text:
User:Fearedbliss/Installing Gentoo Linux On ZFS
- 1 Install Gentoo Linux on ZFS
- 1.1 Preface
- 1.2 Required Tools
- 1.3 Assumptions
- 1.4 Confirm that you booted in UEFI Mode
- 1.5 Partition
- 1.6 Format your drives
- 1.7 Installing Gentoo
- 1.8 Inside the chroot
- 1.8.1 Generate locales
- 1.8.2 Fix your date
- 1.8.3 Set hostname
- 1.8.4 Create mtab symlink
- 1.8.5 Edit fstab
- 1.8.6 Modify make.conf
- 1.8.7 Get the portage tree
- 1.8.8 Compiling your own kernel
- 1.8.9 (Optional) Add bliss-overlay and install a pre-compiled/configured ZFS-ready kernel
- 1.8.10 Install required applications
- 1.8.11 Update your system
- 1.8.12 Installing the bootloader onto your drive
- 1.8.13 Make the GRUB 2 configuration file
- 1.8.14 Generating new zpool.cache file before/after reboot
- 1.8.15 Generate the initramfs and move the file to its correct location
- 1.8.16 Final steps before reboot
- 1.9 After you reboot
Install Gentoo Linux on ZFS
Author: Jonathan Vasquez (fearedbliss)
Contact: email@example.com
This guide will show you how to install Gentoo Linux on AMD64 with:
* UEFI-GPT (EFI System Partition) - This will be on a FAT32 unencrypted partition as per UEFI Spec. * Everything on ZFS (/, /home, /boot, swap) * ZFS 0.7.8 * GRUB 2.02 (Bootloader) * OpenRC * Gentoo Stable (amd64)
Notes on other stuff you can do in the ZFS install can be found here.
Required Tools
Download my modified System Rescue CD that includes ZFS from one of the mirrors on the front page.
If you have a Linux system, you can create a bootable usb from it by doing the following:
root #
mkdir /tmp/iso
root #
mount -o loop,ro <system rescue iso> /tmp/iso
root #
That should open up a screen where you can select your plugged in flash drive. If script show
dialog: loadlocale[...] try set your locales to
You can download "Rufus" and select the ISO by clicking the little image next to the "Create a bootable disk using <>". After that, you can use the default settings and press "Start".
Mac OS
If you have a Mac OS system, you can create a bootable usb by doing the following:
- Insert the USB device.
- Using Disk Utility, format the USB device. Make sure to set the file system to FAT.
- Mount the ISO. Both the freshly formatted USB and the ISO should appear mounted under /Volumes
- From a terminal, run: cp -R /Volumes/sysresccd_zfs /Volumes/<USB name> (Chances are your USB drive name is UNTITLED.)
- Unmount the USB using Finder or the command line diskutil application.
- Reboot, holding down Left-Option when you hear the chime. Release after a few seconds.
- You should see a removable device you can now boot from. After selecting, it should load GRUB.
- Only installing Gentoo on one drive called /dev/sda. Note, this means that this guide does not support NVMe SSD drives - if you wish to install Gentoo with ZFS on such a drive follow this guide instead.
- You are using my modified System Rescue CD that includes ZFS.
- bliss-initramfs and bliss-kernel are used. You can also use genkernel and your own custom compiled kernel if desired.
Also, this guide is the way I install Gentoo, not exactly the way the handbook has it.
Confirm that you booted in UEFI Mode
After you booted into the Live CD, make sure that you booted into UEFI mode by typing the following:
root #
ls /sys/firmware/efi
If the above directory is empty or doesn't exist, you are not in UEFI mode. Reboot and boot into UEFI mode.
Continuing the installation without being in UEFI mode will most likely yield an unbootable system. If you want to install in BIOS mode, you will need a different setup.
We will now partition the drive and aim to create the following layout:
/dev/sda1 | 512 MiB | EFI System Partition | /boot/efi /dev/sda2 | 500 MiB | Boot Partition (No Feature Flags) | /boot /dev/sda3 | Rest of Disk | ZFS | /, /home, swap, etc
There are many UEFI motherboard firmwares that are extremely buggy. We will attempt to use a 512 MiB FAT32 partition configuration to increase success.
Open up your drive in GNU parted and tell it to use optimal alignment:
root #
parted -a optimal /dev/sda
Keep in mind that all of the following operations will affect the disk immediately. GNU parted does not stage changes like fdisk or gdisk.
Set the units to Mebibytes
unit mib
Create GPT partition layout
This will delete all partitions and create a new GPT table.
mklabel gpt
Create and label your partitions
mkpart primary 1 513
mkpart primary 513 1013
mkpart primary 1013 -1
name 1 esp
name 2 boot
name 3 rootfs
Set the bootable flag on the ESP partition
set 1 boot on
Final View
Model: ATA VBOX HARDDISK (scsi) Disk /dev/sda: 102400MiB Sector size (logical/physical): 512B/512B Partition Table: gpt Number Start End Size File system Name Flags 1 1.00MiB 513MiB 512MiB esp boot, esp 2 513MiB 1013MiB 500MiB boot 3 1013MiB 102399MiB 101386MiB rootfs
Exit the application
Format your drives
Create your boot partition
root #
mkfs.fat -F32 /dev/sda1
This partition needs to be FAT32 due to it being an UEFI requirement. If it isn't, your system will not boot!
Create your zpool
Create your zpool which will contain your drives and datasets:
root #
zpool create -f -o ashift=12 -o cachefile= -O compression=lz4 -m none -R /mnt/gentoo rpool /dev/sda3
Create your zfs datasets
We will keep it simple and just create a few datasets for /, and /home. ZFS is extremely flexible and you can easily add or remove datasets in the future.
root #
zfs create rpool/ROOT
root #
zfs create -o mountpoint=/ rpool/ROOT/gentoo
root #
zfs create -o mountpoint=/home rpool/HOME
Create your boot pool
It is safer for us to create a separate zpool that has all feature flags disabled. This is because even though grub currently supports the latest feature flags for the 0.6.5 release of ZFS, new feature flags added to ZFS without proper bootloader support can make your system unbootable. Since GRUB 2 has solid support for a zpool with no feature flags, we will create a separate featureless boot pool. However, our main system will still have all features enabled.
root #
zpool create -f -d -o ashift=12 -o cachefile= -m /boot -R /mnt/gentoo boot /dev/sda2
Do not do a 'zpool upgrade' on your boot pool. Doing this will mostly likely leave your system unbootable.
Create your swap zvol
This creates the zfs block device that we will use for swap.
root #
zfs create -o sync=always -o primarycache=metadata -o secondarycache=none -b 4k -V 4G -o logbias=throughput rpool/swap
root #
mkswap -f /dev/zvol/rpool/swap
root #
swapon /dev/zvol/rpool/swap
- Currently, the performance of the swap zvol isn't that great. This should improve over time.
- You should have a swap even if it is small. Your system could lock up under high load, especially since ZFS uses your physical RAM for its ARC.
Verify everything looks good
You can verify that all of these things worked by running the following:
root #
zpool status
pool: boot state: ONLINE status: The pool is formatted using a legacy on-disk format. The pool can still be used, but some features are unavailable. action: Upgrade the pool using 'zpool upgrade'. Once this is done, the pool will no longer be accessible on software that does not support feature flags. scan: none requested config: NAME STATE READ WRITE CKSUM boot ONLINE 0 0 0 sda2 ONLINE 0 0 0 errors: No known data errors pool: rpool state: ONLINE scan: none requested config: NAME STATE READ WRITE CKSUM rpool ONLINE 0 0 0 sda3 ONLINE 0 0 0 errors: No known data errors
root #
zfs list
NAME USED AVAIL REFER MOUNTPOINT boot 484K 448M 136K /mnt/gentoo/boot rpool 4.25G 91.7G 96K none rpool/HOME 192K 91.7G 96K /mnt/gentoo/home rpool/ROOT 192K 91.7G 96K none rpool/ROOT/gentoo 96K 91.7G 96K /mnt/gentoo rpool/swap 4.25G 95.9G 64K -
Now we are ready to install Gentoo!
Installing Gentoo
Check and set your time
root #
If it's wrong then fix it. Let's say it's August 24, 2014 @ 2:48 PM (will be 14:48 in 24 hour time), we would do the following:
root #
date 082414482014
08 24 1448 2014 (Month, Date, 24 Hour Time, Year). It will be UTC at the moment but we will fix it once we are inside our environment.
Preparing to chroot
First let's mount our efi boot partition in our chroot directory:
root #
cd /mnt/gentoo
root #
mkdir boot/efi
root #
mount /dev/sda1 boot/efi
and now download the amd64 image here and extract it:
root #
wget <file>
root #
tar xpf <file>
Copy zpool cache
root #
mkdir etc/zfs
root #
cp /etc/zfs/zpool.cache etc/zfs
Copy network settings
root #
cp /etc/resolv.conf etc/
Mount required devices
root #
mount --rbind /dev dev && mount --make-rslave dev
root #
mount --rbind /proc proc && mount --make-rslave proc
root #
mount --rbind /sys sys && mount --make-rslave sys
Chroot into your environment
root #
env -i HOME=/root TERM=$TERM $(which chroot) . bash -l
Inside the chroot
Generate locales
I'm using US English w/ UTF-8, so I will enable that locale on my system:
root #
nano /etc/locale.gen
root #
root #
eselect locale set en_US.utf8
root #
root #
. /etc/profile
Fix your date
I'm in New York and the time currently is August 24, 2014 @ 2:48 PM (will be 14:48 in 24 hour time)
root #
ln -sf /usr/share/zoneinfo/America/New_York /etc/localtime
root #
date 082414482014
Set hostname
Open up /etc/conf.d/hostname with your text editor and add your desired hostname in there.
root #
nano /etc/conf.d/hostname
For example, my /etc/conf.d/hostname file could be:
Create mtab symlink
root #
ln -sf /proc/self/mounts /etc/mtab
Edit fstab
Everything is on zfs so we don't need anything in here except for the boot and swap zvol entries. My fstab looks as follows:
root #
nano /etc/fstab
/dev/sda1 /boot/efi vfat noauto 1 2 /dev/zvol/rpool/swap none swap sw 0 0
Do not auto-mount the /boot/efi partition. If you do, the init system will create the /boot/efi folder if it doesn't exist, and that will prevent the 'boot' zpool from mounting.
Modify make.conf
Let's modify our /etc/portage/make.conf so we can start installing stuff with a good base (Change it to what you need):
root #
nano /etc/portage/make.conf
CPU_FLAGS_X86="mmx sse sse2" USE="-branding" # This should be your number of processors + 1 MAKEOPTS="-j5" EMERGE_DEFAULT_OPTS="--with-bdeps=y --keep-going=y" FEATURES="buildpkg" LINGUAS="en en_US" # This is required so that when we compile GRUB later, EFI support is built. GRUB_PLATFORMS="efi-64"
Get the portage tree
Copy the default example portage config
root #
cp /usr/share/portage/config/repos.conf /etc/portage/repos.conf
root #
emerge --sync
Compiling your own kernel
Using the 'bliss-kernel' configuration as a base
If you don't want to use the pre-compiled "bliss-kernel" in "bliss-overlay" below, I've made available the 'bliss-kernel' configs so that you can compile the kernel yourself using that configuration as a base. The configuration is ready to go for general desktop, laptop, and server use, and it is also configured to work with ZFS.
You can download the 'bliss-kernel' configuration here.
Using your own configuration
If you are not going to use the above configuration, then you will need to make sure your kernel has the following options enabled or ZFS will not work:
This list is still being updated, and is using the options listed in the sys-kernel/spl ebuild as a base.
Disable anything marked [ ], Compile in [*], Options marked [M] may either be separate modules or compiled into the kernel:
(Optional) Add bliss-overlay and install a pre-compiled/configured ZFS-ready kernel
bliss-kernel is a vanilla kernel (directly from kernel.org, no patches) that fearedbliss maintains. It uses a slightly tweaked fedora config. The kernel is pre-configured and tested for ZFS. Use this if you don't want to spend time tweaking/debugging the Linux Kernel.
Open repos.conf and add bliss-overlay
root #
nano /etc/portage/repos.conf
[bliss-overlay] location = /var/lib/overlays/bliss-overlay sync-type = git sync-uri = https://github.com/fearedbliss/bliss-overlay.git auto-sync = yes
Compile git
root #
emerge dev-vcs/git
Sync once more to get bliss-overlay
root #
emerge --sync
Install the kernel
root #
emerge bliss-kernel
Consider pinning the kernel version that was installed so that when we do a future kernel upgrade, an emerge --depclean won't delete our kernel files from the /boot directory. If the bliss-kernel ebuild that emerge pulled to install was: bliss-kernel-4.1.8, we would do:
root #
emerge --noreplace =bliss-kernel-4.1.8
Install required applications
Enable ZFS support in GRUB
root #
echo "sys-boot/grub libzfs" >> /etc/portage/package.use
Now install the apps:
root #
emerge bliss-initramfs grub
Update your system
root #
emerge -uDNav @world
Installing the bootloader onto your drive
We will need to install the bootloader onto the drive. Before we do that however, let's see if GRUB can detect our /boot and /boot/efi filesystem types:
root #
grub-probe /boot
This should say 'zfs'. If it doesn't, then something is wrong and your system will not boot!
root #
grub-probe /boot/efi
This should say 'fat'. If it doesn't, then something is wrong and your system will not boot!
Before we install the bootloader, we will need to have read/write access to the efi nvram variables. Let's remount our efivars now:
root #
mount -o remount,rw /sys/firmware/efi/efivars/
Now run the following to install the bootloader to the drive:
root #
grub-install --efi-directory=/boot/efi
The above command will install the grub bootloader files into /boot and the efi files into /boot/efi. It should return a "Installation finished. No error reported." message. If it doesn't, then something is wrong and your system will not boot!
If after a reboot your UEFI motherboard doesn't detect your bootloader, it might be because of a buggy motherboard firmware. Try to pass the "--removable" flag to grub install and try again. This will install the efi file to the EFI specification's fallback directory.
Make the GRUB 2 configuration file
You can use the following configuration file as a basis for your system:
root #
nano /boot/grub/grub.cfg
set default=0 set timeout=1 insmod part_gpt insmod fat insmod efi_gop insmod efi_uga menuentry "Gentoo - 4.1.8-FC.01" { linux /@/kernels/4.1.8-FC.01/vmlinuz root=rpool/ROOT/gentoo by=id elevator=noop quiet logo.nologo triggers=zfs refresh initrd /@/kernels/4.1.8-FC.01/initrd }
Generating new zpool.cache file before/after reboot
ZFS is very sensitive about the data that is contained in the zpool.cache file and at this point, when we reboot, the information in it might not be completely accurate. To ensure we have a good cache file, we have instructed bliss-initramfs in the bootloader config above, to ignore the current cachefile on the system, and make a new one that is up-to-date. We only need to do this once.
Generate the initramfs and move the file to its correct location
root #
bliss-initramfs zfs 4.1.8-FC.01
root #
mv initrd-4.1.8-FC.01 /boot/kernels/4.1.8-FC.01/initrd
Final steps before reboot
root #
rc-update add zfs-import boot
root #
rc-update add zfs-mount boot
root #
rc-update add zfs-share default
root #
rc-update add zfs-zed default
root #
root #
root #
After you reboot
Remove zpool.cache refresh flag from bootloader configuration
Open up your grub.cfg and remove the 'refresh' flag from the kernel line.
root #
nano /boot/grub/grub.cfg
Take a snapshot of your new system
Since we now have a working system, we will snapshot it in case we ever want to go back or recover files:
root #
zfs snapshot boot@2016-03-25-0000-01-INSTALL
root #
zfs snapshot rpool/ROOT/gentoo@2016-03-25-0000-01-INSTALL
root #
zfs snapshot rpool/HOME@2016-03-25-0000-01-INSTALL
You can view the contents of these snapshots by checking their respective and hidden .zfs directories:
root #
ls /boot/.zfs/snapshots
root #
ls /.zfs/snapshots
root #
ls /home/.zfs/snapshots
We are naming the snapshots in this format because it's the format that my 'bliss-zfs-scripts' use, and it is also very readable.
Other stuff you might want to install
Tools in Portage
root #
emerge sudo htop gentoolkit eix app-misc/screen app-text/tree
Bliss ZFS Scripts
The following scripts allow you to automatically:
- Take snapshots of your pool
- Replicate the pool to another pool (Full and Incremental Backups)
- Clean the old snapshots on your pools.
You can download, customize, and install the scripts into your /usr/local/sbin directory. Github.
And that's it. Enjoy!


text:
The Beaux-Arts home, located on 74th Street between Madison and Fifth avenues, dates back to 1898. It was designed by architect Alexander Welch and it has housed many famous names over the year, including artist Marc Chagall and the late Michael Jackson, who rented the house in the 1990s and 2000. Here is your chance to discover this astonishing New York house design.
The house is close to both Central Park and the Met Breuer and its exteriors and some of the interiors were used as a stand-in for Nate Archibald's rich house on Gossip Girl. The Michael Jackson townhouse was also used as the office of therapist Susan Lowenstein (aka Barbra Streisand) in the 1991 movie The Prince of Tides.
Current owner Marc Lasry, the billionaire co-owner of the Milwaukee Bucks, has listed the limestone and red brick house for $39 million. The "King of Pop" lived there from the middle of 1999 through early 2000, and Lasry paid $11 million for it in 2011, according to public records.
You might also like: 5 Tips to Create an Elegant and Comfortable Dining Room Design
This amazing mansion crosses 12,745 square feet, with at least eight bedrooms, nine bathrooms, three sitting rooms, a media room, a cellar, a home gym, and many of its original details remain intact.
It also has modern conveniences, including a massive eat-in kitchen, an elevator, and central air conditioning.
There are three outdoor spaces: a main floor deck, third-floor terrace and a landscaped roof deck with views.
This house design is a perfect balance of keeping all the original details intact, yet the furnishings, art and color selection give it an inviting, contemporary look.
See more: Pantone Color of The Year: Ultra Violet in Home Decor

subset: commoncrawl


text:
The team has found that carbon nanotubes measuring less than one micron in length made by rolling graphene sheets can be used in targeted photodynamic therapy where cancer cells are killed by inducing heat without any toxic effect to the body. The therapy involves targeting cancer cells embedded with the nanotubes which alone get heated during the process.
IITM has been doing the research in collaboration with National University of Singapore.
Graphene, an atomic layer of graphite in the form of sheet, is rolled to make carbon nanotubes. The inner diameter of these tubes measure about 20 to 30 nanometre (1 nanometre is one-billionth of a meter) and the outer diameter 50 to 60 nanometres. Their length could be up to 1,000 nanometre.
Professor and head of alternative energy and nanotechnology laboratory at IIT-M, Sundara Ramaprabhu said these carbon nanotubes have excellent thermal conductivity and have a large surface area to support and carry a drug.
In photodynamic therapy, the professor said, these carbon nanotubes are coated with vitamins like folic acid and injected into the patient. The folate receptors, a protein in the body, then take both the vitamin and the carbon nanotubes to the nucleus of the cell including the cancerous cells. Therapists pass infrared radiation in pulses at a wavelength of about 800 nanometre. "A good amount of energy in pulses will be stronger," the professor said.
With the carbon nanotubes possessing good thermal conductivity, the heat from the radiation passes through the nanomaterial present in the cells. Once the temperature rises to 42 degrees Celsius, it kills the cancerous cells. The surrounding cells, which have not absorbed the nanotubes, will remain unaffected.
"Much research is being conducted world over with various nanomaterials like iron oxide, gold nanoparticles and metal nanoparticles for photodynamic therapy. We found that carbon nanotubes made by rolling grapheme sheets are more effective in cancer therapy," Ramaprabhu said.
Studies were also conducted to check the toxicity of the nanomaterial by evaluating few parameters like the tubes' length, solubility, concentration and purity. They found that the carbon nanotubes were less toxic in a range of concentrations that are normally used, but also are 'ultrapure', which means it guarantees safe application.
The scientists say that more research is needed before applying the process on different types of cancers.


text:
Trouble in the Education "Business"
The University of Phoenix is having trouble (subscription) says The New York Times in an article titled "Troubles Grow for a University Built on Profits."
As if that should be a surprise.
Many of us in education have been watching the growth of for-profit institutes of higher education with concern for quite some time. Our college and university system, after all, was built on a non-profit foundation for reasons—and those reasons were being swept aside in a rush for results—i.e., profits. As a result:
students spend 20 to 24 hours with an instructor during each course, compared with about 40 hours at a traditional university. The university also requires students to teach one another by working on projects for four or five hours per week in what it calls "learning teams."
Do it faster and with less supervision—yes! And profits shall descend on one!
That's not the only reason for-profit scares many educators. The fact of different constituencies, all with power in the institution, has long kept more traditional educational institutions from straying too far from concentration on their primary mission. Faculty power, for example, has kept administrations from centralizing structures. At the University of Phoenix, the role of the faculty is diminished, taking them completely out of the decision-making process:
Courses are written at university headquarters, easing class preparation time for instructors.
Though these are the words of Times writer Sam Dillon, they reflect the U of P attitude: reduce the workforce and increase the profit. The attitude completely bypasses recognition of the importance of teaching (in its full extent of preparation, class time, and evaluation) to education, recognition that the essence of teaching, the personal interaction of student and teacher, cannot be reduced to a role of "facilitation" of a program created at a distance. This was demonstrated a generation ago by the psychologists who developed the first teaching machines—almost all of whom eventually abandoned their projects to return to direct interaction with students.
On some level, even the people at U of P know this, as the following passage indicates:
Although Phoenix is regionally accredited, it lacks approval from the most prestigious accrediting agency for business schools, the Association to Advance Collegiate Schools of Business.
John J. Fernandes, the association's president, said the university had never applied. "They're smart enough to understand their chances of approval would be low," Mr. Fernandes said. "They have a lot of come-and-go faculty. We like institutions where the faculty is stable and can ensure that students are being educated by somebody who knows what they're doing."
And not, the implication is, by a team of course constructors at a distant location.
Having taught, myself, for one of U of P's competitors (though only for a couple of their short terms), I understand completely why one of the problems for all of the for-profit colleges is retention of qualified faculty. This little incident recounted in the article did not surprise me at all:
Robert Wancha, 42, a former National Guard commander who is pursuing a bachelor's degree in information technology at the university's Detroit campus, said that in a computer course last fall his instructor, Christopher G. Stanglewicz, had boasted that he had a doctorate but did little teaching, instead assigning students to work in learning teams while he toyed with his computer.
Mr. Stanglewicz, reached at his home, acknowledged that he had covered only a fraction of the syllabus , partly, he said, because the university required him to cram too much information into too few sessions.
"Students get overwhelmed," he said. Mr. Stanglewicz asserted in the interview that he had earned a doctorate in economics from the University of Kentucky. But the authorities there said his name was not in their records. (Dr. [William] Pepicello [U of P's President] said that Mr. Stanglewicz had never told the university that he had a doctorate, and that he was qualified to teach.)
If U of P is anything like the school where I was teaching, "qualification" is based solely on administrative evaluation of credentials along with a short course (mine was online) familiarizing the "facilitators" with institutional procedures.
What we have, ultimately, in all of these institutions is a Wizard-of-Oz educational structure, where the receipt of a degree conveys knowledge and ability. "Give me the money and I'll give you the degree—and who's to be the wiser" seems to be the attitude. The assumption is that a real education isn't good for much anyway, that the only difference between the person with a diploma and the one without is that sheet of paper.
Like all of those who have benefited from a real education, I beg to differ.


text:
It is a well known scientific fact that water always flows downhill by the easiest possible route! The basic concept of a French drain is a slightly sloped trench filled with round gravel and perforated pipe to divert underground water away from your house. This is a common drainage system primarily used to prevent ground water from penetrating or damaging building foundations by providing an easy channel allowing water to flow.
A French drain is in no way connected with anything French! Henry French, an American Judge and Farmer from Concord, Massachusetts, designed and promoted the French drain in his book in 1859 promoting farm drainage. French´s drains were made with clay tiles, but modern drains use 4-inch diameter plastic pipes. The solution to a persistently wet basement or a soggy lawn could be a French drain, particularly if you live on a slope.
Surface and sub-surface water runs through the spaces between the round gravel and perforated pipe at the bottom of the trench, which should be sloped. Emptying a safe distance from the house, the water has travelled freely through the pipe.
If you have a driveway that is continually water-logged or a soggy lawn for example creating problems with surface water, then a shallow French drain or curtain drain is useful. This type of drain need only be sufficiently deep to divert water.
If the shallow drain doesn´t keep water out of your basement, you may need a deep French drain. This is also called a footing drain and runs round the perimeter of the house at footing level. This is difficult and expensive should it be necessary to add later but can easily be installed during the house construction.
A French drain is dependent on gravity.
A sump pump may be required to lift collected water piped to a basement basin and send it to the storm drain system if the property is built on flat ground.
Interception of water into your basement if it continually enters, can be remedied by building a French drain at the point of entry. Install a perforated pipe all the way round the perimeter of the basement floor after cutting a channel and chipping out the concrete. A collection tank sunk into the floor receives the water via a solid pipe and a pump sends it out to the yard or storm drain.
If you are building a retaining wall on a hillside, incorporate a French drain behind the first course of stones or blocks. Otherwise the wall could be undermined or even tipped by water moving down the hill or building up behind the wall. To avoid clogging with silt, drape landscape cloth across the base or footings and up the slope before adding the pipe and adding the pipe and drain gravel. Nearing the top of the wall, fold the cloth over the top of the gravel and cover with several inches of soil.
French drains in Spain are a common occurrance due to the dry, hard ground and flash flooding makes them a necessity.
One French drain specialist in Spain is M3BT who are an establised construction company working with the major insurance companies throughout Spain.


text:
COOKIE MONSTER. Monster Pet Supplies uses Cookies to ensure we provide a tail-waggingly good service. If you continue we assume that you consent to receive all cookies. Read more.
Back to All Dog Products • Home > Dog > Dog Rainy Days >Pet Head Natural Oatmeal Dog Shampoo
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A Message from Linda Levin
Thanks to the Internet, it's now easy to find quotes, statistics, blogs, cartoons and essays on growing old. While there is an abundance of messages and information proclaiming the downside of aging, you can also find plenty of useful resources and information on how to live your best life after the age of 50.
We are ElderSource, the Area Agency on Aging and Aging & Disability Resource Center for Northeast Florida, equipped to help individuals living in our seven county areas to access the resources they need to face the challenges of aging, as well as find ways to harness the information and tools they need to live out their lives with independence and dignity.
For adults living with disabilities and the loved ones caring for them, ElderSource wants to be there for them, too, helping them to find resources and applying for benefits.
In addition to serving as the key "connector" to these resources, ElderSource is the voice for elders speaking on their behalf to community leaders, government officials and businesses.
As our communities grow and change, we believe it is important to start addressing these changes now. We need to develop the necessary plans to help people age with dignity and independence and keeping them engaged and contributing to our community, society and economy.
When we talk about transportation, the discussion needs to include elders. People are going to need accessible and reliable and affordable transportation; our roads will need to accommodate older drivers, such as adapting signage.
When talking about housing, the discussion needs to include elders. Communities need to be planned so they are walkable and older adults can easily get to places of business and affordable housing need to be designed and built to accommodate the aging population.
When talking about education, we need to think about elders. It is these elders who volunteer in the schools and more of them will be needed. Many grandparents are raising their grandchildren, something they didn't expect to have to do and at a time when subjects they never had to learn are being taught or are being taught differently from when they were in school.
When we talk about jobs, we need to talk about elders. People are going to have to work longer than expected, we are going to need more of them to continue working longer as there will not be enough young workers to support them or companies.
When we discuss crime and justice, we need to include elders in the conversation as they are often vulnerable and are easy targets for predators, falling victim to abuse, neglect and exploitation.
An aging community is not good or bad, it's just a fact and one we need to start preparing for now so that we not only address the needs of a growing aging community, but also ensure that elders remain engaged.
The staff and Board at ElderSource are working diligently every day to ensure that seniors, adults living with disabilities and their caregivers are included in the important discussions happening in our community. I hope you will work with us to be part of that discussion, and work with us to action, too.
Linda Levin
Chief Executive Officer
11 Of The Smartest Things Anyone Ever Said About Getting Older


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In the real world, we avoid stress whenever possible but in writing, the opposite is true. Why? Because in the land of the Three Act Structure (TAS), STRESS = CONFLICT.
In the TAS, our main character has a need or desire which outside forces try to prevent, challenging him to overcome adversity to achieve his goal. Sounds like a nice, simple formula, doesn't it? However, without the critical element of STRESS, the storyline falls flat.
Little Johnny wants a cupcake. His mother says no. He waits until she turns her back and then takes one anyway.
Can you see it? No Stress = BORING. Johnny wants something, but he isn't stressed about it. The reader could care less whether he achieves his goal or not, because the stakes are nonexistent.
But let's look again, this time applying factors to cause stress:
Little Johnny has diabetes, and hypoglycemia is setting in. He doesn't want that cupcake, he freaking needs that cupcake. He's shaky, sweating, and his limbs aren't cooperating like they should, and he knows that if he doesn't get sugar STAT, it'll be lights out.
As readers, this situation has our blood boiling. Mom's obviously sadistic, not handing the sweet over. If it was up to us, we'd call Social Services and cheer as she's charged with neglect and failing to provide the necessities of life. Sadly, it isn't up to us, so all we can do is watch, helpless, and pray Johnny is strong enough to figure out what to do.
The application of stress has achieved something incredible: the reader now cares about Johnny. They are invested in his situation and riveted on the outcome.
With the stress level maxed, the stage is set for action. The stakes are high. What will Johnny do to survive? How will he overcome his weakness and defeat this older, stronger mother-villain? How will he push aside the emotional connection of being her son to save his own life?
It doesn't matter what the situation is, STRESS is what pushes your main character to ACT. It can force them to go against their own nature or beliefs, to meet challenges, overcome obstacles and face danger. Stress creates tension, which leads to conflict, so don't be afraid to amp up the stress. The higher the stress, the more elevated the stakes are, laying the groundwork for a compelling story that will captivate your reader and make them feel invested in the outcome.


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The Truth about Growing Your Own Marijuana
Learning the basics about growing marijuana is important for all growers because cannabis isn't a simple plant for anyone to grow. There are marijuana strains that are sensitive and will require a lot of attention during its growing stage. So before you think of growing your own marijuana, you have to figure out if you


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Can We Change Name In PPF Account?
Public Provident Fund or PPF is a plan of the Government of India, confined under PPF Act, 1968. In brief, the Public Provident Fund is a central government sponsored, long term small investment scheme that was first begun by the Central Government on the grounds that it needed to give retirement security to independently employed people and workers of the private sector.
At present the Public Provident Fund is the most popular investment plan of the Indian citizens. In the event that you are enthused about a sheltered amount, a decent return rate, tax cuts (on the cash contributed, tax free premium and tax free full maturity amount) and want a long time investment plan, then the Public Provident Fund is best for you. Bear in mind that you should be restrained with the Public Provident Fund to benefit as much as possible from it, furthermore meet your liquidity requirements somewhere else, in light of the fact that with this plan your cash will be blocked for a long time.
The PPF likewise offers credits against your account that can assist you amid events such as a wedding, facilitate education of your kids, and so forth. Over all, it gives you a significant serenity as your cash is safe.
One of the main important things to know about PPF is the process of changing name. Many people want to change name in their account due to marriage or some other reason. Given below are steps given to change name in a PPF account, which should be read properly.
Write an Application: you must write a specific application portraying the solicitation, reason and documents attached.
Form: you will be given a form, which is used only to change name and address.
PAN Card: You must provide a Copy of your PAN card.
Proof of Address: Copy of your Address proof, DL, Voter Card, Passport, Aadhar Card.
Certificate of Marriage: Copy of your marriage Certificate.
All documents must be attested by the applicant.
You must carry all the original documents while submitting the application.
Submit the above applications at the PO in which you got the PPF account opened. Your PPF account is not centralized; consequently application will be gotten at your branch which opened your record. Extensively, the PPF record is something that is very beneficial to have, particularly for those people who don't work in the public sector and henceforth don't possess an EPF account.
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Pre & After School Care
Getting through the morning routine and everyone out the door for a certain time is no mean feat. To make things a little simpler, why not take advantage of our pre and after school care?
Available during term times
Pe and after school care is available during each term and is provided at either end of the core school day.
- Available to children from Reception up to Year 6
- Open from 8am and up until 6pm as part of the standard monthly fee
- Supervised and managed by a dedicated After School and Holiday Club Co-ordinator and Our Lady's staff
- Designed to include both fun activities and relaxed learning opportunities
Extended hours available
For added flexibility, you can also take advantage of extended hours. For an additional fee, book care from as early as 6am and up to as late as 8pm. All we ask is that you book in advance so that we can ensure sufficient teachers and facilities are made available.


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SHEBOYGAN FALLS — The Carnegie Hero Fund Commission awards the Carnegie Medal to individuals from throughout the United States and Canada who risk their lives to an extraordinary degree saving or attempting to save the lives of others — and 11-year-old Natalie Martin, who died in a fire in Sheboygan Falls on January 26th, 2016 is one of 21 people to receive one of these medals.
Natalie Martin and her two brothers, 10-year-old Benjamin Martin and seven-year-old Carter Maki died as a result of the fire that happened at their family home near 5th and Western in Sheboygan Falls.
Carter Maki died at St. Nicholas Hospital. Natalie Martin was airlifted to Children's Hospital — where she later died.
Benjamin Martin died days later at Children's Hospital.
Nine-year-old Jenna Martin was able to get out of the home safely thanks to what is being described as the heroism of her big sister, Natalie.
When crews arrived on scene, they made contact with Jenna, who was able to escape the home safely after being alerted by Natalie.
Authorities said Natalie Martin re-entered the home in an attempted to locate two other children — Benjamin and Carter.
Firefighters found heavy smoke pouring from the structure upon their arrival. They entered the home while attacking the fire — and then, with the help of thermal imaging cameras, located all three children in an upstairs room.
"The nine-year-old girl was told to get out, by Natalie, who's the oldest, to get out — and then (Natalie) ran back in to try to get the rest of them out," a family spokesman said.
The family spokesman said nine-year-old Jenna was safe because Natalie was caring for her siblings while their parents, Chris and Kristi Maki, attended a church group meeting.
Investigators determined the fire started in the basement playroom in an area under a light fixture next to a couch. One of the children discovered the fire, called out to her siblings and ran next door to call 911.
There was so much smoke and heat the neighbor and Sheboygan Falls police couldn't enter. Firefighters eventually got in and yelled for occupants — one heard a girl's voice saying, "Help me."
The Maki family later donated the land where the house once sat to Habitat for Humanity.
Habitat officials are searching for a new family to live on that land, in an effort to bring happiness back to a place that once held so many memories.
Building won't begin until a new family is selected for the property. Habitat officials expect to break ground in the summer of 2017.
Below is the complete list of the 21 Carnegie Medal recipients. A total of 93 Carnegie Medals have been awarded in 2016. Again — the medal is given to those who risk their lives to an extraordinary degree while saving or attempting to save the lives of others. Four of the awardees, including Natalie Martin, died in the performance of their heroic acts.
Each of the awardees or their survivors will also receive a financial grant.
Peter F. Pontzer — Fairfax, Va.
Duncan O. C. Harris — Buffalo Grove, Ill.
Ryan P. Rollinger — Harrisburg, S.D.
William E. Ramirez — Pembroke Pines, Fla.
Natalie Renee Martin, deceased — Sheboygan Falls, Wis.
Nathan Ryan Reynolds — Claremont, Calif.
R. Wayne Trivette — Metamora, Ohio
Timothy A. Holtz II — Sand Creek, Mich.
Clark Whitecalf Gallivan — Sask.
Cody Fields — Silver Spring, Md.
Brian Nesbitt — Germantown, Md.
Shei'Londa S. Brooks — Houston, Texas
Lori Doppelheuer Kearney, deceased — Maysville, Ky.
Ivan R. Romero, Jr. — Antelope, Calif.
Dennis D. Swenson, deceased — Cornucopia, Wis.
Calvin Bradley Stein — Madoc, Ont.
Kiera Vera Larsen, deceased — El Cajon, Calif.
Damian Bell — Pleasant Hill, Iowa
Joseph Kolanchick — Albany, N.Y.
Joshua Gary Grimmett — Boise, Idaho
Robert M. Shaw, Jr. — Wallingford, Conn.
Below is the description of Natalie Martin's heroism, as provided by Carnegie officials:
Natalie Renee Martin saved Jenna J. Martin and died after attempting to save Benjamin L. Martin and Carter J. Maki from burning, Sheboygan Falls, Wisconsin, January 26, 2016. At night, siblings Jenna, 9; Benjamin, 10; and Carter, 7, were in bedrooms on the second floor of their family's two-story house after fire broke out in the basement. Their sister, Natalie, 11, student, discovered the fire and woke Jenna. She led Jenna downstairs, where fire from the basement was starting to enter the kitchen. Taking Jenna to a side door, Natalie opened the door, allowing Jenna to escape the house, and then she turned back into the structure, despite deteriorating conditions. Jenna fled to a neighbor's house to alert help. First responders were initially thwarted by smoke and heat from entering the burning house. In the dense smoke, firefighters then located Natalie, Benjamin, and Carter together in another second-floor bedroom. All were unresponsive and later died of complications from smoke inhalation: Carter that night, Natalie the next morning, and Benjamin one day later.


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"Everybody's getting younger
It's the end of an era, it's true."- LCD Soundsystem-"Dance Yrself Clean"
Much ado has been made about the fact that this is reportedly, LCD Soundsystem's final album. Thankfully, This Is Happening is a fantastic record, and frankly for James Murphy to have followed up 2007's seminal Sound Of Silver with such a strong album deserves applause. This Is Happening feels to Sound Of Silver much how Sound Of Silver felt to Murhpy's self-titled debut; different, but no less essential.
Murphy's vocals have always taken a back seat in his music; it's always seemed that that's how he wanted it. Throughout This Is Happening the vocals are a force to be reckoned with, as the music has always been. Generally speaking the whole record is more of an ironic hipster meltdown than the defiant punk of Sound Of Silver, (North American Scum, Us V Them) whether it's on the college anthem of "Drunk Girls" (Drunk girls know that love is an astronaut/It comes back but it's never the same) or the 80's shimmer of "I Can Change" (Never change/It's why I fell in love – I can change/If it helps you fall in love).
And the tunes are here, aplenty. Murphy's glorious return comes in the shape of "Dance Yrself Clean", what at first seems like a quiet opener but which actually hides ravenous synths waiting to jump you at the next corner. "One Touch" is an extended funk trip; Murphy doing his best Kraftwerk vocal impression, cold and distant while the nagging yet chirpy electronics skitter in the background. The most dubious territory vocally on This Is Happening comes from "You Wanted A Hit". Like the opening track, it's great because it's linear, albeit bookended by gentle electronics. You've got to wonder though who exactly Murphy is singing for/at (You wanted a hit/but maybe we don't do hits). Is it his own record label that he's giving a telling off? If that's the case it doesn't make sense, and if it's not, it still doesn't make sense. Regardless, the style of vocals fit well with the Stevie Nicks style guitars and simple bleeps and chirps happening throughout.
Comparing "All I Want" to David Bowie's "Heroes" is fast becoming cliché, but it's by far the most apt comparison. Not that this needs comparing; Murphy's vocal performance whilst the quietest on the album lend the number something that couldn't be achieved were he screaming at the top of his lungs. By the time the grand climax of guitar, distorted brass and drums kick in, Bowie will be the farthest thing from your mind. "And all I want is your pity/Oh all I want are your bitter tears"
The Talking Heads funk of "Pow Pow Pow" at first got on my nerves, but like Murphy's work in general it soon grows on you. The insistent beat and Murphy's stream of consciousness meets witty hipster vocals actually make it one of the tracks that you'll come back to, very reminiscent of breakthrough single "Losing My Edge". "From this perspective, from this position, I have a good grip on both of them/Because I have stayed home and learned a little bit more about my neighbourhood, which is important."Weak link in the chain has probably got to be "Somebody's Calling Me", it's steady piano beat is neither catchy, nor is the vocal content that amusing. It has the potential to be a grower though, but it's definitely the least accessible track on the album.
This brings us to epic closer, "Home". Personally speaking my favourite track of the year, at approximately 9 minutes it isn't (like most of the album) a brief affair, but it has exactly the kind of chirpy melancholy displayed in Talking Heads classic, "This Must Be The Place". Obviously carrying added poignancy being Murphy's swansong, it's the best bow out fans could have hoped for "Just do it right, make it perfect and real/Because it's everything, no everything/Was never the deal". Another superb vocal performance, (Afraid of what you neeeeeeeeeed!) it's a fantastically realised final performance.
As a massive LCD fan it's hard to come down on This Is Happening in the same way other critics might, but even after a stab at objectivity it stands out as my album of the year thus far. "Somebody's Calling Me" may not be as on form as the others, but Sound Of Silver was hardly 100% consistent, (Watch The Tapes, anyone?) and with the rest of the songs being as strong, it's still a fantastic album. At the tender age of 40, Murphy follows the golden rule: Always leave them wanting more.
"To tell the truth, this could be the last time
So here we go, like a sales force into the night"- LCD Soundsystem – "All My Friends"LCD Soundsystem - This Is Happening,


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The genus Nepenthes, a carnivorous plant, has a pitcher to trap insects and digest them in the contained fluid to gain nutrient. A distinctive character of the pitcher fluid is the digestive enzyme activity that may be derived from plants and dwelling microbes. However, little is known about in situ digestive enzymes in the fluid. Here we examined the pitcher fluid from four species of Nepenthes. High bacterial density was observed within the fluids, ranging from 7×106 to 2.2×108 cells ml−1. We measured the activity of three common enzymes in the fluid: acid phosphatases, β-d-glucosidases, and β-d-glucosaminidases. All the tested enzymes detected in the liquid of all the pitcher species showed activity that considerably exceeded that observed in aquatic environments such as freshwater, seawater, and sediment. Our results indicate that high enzyme activity within a pitcher could assist in the rapid decomposition of prey to maximize efficient nutrient use. In addition, we filtered the fluid to distinguish between dissolved enzyme activity and particle-bound activity. As a result, filtration treatment significantly decreased the activity in all enzymes, while pH value and Nepenthes species did not affect the enzyme activity. It suggested that enzymes bound to bacteria and other organic particles also would significantly contribute to the total enzyme activity of the fluid. Since organic particles are themselves usually colonized by attached and highly active bacteria, it is possible that microbe-derived enzymes also play an important role in nutrient recycling within the fluid and affect the metabolism of the Nepenthes pitcher plant.
Citation: Takeuchi Y, Salcher MM, Ushio M, Shimizu-Inatsugi R, Kobayashi MJ, Diway B, et al. (2011) In Situ Enzyme Activity in the Dissolved and Particulate Fraction of the Fluid from Four Pitcher Plant Species of the Genus Nepenthes. PLoS ONE 6(9): e25144. https://doi.org/10.1371/journal.pone.0025144
Editor: Wendy A. Peer, Purdue University, United States of America
Received: March 16, 2011; Accepted: August 26, 2011; Published: September 16, 2011
Copyright: © 2011 Takeuchi et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was funded by the University Research Priority Program in Systems Biology/Functional Genomics of the University of Zurich (http://www.sysbio.uzh.ch/index.html) and a Japan Science Promotion Society Research Fellowship for YT (http://www.jsps.go.jp/english/). These funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. BD performed the experiments and is an employee of Sarawak Forestry Corporation (http://www.sarawakforestry.com/), which permitted and supported our field research in Lambir Hills National Park in Sarawak, Malaysia.
Competing interests: BD is an employee of Sarawak Forestry Corporation, which permitted and supported the authors' field research in Lambir Hills National Park in Sarawak, Malaysia. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials.
As already described by Darwin [1], carnivorous plants possess special organs to consume insects and other small invertebrates. This carnivory reflects an adaptive trait to grow in low-nutrient habitats where there is a particular lack of nitrogen, phosphorous, and potassium [2]–[4]. Such plants compensate for the lack of these nutrients in the soil by catching prey, and then digesting and absorbing nutrients from it.
The pitcher plants develop jug-shaped leaves or leaf extensions to trap animals. Among the seven genera of pitcher plants, Nepenthes is the largest genus in the tropics and is mostly distributed in the Malesia region, with its highest diversity occurring in Borneo [5]. The pitchers have nectar that is attractive to ants and other arthropods [6]. Because the inner wall of the upper pitcher is waxy and slippery, insects lose their footing while foraging for nectar and slip down the steep walls, thus becoming trapped in the fluid of the lower pitcher. It is reported that more than 60% of the nitrogen of the plant body is derived from prey in Nepenthes mirabilis (Lour.) Druce [7], which suggests a relatively high degree of reliance on animal-derived nutrient in this genera. The efficiency of prey capture and digestion is thus crucial for plant growth and survival.
The proximate agents in digesting or decomposing the organic matter are extracellular enzymes within the pitcher fluid. These enzymes in part originate from the plant itself, where they are secreted by digestive cellular glands that are located in the surface of the lower zone within the pitcher [8], [9]. The characteristics of the enzymes derived from the Nepenthes species have been investigated in previous studies, and the enzymes include at least proteases [10]–[12], chitinases [12], [13], phosphatases [8], [14], and glucosidases [14].
On the other hand, digestive enzymes might also be produced by microbes such as fungi and bacteria that, for example, colonize insect carcasses. So far, such putatively microbial-derived enzymes have not been examined in the pitcher fluid of Nepenthes, even though it is known that bacteria may form in high abundance in this habitat [3], [15], [16]. Microbial-derived enzymes such as phosphatases, glucosidases, and aminopeptidases typically play a critical role in the processing of carbon, nitrogen, and phosphorus in a variety of ecosystems (e.g., fresh water [17]–[24], sea water [25], [26], and soil [27]–[31]), and microbes substantially contribute to ecosystem functioning, especially in terms of nutrient cycling. In addition, microbes that dwell on organic solids such as dead zooplankton, animal feces, and algal aggregates are often metabolically more active than their free-living counterparts, and the degradation activities of enzymes that are attached to bacterial cells and aggregated organic matter are often substantially higher than the respective activities of the dissolved enzymes in natural waters [32]. Likewise, it is conceivable that enzymes associated with the particulate fraction of the pitcher fluid of Nepenthes, which is composed of free-living microbes and complexes of bacteria and organic solids, might also substantially contribute to organic matter decomposition.
In this study, we aimed to profile the in situ enzyme activity and the abundances of free-living microbes within the pitcher fluid of four Nepenthes species. We also assessed enzyme activity before and after filtration of the fluid to establish whether the digestive enzymes were mainly dissolved, or if a substantial fraction was also associated with particles such as bacterial cells and other organic solids. We addressed the following specific questions.
- What is the density of free-living microbes in the pitcher fluid of Nepenthes?
- How prevalent is the digestive enzyme activity in the pitcher fluid?
- What is the proportion of particle-bound enzyme activity in the fluid as compared with dissolved enzyme activity?
Materials and Methods
Study site and plant species
We used four Nepenthes species, Nepenthes albomarginata T. Lobb ex Lindl, Nepenthes ampullaria Jack, Nepenthes bicalcarata Hook. F., and Nepenthes gracilis Korth, which usually occur in lowland heath forest, in peat swamp forest, or at forest margins in Borneo [5]. The lid of the pitcher in N. ampullaria is held away from the pitcher mouth, thus allowing rainwater and fallen leaves to enter the pitcher; for the other three species, lids cover the pitcher mouth. The samples were collected within or around the Lambir Hills National Park (LHNP), Sarawak, Malaysia (4°2′N, 113°50′E; 150 m a.s.l.). N. albomarginata was collected near the summit of the Bukit Pantu hill (approximately 300 m a.s.l.). N. ampullaria was collected near a pond or at the edge of the LHNP in a relatively open and bushy area. N. bicalcarata and N. gracilis were also collected at the edge of the LHNP.
We also used the fluid of cultivated N. ampullaria, N. bicalcarata, and N. gracilis (a possible hybrid) from the greenhouse in the Botanical Garden of the University of Zurich, Zurich, Switzerland (47°21′N, 8°33′E; 440 m a.s.l.).
Collection of pitcher fluid, pH measurement, and filtration procedure
Collection of field samples in Borneo took place four times, in Feb., Jun., Aug., and Nov. 2009, and cultivated plants in Zurich were sampled once, during Nov. 2009. The sample size and species varied each time because of the availability of plants on each date/site; in Borneo, 6 pitchers of N. albomarginata in Jun.; 2, 6, 5, 6 pitchers of N. ampullaria in Feb., Jun., Aug., Nov., respectively; 3, 5 pitchers of N. bicalcarata in Jun., Aug., respectively; 3, 6, 6 pitchers of N. gracilis in Feb., Jun., Nov., 2009, respectively; in Zurich, 6, 5, 4 pitchers of N. ampullaria, N. bicalcarata, and N. gracilis, respectively.
First, we carefully transferred the pitcher fluid to Falcon tubes using a 5–10 ml pipette in the field. All the pitchers of the Borneo plants contained prey, such as ants, termites, and flies, but also living mosquito larvae, and debris. The pitchers from cultivated plants also contained prey such as flies, but no living mosquito larvae. Because of logistic restraints in the field, the samples were stored in the dark at 4°C for up to three days prior to the enzyme assay.
Each sample was split into two subsamples, one of which was left unmanipulated (subsequently termed 'untreated') while the other one was filtered through a membrane syringe filter (pore size, 0.2 µm; subsequently termed 'filtration'). Because such a filtration treatment should remove both large particles and free-living microbes from the fluid, we assumed that the filtered fluid contained only dissolved enzymes. By contrast, the untreated fluid contained both dissolved and particle-bound enzymes (subsequently termed 'total enzymes'). Thus, any difference in enzyme activity between the untreated and filtered samples should be caused by particle-bound enzymes that, at least in part, also originate from microbes. In addition, in contrast to sterile fluid of unopened pitcher in Sarracenia, fluid in the unopened Nepenthes pitcher contains bacteria [15]. Thus, unopened pitchers were not treated as a negative control. We also initially attempted fluid sterilization by adding antibiotics such as kanamycin, ampicillin, and spectinomycin, in order to distinguish between the enzyme activity in the fluid with and without microbes. However, the pH of the fluid changed significantly after addition of these antibiotics, possibly because they induced microbial cell burst. We therefore used the filtration treatment exclusively to obtain sterile fluid.
The pH values of untreated and filtered fluid were measured using a pH meter (Huber & Co. AG, Switzerland), while we used pH paper (PANPEHA, pH 0–14, Schleicher & Schuell, USA, and pH 2.8–4.4, Toyo Roshi Co. Ltd., Japan) when the amount of the sample fluid was too small for the pH meter (N = 2 for N. bicalcarata in Zurich, and N = 4 for each of N. gracilis in Borneo (Nov. 2009) and Zurich). No significant difference between the two methods was found in the pH values (data not shown).
Enzyme assay
We used three chromogenic substrates, 4-nitrophenyl phosphate, 4-nitrophenyl β-d-glucoside, and 4-nitrophenyl N-acetyl-β-d-glucosaminide, to estimate the activity of acid phosphatases (AP), β-d-glucosidases (BG), and β-d-glucosaminidases (NAG), respectively. We followed up by using a common colorimetric method for 4-nitrophenol linked substrates [33] to determine the enzyme activity in the pitcher fluid samples. All substrates were prepared in 50 mM (pH 5.0) acetate buffer, and the concentration of each was 5, 5, and 2 mM, respectively.
In Feb., Jun., and Aug. 2009, the assays were conducted for both untreated and filtered fluid in the laboratory of the LHNP field station as follows. Portions of 100, 150, and 100 µl of fluid from N. albomarginata, 200, 400, and 400 µl from N. ampullaria, 100, 400, and 400 µl from N. bicalcarata, and 100, 200, and 200 µl from N. gracilis were used for assessing the activity of AP, BG, and NAG, respectively. Each sample was added to a 1.5 ml tube, diluted, if necessary, to 400 µl with 50 mM acetate buffer, and mixed with 400 µl of each substrate. As controls, 400 µl of each substrate solution was mixed with 400 µl of 50 mM acetate buffer. Mixed samples were incubated at 25°C for two hours. Thereafter, 160 µl of 1.0 N NaOH was added to terminate the reaction. Absorbance was measured at 410 nm for AP, BG, and NAG with a Shimadzu UV-1200 spectrophotometer (Shimadzu, Kyoto, Japan). In Nov. 2009, the same treatment was carried out with fluid from cultivated plants in the laboratory at the University of Zurich. On that occasion, we reduced the total volume of measured sample to one-tenth of the volume that was required in the LHNP field station because of the availability of superior instrumentation. Absorbance was then measured using a Beckman DU800 microplate spectrometer (Beckman, Fullerton, California, USA), which has the same accuracy in terms of wavelength and photometrics as the UV-1200 according to the manufacturer's specifications.
Standard curves were determined for each set of measurements using 4-nitrophenol for AP, BG, and NAG. The activities were recalculated into mmol l−1 h−1. If the estimate of particle-bound activity as derived from the difference of untreated and filtered samples yielded a negative result, it was set to zero (i.e., no difference). If the activity measurement in a particular sample was already below the detection limit before filtration, it was excluded from the subsequent statistical analysis.
Total bacterial abundance
Samples of pitcher fluid were fixed with formaldehyde (2% final concentration) and stored at 4°C until further processing. Five to 300 µl of formaldehyde-fixed samples were stained with DAPI (4′, 6-diamidino-2-phenylindole, 1 µg ml−1 sample [34], and filtered onto black polycarbonate filters (0.22 µm pore size, Osmonics) placed on cellulose nitrate support filters (0.45 µm pore size, Sartorius). Images of preparations were automatically recorded with a CCD camera (Zeiss Axiocam) mounted on an epifluorescence microscope (Zeiss AxioImager.Z1) equipped with a motorized stage, and analyzed by a custom-made automated counting routine programmed in Visual Basic for Applications [35].
Statistical analyses
We examined the differences in mean pH values and enzyme activity among dates/sites within species and among species within dates/sites by applying Kruskal–Wallis tests. When the difference was significant, we also performed multiple comparisons with a post hoc Scheffé-type test (nonparametric). The differences in pH values and enzyme activities between untreated and filtered fluid were examined by a Wilcoxon signed-rank test for paired observations. The ratio of enzyme activity in untreated and filtered fluid was calculated and examined by a Kruskal–Wallis test. In all cases of multiple comparisons, we adjusted the p-values by a false discovery rate control method [BH method; 36]). These statistical analyses were performed using R 2.13.0 [37]. Furthermore, to explore factors that affect enzyme activity in the Borneo samples, we conducted a generalized linear mixed model (GLMM) for each enzyme using the lmer function in R (the restricted maximum likelihood (REML) approach, package lme4. Enzyme activities were log-transformed and were included as the response variable (N = 86 for AP, N = 68 for BG, N = 52 for NAG). If the activity of the filtered sample was zero, we added a small positive value, 10−2, because of log-transformed values (N = 7 for BG, N = 5 for NAG, i.e., closed symbols in Figures 1 and 2). We used the filtration treatment (untreated and filtration), pitcher plant species and pH (log-transformed) as explanatory variables. We included random intercepts for the pitcher ID and date that allowed intercepts to vary by them, as well as resulting in a random slope with respect to the treatment that allowed the effect to vary by pitcher ID. To evaluate the significance of parameter estimates in the fitted GLMM, we obtained a 95% confidence limit (CL) using the Markov Chain Monte Carlo (MCMC) method using the mcmcsamp function in R (package lme4). When the CL of the parameter does not include zero, this indicates that the factor differs significantly from zero at the 5% level. This multilevel modeling and MCMC procedure work reasonably well when the data contain groups with a small sample size [38].
(a) Acid phosphatases (AP), (b) β-d-glucosidases (BG), and (c) β-d-glucosaminidases (NAG) in each species. A closed symbol represents that dissolved enzyme activity was not detectable and was treated as zero. The differences in mean values between untreated and filtered fluid were examined by Wilcoxon signed-rank test, and p-values were adjusted by the BH method for multiple comparison and were shown in the upper box for each species (unadjusted p-values are shown in brackets). ns: not significant.
(a) Acid phosphatases (AP), (b) β-d-glucosidases (BG), and (c) β-d-glucosaminidases (NAG) in each species. A closed symbol represents that dissolved enzyme activity was not detectable and was treated as zero. The differences in mean values between untreated and filtered fluid were examined by the Wilcoxon signed rank test, and p-values were adjusted by the BH method for multiple comparison and were shown in the upper box for each species (unadjusted p-values are shown in brackets). ns: not significant.
Estimated numbers of bacterial cells per ml were log-transformed. An ANCOVA was performed to examine whether the pH and species/site were associated with the density. Posterior comparison of means after the ANCOVA used the Tukey–Kramer HSD test. ANCOVAs were implemented using the REML formula, JMP 5.0 (SAS Institute). We also tested the correlation between bacterial densities and the mean ratio of dissolved enzyme activity to total enzyme activity (filtration/untreated) by using a generalized linear model (GLM) for each enzyme.
The characteristics of the Nepenthes fluid
The pH values of Nepenthes fluid were acidic in all the samples (Figure 3), and did not differ for the dates of collection within the species sampled in Borneo. N. gracilis tended to have a lower pH compared with other species but this was not always significant through the date sequence (Figure 3). The pH did not change after the filtration treatment (Wilcoxon signed-rank test, p = 0.97).
The differences in mean value of the pH between the dates/sites within a species were examined by the Kruskal–Wallis test (p-values adjusted by the BH method, shown above each species). Differences of a species/site between dates were also examined by the Kruskal–Wallis test (X-axis legend; Feb. and Aug.; not significant, Jun. and Nov.; p = 0.005). Results that share the same letters are not significantly different (lower case for species examined at the same date, upper case for dates between species, Scheffé-type test, p<0.05).
In situ enzyme activity
The averaged total enzyme activity (untreated) and dissolved enzyme activity (filtration) was calculated for the four Nepenthes species (Table 1, Figures 1 and 2). AP activity was always detectable in all the pitchers of all species in both Borneo and Zurich (Table 1). Likewise, BG and NAG activity was also found in all species, although there was no measurable activity of this enzyme in some pitchers of all the examined species (Table 1). Altogether, total enzyme activities did not differ significantly between the dates for either enzyme in either species (Kruskal–Wallis test, p<0.05); therefore, they were pooled for further statistical analysis.
To assess the contribution of particle-bound enzyme activity, the mean ratio of dissolved enzyme activity to total enzyme activity was calculated. The values varied from 0.2 to 1.0 in the Borneo samples and from 0.3 to 1.0 in the ones from Zurich (Table 1). Total enzyme activity was significantly lower in the Zurich samples than in the Borneo samples for all the enzymes (Mann–Whitney test, p<0.05). Similarly, the Zurich samples showed a lower dissolved enzyme activity than the Borneo samples (Mann–Whitney test, p<0.05).
Dissolved vs. particle-bound enzyme activity
First, we tested whether the filtration reduced the enzyme activity significantly. In the Borneo samples, AP activity significantly decreased after filtration for N. gracilis, whereas this decrease was only marginally significant for N. ampullaria and N. bicalcarata (Wilcoxon signed-rank test, Figure 1a). BG activity decreased after filtration in N. ampullaria and N. bicalcarata (Figure 1b). For NAG, we found a marginally significant decrease in activity after filtration in N. ampullaria, N. bicalcarata, and N. gracilis (Figure 1c). These data support the contribution of particle-bound enzyme activities in Borneo samples. In the Zurich samples, we did not find any significantly decreased enzyme activity after filtration (Figure 2), with the possible marginal exception of AP activity in N. bicalcarata (Figure 2a), which might be partly because of the lower enzyme activity in the Zurich samples (also see Discussion).
Second, we tested whether the mean ratio of enzyme activity in untreated and filtered fluid differed among species. The ratios did not differ for any enzyme among the species from the Borneo samples (Kruskal–Wallis test, p>0.05 after p-value adjustment by the BH method), nor among the collection dates for the pooled species (Kruskal–Wallis test, p>0.05 after p-value adjustment by the BH method). Similarly, there were no differences among the species sampled in Zurich, except for the ratio of AP activity (Kruskal–Wallis test, p<0.02 after p-value adjustment by the BH method), which was in the order N. gracilis≤N. bicalcarata≤N. ampullaria (Scheffé test, p<0.05). The total enzyme activity differed among sites within the same species sampled on the same date (Nov. 2009). The activities in the Borneo samples were higher in N. ampullaria for AP, and in N. gracilis and N. ampullaria for BG (Kruskal–Wallis test, p<0.05 after p-value adjustment by the BH method).
To investigate the contribution of various factors including the filtration treatment, pH and species on enzyme activity, the GLMM was conducted and then confidence intervals were estimated using MCMC. The parameter estimates of the filtration treatment were negative in all three enzyme activities (−0.77 to −0.06 for AP, −7.48 to −2.13 for BG, −7.87 to −1.84 for NAG), which further supports the observation that filtration treatment decreased the activity in all enzymes (Table 2). The pH was not correlated with the enzyme activity in the three enzymes, and furthermore, the Nepenthes species did not have any effect to enzyme activity mostly (Table 2).
Bacterial abundance in the fluid
The mean bacterial density in pitchers was 7.1×107 cells ml−1. The density in N. ampullaria in the Zurich samples was the lowest (7×106 cells ml−1, Table 3), whereas that of N. gracilis in the Zurich samples was the highest (2.2×108 cells ml−1, Table 3). There were no significant correlations between bacterial densities and pH (ANCOVA, F = 0.11, df = 1, p = 0.74). Bacterial densities were positively correlated with the mean ratio of dissolved enzyme activity to total enzyme activity in BG (GLM, t = 2.09, p<0.05) and NAG (GLM, t = 2.349, p<0.05), but were not correlated with that in AP (GLM, t = −1.911, p>0.05).
High in situ enzyme activity in the pitcher plant fluid
In this study, we profiled the in situ enzyme activity of the fluid in four Nepenthes species growing in Borneo (wild) and Zurich (cultivated). We detected activity for all of the enzymes examined in this study, AP, BG, and NAG, in the liquid of the pitchers of all four Nepenthes species. Compared with other environments such as freshwater, seawater, and soil (Table 4), the enzyme activity was surprisingly high in the liquid of Nepenthes species, up to five orders of magnitude greater than in the others. Moreover, the activity of all the studied enzymes in Nepenthes was much higher than that in another carnivorous plant species, Utricularia Table 4, [39]. Prey in the pitchers could be a most important nutrient source for plant growth and fitness in Nepenthes, for which it is reported that 60% of nitrogen is derived from prey [7]. A high enzyme activity in the fluid would help in the rapid decomposition of prey to maximize the efficient nutrient use within the pitcher.
Enzyme activity was generally higher in Borneo, which might be related to selective pressure for high enzyme production in the wild plant but also to higher activity by the indigenous microbial consortia. Moreover, the level of enzyme activity varied substantially between individual pitchers of a single species at each site and for single sampling dates. This implies that the enzyme secretion by plants and/or microbes may depend on the local condition within each pitcher; e.g., the pitcher age, the presence of plant debris, and the amount of prey could all affect the metabolic secretion of digestive enzymes. Currently, the mechanisms of accumulation or consumption of enzymes within the pitcher remain uncertain.
All investigated species exhibited high AP enzyme activity. APs are common plant enzymes of low substrate specificity that appear to be important in the production, transport, and recycling of phosphorus [40]. It has been reported that phosphorus may be the limiting nutrient in Nepenthes as well as in other carnivorous plants [2]. In view of such findings and our own observation of a possibly constitutive phosphatase activity in all pitchers, phosphorus may be an essential requirement in Nepenthes.
By contrast, we found that BG activity was not always detectable in each pitcher, especially in N. albomarginata and N. gracilis. BG is required in the degradation process of cellulose, which is a structural component of plant cell walls. Plant material such as fallen leaves could drop into the pitcher, especially with regard to N. ampullaria [41], which is an open-lid species, or N. bicalcarata, which has a large pitcher with a lid relatively distant from the pitcher mouth. For such species that are likely to receive plant material into their pitcher, BG may be essential for cellulose decomposition.
NAG is linked to the cycling of nitrogen, which is an important nutrient for Nepenthes [2]. Interestingly, not all examined pitchers scored positively for NAG. These enzymes are centrally involved in the breakdown of chitin, which is the hard amino sugar component of insect carapaces and microbial cell walls. The degradation of this material might also increase the ability of other enzymes to gain access to the soft body tissue within [3]. NAG should therefore be a key enzyme to obtain nitrogen and other nutrients for the plants, and it is, therefore, unexpected that some pitchers had no measurable NAG activity. Moreover, it should be noted that some of the observed NAG activity might even be related to the molting of specialized arthropods [24], as some Nepenthes species, for example, are known to host live mosquito larvae that complete their life cycle inside the pitchers [42].
Bacterial abundance in the pitcher plant fluid
A high bacterial density was confirmed here even in the pitcher fluid of N. gracilis at an extremely low pH (Table 3), in agreement with previous findings in another Nepenthes species, Nepenthes alata Blanco [15]. The cell densities of free-living microbes were as high as, or higher than, those in productive natural aquatic environments [43]. Moreover, because we did not specifically count cells attached to larger particles, it is conceivable that the total bacterial densities in the pitchers were even higher.
Importance of particle-bound enzyme activity
We found that the activity of all three studied enzymes decreased after filtration in various samples collected in Borneo (Tables 1 and 2, Figure 1). In addition, the ratio of dissolved to particulate BG and NAG activity increased with higher numbers of bacterial cells in the fluid. While this observation does not prove that the enzyme activity in the particulate fraction of the fluid was exclusively of microbial origin, it nevertheless suggests that both free-living and particle-bound microbes might have contributed substantially to the total enzyme activity. We also found that not all three types of enzyme activity decreased following filtration in N. ampullaria and N. gracilis (Table 1, Figures 1 and 2). Moreover, the ratio of enzyme activity between the untreated and filtered samples was highly variable throughout all of the pitchers, species, and enzymes. These data suggest that other factors related to the plant species or individual pitchers might also affect the contribution of particle-bound enzyme activity.
It has been suggested in other carnivorous plants that the microbial community would play a significant role in digestion or in the attraction of prey [3], [44]. For example, Utricularia species harbored plant-associated algae and bacteria in the traps; the bacterial density in the trap was also at a similar level as Nepenthes, e.g. 107 cells ml−1 (Table 4, [45]). They produced significant amount of phosphatases, especially in old pitchers [45]. This might indicate that these microbes would control the nutrient flow to other components. The algae community occurring in traps of Genlisea species [46] also produced enzymes, but might compete for organic phosphorous with the host plant. Another example is pitchers of Sarracenia, which also harbored algae and bacteria producing enzymes [47]. Considered with other evidence from a nutrient tracing experiment in Sarracenia species [48], these microbial communities might significantly affect the nitrogen availability within the pitcher. The taxonomic composition of the bacterial community in the fluid of Sarracenia species has partly been investigated recently [49], [50], and it has been suggested that this is similar to an animal gut-like bacterial community. Thus, the role of the community might be related to digestion of prey, although their functions are still uncertain.
Our study also suggests, albeit indirectly, that there may be activity of enzymes derived from microbes in the investigated Nepenthes species. Higashi et al. [14] isolated gram positive and gram negative bacteria in the fluid of N. hybrida. They also suggested that these microbes would also produce digestive enzymes. Moreover, microbe-derived enzymes would have different roles on degradation of prey from plant-derived enzymes because these two kinds of enzymes had different optimum pH-ranges of enzymes. These results could indicate that, to some extent, the microbial community might play a role in the nutrient cycle and metabolism of the Nepenthes pitcher and act cooperatively to the host plant. To substantiate this hypothesis, further evidence is required, such as that from a proteomics study or protein sequencing, to assess the origin and relative amounts of enzymes derived from microbes. Presently, we still do not know the diversity and abundance of different specialized populations within the microbial communities in the pitcher fluids. While the high concentrations of nutrients and organic material, as well as the prey variety might foster high microbial community diversity, the plant-produced antibacterial enzymes [12] and low pH conditions might, in turn, inhibit the growth of some microbial species.
In this study, we did not focus on the effect of pitcher age and prey species on enzyme activity. In fact, it is known that fluid in the unopened Nepenthes pitcher contains bacteria [15]. Consistently, fluid in unopened pitchers had the same level of phosphatase activity as that in opened and matured pitchers (Takeuchi et al., data not shown, also see [14]). This might indicate that the effect of pitcher age on enzyme level would not be strong during the younger to matured stage. Because it is suggested that enzyme secretion decreased with plant age in Utricularia species [45], the possibility that older pitchers could also decrease enzyme secretion more or less in Nepenthes might not be excluded. The prey species composition and the structure of the food web in the pitcher would change in space and time as well as pitcher age [6], [15], [51]. An experimental study on these effects would be helpful for understanding plant-microbe interaction within a pitcher. In addition, future research should aim at elucidating the causes of the observed variability in particle-bound enzyme activity to provide additional evidence for the specific contribution of microbes to this activity. Eventually, a study is required of the community composition and function of the different microbial species in the pitcher fluid.
The authors thank Ms. Lucy Chong (Forest Research Center, Sarawak) and Mr. Mohd. Shahabding Sabki (Sarawak Forest Department) for their permission for, and assistance with, this work in Sarawak, Malaysia. We are also grateful to Mr. B. Jingan for his help in the fieldwork.
Author Contributions
Conceived and designed the experiments: YT MJK KKS. Performed the experiments: YT MMS MU RSI MJK BD. Analyzed the data: YT MMS CvM JP. Contributed reagents/materials/analysis tools: YT MU BD CvM JP KKS. Wrote the paper: YT MMS MU JP KKS.
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(c) Finally, you can
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From Flickr Hive Mind. To banish yourself from Flickr Hive Mind, you must first authenticate to Flickr, which lets Flickr Hive Mind identify who you are. This is necessary to make sure you are the user in question, and also because people use such crazy characters in their Flickr user names. You can revoke access when finished. Once logged in, come back to this area
and there will be a link to banish you. If there are any problems email me at nosflickrhivemind ... at ... fiveprime ... dot ... org. (a few people with weird characters in their user names may have had problems)
Log In To Flickr Now
Flickr Hive Mind is a data mining tool for the Flickr photography database, allowing search by: tags(keywords); Flickr photography groups; Flickr users, their contacts, and favorites; free text; the Flickr Explore algorithm for interestingness. Flickr Hive Mind can also be an effective tool to identify photography with licenses that allow non-commercial (and sometimes) commercial use. Inspired by (and thanks to) Flickr Leech. Dedicated to Anna and her Flickritis. Many thanks to Lokesh Dhakar for his lightbox2 image viewer. If you find your login does not work, perhaps your browser has disabled the cookies that I use to track if you are logged in or not. Try telling your browser to accept them. Flickr Hive Mind stores no personal information nor photos, but some general time and IP address information ends up in my apache server logs. For additional details see the privacy policy. Any revenue generated through advertisement on this site is used only to cover the cost of keeping the site online, beyond that Fiveprime donates to charitable organizations (also, there are no ads on User pages). Flickr Hive Mind is currently consuming about 13.6 terabytes of network bandwidth per year (not including the photos themselves)!