cycles of 94uC for 30 s, 60uC for 30 s, and 72uC for 30 s; 72uC for 10 min for the final extension. All PCR products were detected by agar gel electrophoresis. The Quantity 1 (version 4.4.0) software from Tanon Science & Technology Co., Ltd was used to analyze the relative expression quantities in different tissues.

In situ hybridization (ISH)
Tissue preparation. The testes of the caridean shrimp were embedded into an optimum cutting temperature (O.C.T) compound and sectioned at 6 mm thickness using a sliding microtome. The tissue sections were mounted on RNase-free poly-l-lysinecoated slides and the samples were rapidly stored at 280uC.
Riboprobe synthesis. wo special primers were designed and used to amplify the target cDNA fragment (662 bp) for riboprobe synthesis. The PCR was run as follows: 94uC for 4 min; 35 cycles of 94uC for 30 s, 60uC for 30 s, and 72uC for 40 s; 72uC for 10 min for the final extension. The target cDNA products were sequentially inserted into the PGEM-T EASY Vector (Promega, Fig. 3. The phylogenetic tree of KIFC1 protein and its homologues. This figure shows the phylogenetic tree of KIFC1 and it homologues that were constructed by the neighbor-joining method using Mega 5 (version 5.0) software. The KIFC1 homologues from Eriocheir sinensis, Gallus gallus, Xenopus laevis, Homo sapiens, Danio rerio, Salmo salar, Rattus norvegicus, and Cynops orientalis were examined. The putative E. modestus KIFC1 protein is most closely related to Eriocheir sinensis. doi:10.1371/journal.pone.0076065.g003 Beijing, China), propagated into competent cells (Escherichia coli DH5a), and sequenced by Biosune Company (Shanghai, China). The plasmid with 662 bp nucleotides of kifc1 was linearized with incision enzyme and transcribed in vitro with T7 promoter. The riboprobes were precipitated with ethanol and LiCl and were resuspended in DEPC-treated H 2 O. The riboprobe concentration and quality were assessed by spectrophotometer and nucleic acid electrophoresis, respectively.
Prehybridization and hybridization. The testis sections were set at room temperature for 10 min and fixed with 4% paraformaldehyde (PFA, pH 7.4) for 10 min. Subsequently, the sections were rinsed for 10 min in 0.1% diethylpyrocarbonate (DEPC)-activated 0.1 M phosphate-buffered saline (PBS, pH 7.4) at room temperature twice and equilibrated for 15 min in 56SSC (sodium chloride 0.75 M, sodium citrate 0.075 M, pH 7.0). Then the prehybridization buffer consisting of 50% deionized formamide, 40 mg/mL denatured salmon sperm DNA, and 56SSC solution was placed on the sections for 2 h at 55uC. The hybridization buffer consisting of approximately 300 ng/mL of denatured and digoxigenin (DIG) -labeled riboprobes was placed on the sections overnight at 57uC. Then, the sections were sequentially rinsed for 30 min in 26SSC solution (pH 7.0) at room temperature; 1 h in 26SSC solution (pH 7.0) at 65uC water bath; and 1 h in 0.16SSC solution (pH 7.0) at 65uC water bath.
Detection of the product signal. After equilibrating for 5 min in Buffer I (0.1 M Tris-hydrochloride, 0.15 M NaCl, pH 7.5), the sections were incubated for 2 h at room temperature in DIG Buffer I consisting of 1:4000 anti-DIG alkaline phosphatase conjugated Fab fragments (Roche, Branford, USA) and 0.5% (w/v) blocking reagent (Roche, Branford, USA). Then, the sections were washed three times (each for 15 min) in Buffer I and equilibrated for 5 min in Buffer II (0.1 M Tris-HCl, 0.1 M NaCl, 0.05 M MgCl2, pH 9.5). The chromogenic agent (330 mg/ mL nitroblue tetrazolium chlorideand (NBT) and 165 mg/mL 5bromo-4-chloro-3-indolyl-phosphate (BCIP) in Buffer II) (Pro-mega, Beijing, China) was added onto the sections for coloration in the dark for 2 hours at room temperature. The reaction was terminated by rinsing the sections with 10 min in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). The sections were washed in 95% ethanol for 2 hours in order to remove any nonspecific staining. The sections were washed in deionized water for 15 min in order to remove any precipitated Tris crystals. The sections were dehydrated with 50, 75, 95, and 100% ethanol in succession, each for 15 min. Finally, the sections were infiltrated in xylol, mounted in neutral resin, and cover slipped. The hybridized sections were observed by using a Nikon Eclipse E80i microscope (Nikon, Tokyo, Japan) under brightfield illumination. The negative control was performed without adding any probes (data is not shown).

Prokaryotic expression
Sequences encoding kifc1 were amplified by PCR with primers (Table 1) containing a BamHI site added on the 59 end and a EcoRI site added on the 39 end. The resulting amplifications of kifc1 sequence were digested and ligated into pET28a (Invitrogen Life Technologies, California, USA). The recombinant plasmid DNAs were transformed into Rosetta (DE3) pLysS (Novagen, Darmstadt, Germany). A single colony of Rosetta harboring the recombinant plasmids was inoculated into 300 ml of Luria-Bertani medium containing chloramphenicol (100 mg/l) with kanamycin (25 mg/l). The culture was shaken at 220 rpm and incubated at 37C until the OD 600 value reached 0.8. Isopropyl-b-D-thiogalactoside was added to a final concentration of 1 mM and the culture was shaken continually at 220 rpm and incubated at 37C for 10 h. After ultrasonication, the precipitates were resuspended in 8 M urea. Recombinant HIS-KIFC1 protein in the resuspended liquids were purified by nickel-nitrilotriacetic acid agarose affinity chromatography according to the QIA expressionist manual (Qiagen, Frankfurt, Germany) and assessed by 12% SDS-PAGE. The purified recombinant HIS-KIFC1 protein was used to make antibody in Hangzhou HuaAn Biotechnology Co., Ltd. (Hangzhou, China).

Antibodies
The KIFC1 polyclonal antibody was prepared by Hangzhou HuaAn Biotechnology Co., Ltd. (China). Rabbit anti-b-actin was purchased from BIOS (Shanghai, China). FITC conjugated monoclonal anti-tubulin was purchased from Sigma (St. Louis, Mo., USA). HRP conjugated goat anti-rabbit IgG was purchased from Immunology Consultants Laboratory, Inc. Texas Redconjugated affinipure goat anti-rabbit IgG was purchased from Protein Tech Group, Inc. The Mito-Tracker Green FM was purchased from Invitrogen (California, USA).

Western blot analysis
Different tissues from adult E. modestus were homogenized in RIPA Lysis Buffer (Solarbio, Shanghai, China) containing protease inhibitors. The homogenates were centrifuged at (14,000 rpm) at 4C for 15 min and the supernatant liquids were collected. The protein concentrations were detected by the Coomassie brilliant blue method and the protein samples were dissolved in 5*SDS sample buffer. The protein samples with equal amounts of protein were separated on 12% gels (SDS-PAGE) and transferred onto the PVDF membrane (Bio-Rad, California, USA). The membrane was blocked with 5% BSA in PBST (PBS with 0.5% Tween 20) for 1 h. Then, the membrane was incubated overnight at 4C with rabbit anti-KIFC1 antibody (diluted 1:500) in 5% BSA. After washing three times with PBST for 1 h, the membrane was incubated for 1 h with secondary antibody HRP- conjugated goat anti-rabbit IgG (diluted 1:1000) in 5% BSA. After that, the membrane was washed three times with PBST for 45 min, three times with PBS for 30 min and three times with ultrapure water for 15 min. The membrane was developed according to the manufacturer's instructions of SuperSignal West PicoTrial Kit (Thermo, Massachusetts, USA). The emitted light was detected using a chemiluminescence imaging (Chemiscope 3400) (Shanghai, China).

Immunofluorescence staining
The testes of adult male E. modestus were fixed in 4% paraformaldehyde (PFA, pH 7.4) overnight, rinsed in PBS three times for 15 min, and incubated overnight in 0.5 M sucrose in PBS. The testes were embedded in Tissue-Tek O.C.T. Compound. The tissue blocks were stored in the refrigerator (220uC) and cut into 6-mm frozen sections using a cryostat. The sections were washed with PBST (PBS with 0.3% Triton X-100) for 15 min at room temperature and then blocked with 5% BSA in PBST (0.1% Triton X-100) for 1 hour. The tissue sections were incubated with anti-KIFC1 antibodies (1:80 dilution) in blocking buffer 5% BSA at 4C overnight. The sections were rinsed 3 times in PBST for 45 min. The tissue sections were then incubated with Texas Red goat anti-rabbit IgG (1:100 dilution, Jackson Immuno Research Laboratories, West Grove, PA., USA) or FITC-anti-

Electron Microscopy
Sample for transmission electron microscopy (TEM) observation was performed according to our previous publications [27.28]. Briefly, SD rat testis were prefixed with 2.5% glutaraldehyde, cut into 1 mm 3 in size, then fixed in the same fixative at 4uC for 2 hours, and then the samples were post-fixed in 1% osmium tetroxide solution (Electron Microscopy Sciences, Hatfield, PA, USA) at room temperature for 1 hour. Samples were washed twice with sodium phosphate buffer. Subsequently, the samples were prestained with uranyl acetate and dehydrated in graded alcohol and acetone. The Spurr's resin was used to embed the samples. The samples were sectioned with a Leica Ultracut UC6 Ultramicrotome (Leica Microsystems AG, Wetzlar, Germany), counterstained with uranyl acetate and lead citrate. Finally, PHILIPS TECNAI 10 (PhilipsMedical System, Best, Netherlands) TEM operated was used to observe the grids at 80 kV [25].

kifc1 sequence analysis
We used a pair of primitive primers to obtain a 662 bp middle fragment. Subsequently, we designed special primers to amplify a 797 bp sequence and a 1787 by sequence for 59 RACE and 39 RACE, respectively. The full-length cDNA of the kifc1 in E. modestus consists of a 73 bp 59untranslated region, a 952 bp 39untranslated region, and a 2100 bp open reading frame that encodes 700 amino acids (Fig. 1).

KIFC1 protein sequence alignment and phylogenetic analysis
The alignment of KIFC1 and its homologues in different species shows that the sequenced KIFC1 has 69.9, 47.8, 47.9, 49.2, and 46.9% identity with its counterparts in Eriocheir sinensis, Crassostrea gigas, Xenopus laevis, Mus musculus, and Danio rerio, respectively (Fig. 2). Phylogenetic analysis demonstrates that the putative protein of KIFC1 is closer to Eriocheir sinensis than other examined species (Fig. 3).

KIFC1 protein structure
The predicted secondary structure of the E. modestus KIFC1 motor protein contains three domains: a) the C-terminus, b) the stalk region, and c) the N-terminus (Fig. 4A). The 346-700 amino acids constitute the putative C-terminus and contain the conserved motor domain that forms a head for ''walking'' along the microtubule (Fig. 4A). The 154-346 amino acids constitute the  coiled stalk region. The 1-154 amino acids constitute the Nterminus with a divergent tail to carry different cargoes (Fig. 4A). The tertiary structure of KIFC1 motor protein comports with the secondary structure characteristics (Fig. 4B).

kifc1 mRNA expression in different tissues
We used semi-quantitative RT-PCR to detect Kifc1 mRNA expression in various tissues of E. modestus. We amplified a 662 bp kifc1 fragment from the testis, heart, muscle, hepatopancreas, and gill (Fig. 5A). A 297 bp b-actin fragment was used as positive control (Fig. 5A). We found that kifc1 mRNA expression occurs abundantly in all of the above-mentioned tissues, including the testis (Fig. 5B).

Temporal and spatial expression pattern of kifc1 mRNA during spermiogenesis
We used in situ hybridization to track the temporal and spatial expression patterns of kifc1 mRNA. We found that the localization of kifc1 mRNA closely relates to acrosome formation (Fig. 6). During the early stage of spermiogenesis, kifc1 mRNA signals weakly distribute throughout the cytoplasm in round or oblong spermatids ( Fig. 6A and B, arrows; blue signal). Later in spermiogenesis, the cytoplasmic components gather to one side of the spermatid and the LCx gradually develops. At this time, kifc1 mRNA signals increase compared to the early stage and concentrate on the side of the spermatid where the acrosome will eventually form during the middle stage of spermiogenesis (   and D, arrows). During the late stage of spermiogenesis, the AFS appears and the nucleus becomes dish-shaped. In addition, kifc1 mRNA signals strongly distribute at the acrosome cap, display a dot-like pattern on the surface of the dish-shaped nucleus, and are absent at the AFS-derived spike ( Fig. 6E and F, arrows). In the mature sperm, kifc1 mRNA signals dramatically decrease in general. The kifc1 mRNA signals weakly distribute at the acrosome cap, are absent at the dish-like nucleus, and are absent at the AFS-derived spike (Fig. 6G ad H, arrows). A series of schematic diagrams clarify the temporal and spatial kifc1 mRNA expression patterns during spermiogenesis (Fig 6I, J, K, and L).

Identification of KIFC1 protein in various tissues of E. modestus
We used Western blots to identify KIFC1 protein expression in E. modestus muscle, gill, heart and testis using an anti-KIFC1 polyclonal antibody. The anti-KIFC1 polyclonal antibody recognized a 78 kDa band indicative of KIFC1 (Fig. 7, upper panel). The control was treated with anti-b-actin polyclonal antibody (Fig. 7, lower panel). We found that KIFC1 protein expression occurs in E. modestus testis, heart, muscle, hepatopancreas, and gill.

KIFC1 cellular transformations during the spermiogenesis in E. modestus
We used immunofluorescent localization of tubulin and KIFC1 in order to explore the role of microtubules and KIFC1 in cellular transformations during spermiogenesis in E. modestus (Fig. 8, 9 , 10, 11). During the early stage of spermiogenesis, tubulin ( Fig. 8B and E, arrows; 8F; green signal) and KIFC1 ( Fig. 8C and E, arrows; 8F; red signal) localize in the cytoplasm and near the nuclear membrane. During the middle stage of spermiogenesis when the LCx forms, tubulin localizes in sporadic areas within the LCx (Fig. 9B and E, arrows; 9F). However, KIFC1 localizes throughout the entire area of the LCx (Fig. 9C and E, arrows; 9F). During the late stage of spermiogenesis when the AFS forms, tubulin localizes specifically to the AFS (Fig. 10B and E, arrows; 10F). However, KIFC1 localizes mainly to the proacrosome with some staining associated with the AFS (Fig. 10C and E, arrows; 10F). In the mature sperm, tubulin localizes primarily in the center of the acrosome ( Fig. 11B and E, arrows; 11F). However, KIFC1 localizes throughout the whole acrosome including the AFSderived spike ( Fig. 11C and E, arrows; 11F).

Manchette location during rat spermatogenesis
We used electron microscopy to demonstrate the distinction between the role of the manchette in rat spermatogenesis and the AFS in E. modestus spermatogenesis. We did not find proves which show that the manchette in rat spermatogenesis plays a role in acrosome formation (Fig. 12A), but rather plays a major role in nuclear shaping (Fig. 12B). Immunofluorescent staining of tubulin and KIFC1 showed a detached manchette that surrounds the nucleus in a skirt-like manner (Fig. 12C, D).

KIFC1 transport substrates
We used immunofluorescent localization of GM130 (a Golgi complex marker) and MitoTracker (a mitochondria marker) in order to identify KIFC1 transport substrates involved in acrosome formation (Fig. 13, 14, 15). During the early stage of spermiogenesis, GM130 localizes in the cytoplasm near the periphery of the nucleus (Fig. 13A-C: Fig. 15A). During the middle stage of spermiogenesis, GM130 localizes along one side of the nucleus as nucleus gradually deforms (Fig. 13D-F; Fig. 15B, C). During the late stage spermiogenesis, GM130 localizes in the LCx and in the proacrosome near the AFS (Fig. 13G-I; Fig 15D). In mature sperm, GM130 localizes in the acrosome (Fig. 13J-L; Fig. 15E). MitoTracker localizes in the same fashion as GM130 during the different stages of spermiogenesis in E. modestus (Fig. 14, 15).

The audio video interactive of E. modestus spermatid
The audio video interactive shows the stereochemical morphology of E. modestus spermatids during the late stage of spermiogen- esis (Video S1). The video shows a dish-shaped nucleus (blue) and the distribution of the microtubular structure AFS (green).

The comparison of acroframosome and manchette
During mammalian spermatogenesis, the microtubular structure called the manchette appears in the developing spermatids and then disappears in the mature sperm [41][42][43]. The skirt-like manchette functions in nuclear shaping by acting as a scaffold via kinesin and dynein motor proteins in order to transform the nucleus from a spherical-shape to an oblong-shape [44][45][46][47][48]. In this regard, abnormal sperm heads and nuclear morphogenesis occur when the synthesis of tubulin (i.e., microtubules) is blocked [45]. A number of motor proteins are key players in spermatogenesis. During mammalian spermatogenesis, KIFC1 associates with the manchette and a complex containing nucleoporin NUP62 and facilitates nuclear shaping [26]. During spermatogenesis in Octopus tankahkeei, a KIFC1-like motor protein associates with the manchette and facilitates nuclear shaping [29]. In addition, another motor protein called dynein associates with the spermatid nuclear envelope during spermatogenesis in mammals [48][49][50].
During caridean shrimp spermatogenesis, we previously reported that a unique microtubular structure called the acroframosome (AFS) is formed which structure is similar to the manchette [37]. The everted umbrella-shaped AFS may play functions during acrosomal formation in the caridean shrimp, whereas manchette mainly functions in nuclear shaping in the rat. According to previous research in our lab, AFS may also contain microfilaments to maintain the spermatids flexibility, which is one of the main differences between AFS and manchette [37]. The AFS is a permanent structure found in both caridean shrimp spermatids and mature sperm, whereas the manchette is a transient structure found in only in mammalian spermatids.

KIFC1 may participate in acrosomal shaping and biogenesis
A comparison of the similarity of E. modestus KIFC1 with different species and the phylogenetic tree illustrated that E. modestus KIFC1 remains relatively conservative which indicates that KIFC1 maintains its vital function throughout evolution. Furthermore, KIFC1 showed a relatively high expression in the E. modestus testis which adds further weight to the idea that KIFC1 plays a vital function in E. modestus spermatogenesis.
The identification of cDNA sequence revealed the existence of kifc1 isogenous gene in the testis of E. modestus. Concurrently, the identification of kifc1 gene may indicate its vital functions in testis. Multiple sequence alignment showed the conservative functional domains, such as ATP-binding domains and the microtubulebinding domain. Structural prediction reveals the KIFC1 of E. modestus mainly contained C-terminus, the stalk region and the Nterminal [5,25,28]. The putative C-terminus is the microtubulebinding domain formed a head which walking along the microtubules. The function of predicted stalk region is to form an extended coiled-coil stalk. And the predicted N-terminal is a divergent tail to carry different cargoes. The comparison of the similarity with different species and the phylogenetic tree illustrated that the KIFC1 of E. modestus kept the relative conservative which may important for the functions of KIFC1 during the evolution.
Analysis of kifc1 mRNA and KIFC1 protein expression in various tissues showed that KIFC1 has relatively high expression in the testis. This result may indicate the indispensable function of KIFC1 during spermatogenesis in E. modestus. In addition, the KIFC1 also expressed in the other tested tissues, which may suggest the importance of motor protein KIFC1 in crustacean E. modestus. It is worth to mention that there is a different KIFC1 expression pattern in different tissue and organs between mouse and the caridean shrimp E. modestus. Zhang and Sperry (2004) described that the expression of KIFC1 protein was very weak in muscle and heart of mouse [51], while the western blot analysis of E. modestus tissues shows abundant expression in muscle and heart. This may suggest a significant expression pattern between mammalian and crustaceans. The functions of KIFC1 in different tissue and organs in caridean shrimp need to be further investigated.
The acrosome formation is one of the most important events of the spermiogenesis of the caridean shrimp. The everted-embrellalike acrosome of the caridean shrimp mature sperm is a unique structure. However, the complicated cellular motility mechanism of the acrosome formation of the caridean shrimp is still unknown. KIFC1 is a C-terminal kinesin motor, which was proved to play crucial roles during spermiogenesis in rat [25]. Based on the results of ISH and immunofluorescence in this study, the KIFC1 has a close relationship in the acrosome formation. The evertedembrella-like acrosome formation is depended on a microtubular structure AFS, which linked the temporary organelle lamellar complex (LCx) together.
In situ hybridization was performed to track the temporal and spatial expression pattern of kifc1 mRNA in different stages during spermiogenesis of E. modestus, which shows kifc1 mRNA locations and will indicate the expressive locations of KIFC1 protein and the potential functions of KIFC1 (Fig. 6). The previous studies expound that KIFC1 played an important role in acrosome formation in mammalian and crustacean [25][26][27][28][29][30]. The ISH results do show that kifc1 mRNA was closely associated with acrosome biogenesis and nuclear shaping during spermiogenesis of E. modestus (Fig. 6). At the middle stage, kifc1 mRNA signals are increased than the early stage and flurry of signals are concentrated on one side (Fig. 6C, D and J). At the late stage, the kifc1 mRNA signals are visibly strong and highly centralize in acrosome cap and displayed dot-like signatures in the surface of dish-like nucleus (Fig. 6E, F and K). That is to say, kifc1 mRNA signals are rapidly increased in the period of acrosome formation and nuclear shaping which implies the key role of kifc1. At the early and mature stages, the expression kifc1 signals are dramatically less (Fig. 6A, B, G and H). It may provide more evidence of kifc1 role in acrosome formation and nuclear shaping. In addition, it seems that the location of kifc1 is closely associated with the temporary organelle LCx and the microtubular structure AFS during the spermiogenesis of E. modestus. This phenomenon may indicate the mechanism of kifc1 as a motor protein participated in acrosome formation and nuclear shaping (Fig. 6I-L).
The immunofluorescence of KIFC1 and tubulin was performed to explore the role of KIFC1 in cellular transformations during spermatogenesis of E. modestus. Microtubes are consisted of tubulin, the gathered signals of tubulin stand for microtubule. The results of dynamic change of KIFC1 and tubulin in different stages obviously show that KIFC1 signals always distribute in the structure AFS, which linked the temporary organelle LCx together during spermiogenesis. From the early stage to the middle stage, with the formation of LCx and the emergence of AFS structure, KIFC1 signals are transfered to one side of the nucleus (Fig. 8 and  9). Microtubules are sporadically localized in the middle of LCx with dotted distribution (Fig. 8 and 9). At the late stage, the LCx is compressed and promoted the formation of everted-embrella-like acrosome (Fig. 10). The KIFC1 signals always appear in LCx and they overlap with microtubules (Fig. 10). These results may indicate an underlying mechanism that the formation of LCx is depended on the structure of AFS. KIFC1 transport cargoes, such as LCx, moving on the microtubules of AFS to facilitate the acrosomal morphogenesis.
The acrosomal infilling contains some organelle components, such as Golgi apparatus and mitochondria [34,36]. As the temporary organelle LCx is the precursor of acrosome, the compositions of them are coincident [34,36]. The immunofluorescence of GM130 and Mito-Tracker Green are to explore the role of KIFC1 in transportation of acrosomal infilling (Fig. 13, 14  and 16). In early spermatids, both GM130 and Mito-Tracker are equably distributed around the nucleus (Fig. 13B, Fig. 14B). However, with the formation of LCx, the GM130 and Mito-  Tracker are almost gathered in a point of the spermatids, where is the AFS formed position in middle spermatids (Fig. 13E, Fig. 14E). Moreover, combining with the immunofluorescence of KIFC1 and microtubules, the signals of GM130 and Mito-Tracker have part overlapped position with them. In later spermatids, the phenomenon is even more pronounced. In the mature sperm, KIFC1, GM130 and Mito-Tracker are found in the acrosome (Fig. 13K, Fig. 14K). These results show a transported mechanism that KIFC1 transport organelles of dismissed protein as cargoes moving on the microtubules of AFS to facilitate the acrosomal formation during spermatogenesis in E. modestus (Fig. 16) Conclusion KIFC1, the acroframosome, and the LCx all play a role in the formation of the everted umbrella-shape acrosome and nuclear shaping in caridean shrimp. The dynamic changes in tubulin and KIFC1 localization during different stages of spermiogenesis show that KIFC1 always distributes in the AFS which links to the LCx. This indicates that KIFC1 probably transports various cargoes along the microtubules of the AFS to facilitate acrosome formation and nuclear shaping. The transported cargoes include but are not limited to the Golgi complex and mitochondria.
This study provides a model to explore the molecular mechanisms of spermatogenesis in different crustacean species which will lead to further understanding of the fertilization mechanisms in crustaceans.

Supporting Information
Video S1 The audio video interactive of E. modestus spermatid. The video shows the stereochemical morphology of E. modestus spermatids during the late stage of spermiogenesis. The video shows a dish-shaped nucleus (blue) and the distribution of the microtubular structure AFS (green). (AVI)