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A Guide To Importing From New Zealand For Australian Businesses
If you're starting to look into importing from New Zealand, this guide will help you discover how to identify, source, and import goods from New Zealand to sell in Australia.
For small business owners looking to import from New Zealand, this option may turn out to be a more cost effective solution than sourcing for products in Australia. Major imports from New Zealand include tobacco, edible products, gold and crude petroleum.
According to The Department of Foreign Affairs and Trade, Australia's import trade with New Zealand averages over AUD 7 billion annually. This ranks the country 6th in terms of merchandise trade in Australia
How to import from New Zealand
Here's what you need to do if you want to import from New Zealand.
- Obtain information on the types of goods you are allowed to import and carry out market research.
- Explore online marketplaces of manufacturers and suppliers to source the goods you want.
- Carry out due diligence on your supplier and sign a contract with them.
- Arrange for shipping and logistics from New Zealand to Australia.
- Understand the various regulations, tariffs, and licenses you need to export.
- On arrival of your goods in Australia you pay the necessary duty, tariffs, and any other fees.
- Market your goods for sale in Australia.
Dealing with importing, international shipping, tariffs, licenses and more can be tricky. We strongly recommend bringing someone into your business who has experience and knowledge in the field
What can you import from New Zealand to Australia?
It's important to consider market trends before making a decision to import. Conduct market research to understand if there is a demand for the product in Australia. Be sure to analyse your competitors and survey existing customers
Here are some hints and tips:
- Avoid food and perishables — It's very expensive to import goods that can be spoiled, as you'll need specialised containers. Additionally, Australia has strict regulations on importing produce.
- Avoid importing products that are widely available already — It can be hard to wiggle your way into the market when the supply for products are widely available. Plus, larger companies tend to order in bulk at wholesale prices.
- Source items based on your prospective market, not the other way around — Find out what prospective customers want and source items from China based on that. Don't source items hoping you'll then find an audience (you probably won't).
- Understand your margins and pricing — Work out your pricing strategy by calculating your fixed and variable costs.
Sourcing goods from New Zealand
There are a selection of Kiwi suppliers and manufacturers to choose from. You can find them below:
- Aus Trade New Zealand
- New Zealand Customer Service – Supplier codes and names
- GMP Pharmaceuticals
- Caelera
- Many more…
Once you know the types of products you're after, make a shortlist of suppliers. Next, you'll want to:
- Read reviews on the suppliers including their customer service, the quality of the goods they provide, and any issues others have had with them.
- Contact the suppliers and request a sample of the goods you're interested in. When you receive the sample have it independently appraised to make sure you're getting what you pay for.
- Ask the supplier about their terms for payment, how long it will take them to send the product to you, what protections and guarantees you have, and what their policies are on returns.
- Complete any other due diligence, and if you're satisfied, sign a contract with them.
Typical container costs — shipping from New Zealand to Australia
If you're only purchasing a small quantity or your items aren't too heavy or bulky, consider air freighting them back to Australia. If you're buying larger quantities, you'll probably have to use a shipping container.
The Sea Freight Calculator lists the cost of renting a full container New Zealand to Australia as approximately:
- 20 ft container — $1,000 USD
- 40 ft container — $1,300 USD
And according to Australia Trade, renting part of a container will cost you between $150 and $250 AUD per cubic meter or 1,000 kilos, whichever is greater.
However, these are purely the costs to ship goods from New Zealand to Australia. Once you get them here you'll need to pay for getting your goods to a warehouse, storage, insurance, duties, and other fees.
Government regulations on imported goods from New Zealand into Australia
It's important to follow trade regulations when importing goods into Australia. To learn more, read this comprehensive guide.
Some of these regulations include:
- A special permit may be required for certain types of goods. This include some chemicals, pharmaceuticals, produce, and more.
- Plant and animal goods may be quarantined.
- Tariffs, duties, and taxes may apply— More on that below.
- Some importers may be eligible for government concessions.
- Free trade agreements could work to your advantage.
- Be aware of several other costs.
- Ensure that your goods are labeled correctly..
For full details, please check out our guide to importing into Australia.
Understand costs to import from New Zealand
When you're importing, it's vital to understand your overall costs. These include:
- The cost of buying the product.
- Shipping, logistics, and distribution costs.
- Tariffs and duties due to New Zealand on export and Australia on import.
- Taxes and tariffs on your imported goods.
Generally, you will have to pay import duty and GST on goods valued over $1,000 AUD.
- Import entry costs and processing charges – Customs will charge you a fee (typically under $200 AUD) for processing your goods.
- Customs import duty is calculated as a percentage of the price you paid for the goods. The duty rate can range from 0% to 10%, but the rate for most goods is 5%.
- Goods and services sales tax (GST) will also be charged based on the following:
- The valuation of the goods plus;
- The customs import duty amount plus;
- The cost of insuring the goods and transporting them to Australia.
- GST is charged at 10% of the final amount.
This is just intended as a general guide – These charges can be influenced by what you're bringing in, excise taxes, free trade agreements, and many other factors. Please do check with customs for your individual circumstances.
Some types of goods are eligible for concessions (reducing the amount of duty you need to pay). You can find a factsheet through this page, just look for "List of current tariff concession orders." You'll also find additional details on concession schemes here.
Sell your products
Once you've received your goods from New Zealand, market them and offer them for sale. Continue to research your markets, source goods, and sell them onwards at a profit, Good luck!
Paying for your goods in foreign currency
Doing import business from New Zealand is usually done in US dollars or New Zealand dollars.
If you are bringing goods in from NZ, consider using OFX They specialise in international payments and receipts for Australian businesses. Each client has a personal manager and access to a range of useful products like Forward Exchange Contracts, Options, Limit Orders and Currency Options.
OFX is a trusted partner of The Currency Shop. Click on this link , sign up and never pay a transfer fee for your international money transfers. Ever.
OFX charges no transaction fees. Minimum transfer A$250. Third party intermediary fees may still apply.
Learn more about OFX
What about receiving money in foreign currency?
Until recently, Australian businesses had 3 options to receive money from overseas customers:
- Get paid into a local Australian based, Australian dollar account.
- Open a Foreign Currency Account, usually with a bank
- Use a payment gateway like PayPal, Braintree or Stripe
Unfortunately, these options are usually filled with fees, balance requirements or massive exchange rate mark-ups.
In October 2017, TransferWise released the Borderless account in Australia and it's really impressive. It allows you to receive, hold and transfer 27 different currencies.
A unique feature that is particularly useful for businesses, is the ability to have local bank account details in USD, EUR, GBP or AUD. This makes receiving money from overseas customers exceptionally easy.
For the full review of the TransferWise Borderless Account, click here


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by yhecht on 09 October 2019 - 17:10
Yvonne Hecht - Was in hospital for 5 days, but am back now. I have been fighting a wound (Hole) in the bottom of my foot since Nov,2017, and have been in and out of hospital, but this last time I forgot to take my computer.
Thanks to all of you that worried about.
Thanks to all of you that worried about.
by GSDHeritage on 09 October 2019 - 18:10
Yvonne Hecht, That sounds like a horrible ordeal to go through, glad you are back.
by Hukka on 09 October 2019 - 19:10
Glad you are feeling better!
by hexe on 10 October 2019 - 04:10
Oh, no--glad you're back home, but sorry to hear that you've been having a bad time of it with your foot! Hopefully they'll have been able to get things back on track for you, and this will finally be resolved!
by Gustav on 10 October 2019 - 11:10
Glad you are doing better Yvonne.
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subset: commoncrawl


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Airport Phone: +1 919-840-2123
Hotel direction: 70 miles E
This hotel does not provide shuttle service.
Take Interstate 40 East to Interstate 440 North/Inner Beltline and continue around the city to US-64 East. Proceed east on US-64 approximately 47 miles to Rocky Mount and exit at Winstead Avenue. Turn right off the exit ramp and proceed to the first traffic light. Turn left onto Curtis Ellis then left again onto Gateway Boulevard.
Complimentary on-site parking


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The latest PUMA x ADER ERROR collection plays with imperfections to create something unexpected and beautiful. Titled Under Creativity, the collection represents one combined voice of different subcultures. It features progressive silhouettes accented with bright pops of color, caution sign tags, visible stitching, and deconstructed elements. The result is an under-construction look that blurs the line between past and future.
The RS 9.8 is dedicated to those who defy societal rules and the laws of physics- so it only makes sense we would collab with Ader Error on this one. Inspired by our '80s running line, this version from Ader Error updates the new silhouette with cool colorblocking, an adventure-ready material mix and space-inspired spheres at the midsole.
- Running System (RS): PUMA's retro running technology that offers cushioning from the forefoot through the heel
- 80s running silhouette
- Mesh upper with suede overlays
- Full lace closure
- Padded tongue and collar
- Lightweight PU midsole with RS cushioning
- Rubber outsole for grip and traction
- 3D TPU pieces at the midsole
- Suede PUMA Formstrip at lateral side
- Metal badge stitched at side
- PUMA x ADER ERROR branded tongue tab


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U.S. Rep. Luis Gutierrez (D-IL,4) says educational outreach around President Barack Obama's executive actions on immigration will press ahead, despite a federal judge's ruling on Monday that temporarily stalled the administration's deportation relief plans.
The Obama administration is expected to appeal the judge's ruling, which comes in response to a lawsuit filed by a group of 26 mostly Republican-run states seeking to stop the president's immigration directives. Illinois has not joined the lawsuit, which claims the two immigration executive orders signed by Obama in November are unconstitutional. The federal judge, Andrew Hanen of the Federal District Court for the Southern District of Texas, determined that the states met the minimum requirements needed to proceed with the lawsuit.
Hanen's temporary injunction blocks Obama's two new immigration programs from taking effect before the case is decided. The application process for one of the new immigration policies was slated to launch Wednesday.
At a Tuesday morning press conference in Chicago, Gutierrez and leaders with SEIU* and the Illinois Coalition for Immigrant and Refugee Rights (ICIRR) called the judge's decision a "temporary setback" that won't derail their efforts to help immigrants prepare for administrative relief.
"This process can be delayed, but we as a community will not be deterred," Gutierrez said, adding that he believes Obama's new immigration policies will ultimately prevail after the legal process plays out. "Let's make it absolutely clear that the president's actions are well established in legal precedent."
The latest roadblock in the fight for immigration reform was unsurprising to Gutierrez and the immigrant advocates. That's because the states named in the lawsuit "shopped for the most conservative anti-Obama judge they could find, and they got the decision they were shopping for," Gutierrez said.
"Eventually we will win," the congressman added. "This decision will be appealed to the Fifth Circuit [Court of Appeals], and I fully expect that when it arrives there" that it will be overturned.
The president's executive actions seek to create a Deferred Action for Parents of Americans (DAPA) program, allowing temporary relief from deportation for qualified undocumented parents of children with U.S. legal status who have resided in the country for at least five years. Additionally, Obama's administrative relief extends deferred action to additional undocumented immigrants who came to the United States as children, but were too old to qualify for the 2012 Deferred Action for Childhood Arrivals (DACA) program.
As many as five million undocumented immigrants living in the United States, including up to 280,000 in Illinois, could be covered by the executive orders, according to ICIRR. The federal government was scheduled to begin accepting applications on Wednesday for the expanded DACA program and in May for the DAPA initiative.
Gutierrez and officials with ICIRR and SEIU will not be suspending their various educational campaigns and workshops around the executive orders in light of the judge's ruling.
"We know thousands of Illinois immigrants who grew up in the country were looking forward [to applying tomorrow]," said Lawrence Benito, ICIRR's CEO. "A single judge temporarily blocked this, but ... we expect that this is a temporary setback, and we're going to continue to organize."
Illinois' new Republican Gov. Bruce Rauner, meanwhile, has expressed support for Obama's executive orders. Rauner's response to the president's immigration directives, along with the fact that Illinois is not a plaintiff in the lawsuit to block them, "speaks to the possibility of working together" on issues important to the immigrant community, noted Gutierrez, who said he plans to schedule a meeting with the governor on the matter.
Hanen's order also comes amid a Congressional fight over immigration and a Department of Homeland Security Funding bill.
Last month, the GOP-led House passed a DHS funding bill that would block Obama's new immigration policies and end the DACA program. Since then, Senate Republicans have tried to take up a similar DHS funding measure with immigration riders, but their efforts have been blocked multiple times by Democrats.
If Congress does not pass a bill to fund DHS by February 27-- when the department's current funding runs out -- some 30,000 workers, including many from the Federal Emergency Management Agency (FEMA), would have to be furloughed, according to Homeland Security Secretary Jeh Johnson.
Gutierrez said the ongoing Republican-led efforts to roll back or stall progress on immigration reform show "just how mean and xenophobic you can be to risk and to put in jeopardy the political future that you have as a national party."
"These actions are going to have a huge detrimental effect" on the Republican Party in 2016, he stressed.
Here's more from Gutierrez, Benito and SEIU Local 1 Secretary-Treasurer Laura Garza:
Tuesday's press conference, held at the downtown offices of SEIU Local 1, was one of many events taking place across the country as part of a national day of action on immigration.
SEIU Executive Vice President Rocio Saenz warned members of the Republican Party that the immigrant community "will remember" their words and actions.
"We are mobilizing," she stressed. "We are organizing, and we're determined that we're going to see justice for the immigrant families."
*The SEIU Illinois Council sponsors this website.


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Online Coaching
Amalfi Coast
Swim Camps
VIP Members
Plans & Pricing
August 30, 2019
By Peter Hendriks
Swim speed needs to be earned, you can't just demand it. There you go, I've said it.It's not something that is achieved with one coaching session.
If you were wanting to learn Karate or Judo would it be fair to walk into a Dojo with absolutely no or l...
August 2, 2019
Peter Hendriks
Breathing properly is an important part of any terrestrial activity, but particularly when exerting energy during sports activities especially swimming.
Swimmers need to be aware of the following basic facts:
Breathing in whilst underwater is still NOT possible for h...
May 13, 2019
These tips apply to swimmers of all levels.
1. Plan your swimming week and be prepared to "DO THINGS DIFFERENTLY". Sounds obvious, but many people fail to make time regularly to get into the pool each week and when they do they fail to do anything differently to what th...
March 21, 2019
Wouldn't it be great if we could just attend a swim workshop and or have a few private lessons and "Abra Kadabra" we are great swimmers.
Yes, we all have some immediate improvements in efficiency (which in some cases results in an impressive improvement in lap times or...
March 14, 2019
Deliberate practice is an activity designed specifically to improve performance, often with a coach or mentors help. Most people practice by mindlessly repeating an activity over and over without any clear goal of what they want to accomplish. For example, let's say a...
January 14, 2019
Pam Hendriks
It's been a long time between blog posts and in the spirit of fulfilling a new year's resolution, less procrastination, more doing, let's fix that. When I realised I'd not written a post since well before our September 2018 tour I began to reflect on what an unforgett...
November 6, 2018
The fear of drowning is a rational and logical fear experienced by many who cannot swim. Aquahobic or Thalassophobic individuals, however, experience a great deal of anxiety around all kinds of water bodies ranging from large oceans to small bathtubs.
Aquaphobic or Tha...
May 31, 2018
There is a saying that goes 'If you keep on doing what you've always done, you'll keep on getting what you always got'.
Most swim coaches can stand on the pool deck and develop a fairly good idea of what swimmers are doing...or not doing. For example: they can se...
February 13, 2018
You've made the big decision and come mid-September you'll be holidaying on the beautiful Amalfi Coast - Brilliant! ........ Now where to next? Possibly you have plans in place already for your onward journey from the Amalfi Coast............ but if you're still in the...
June 17, 2017
So you're looking forward to your Amalfi Coast odyssey and wondering how to spend the remainder of your time in Italy? Well you are certainly spoiled for choice, the options are endless. Much will depend on the amount of time you have available and what interests you...
Swim Speed – you must earn the right
Why breathing out is more important than breathing in when swimming
5 Tips to improve your swimming
You've Invested in a TI Workshop or had some One on One Lessons….
BUT where to from here??
Deliberate Practice
Follow your dream...
Is Aquaphobia holding you back?
The Benefits of Video Stroke Analysis
So Excited about the Amalfi Coast ........ now where to next
Where to from the Amalfi Coast ???
August 2019 (2)
May 2019 (1)
March 2019 (2)
January 2019 (1)
November 2018 (1)
May 2018 (1)
February 2018 (1)
June 2017 (1)
May 2017 (1)
April 2017 (1)
March 2017 (2)
January 2017 (1)


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IWSCET 2017 : International Workshop on Smart Cities Enabling Technologies
|Link: http://www.cyberc.org
Call For Papers
International Workshop on Smart Cities Enabling Technologies
Nanjing, China, October 12-14, 2017
(Former International Workshop on Smart Sensor Network, IWSSN 2012-2016)
By 2050, 70% of the world's population is expected to live in cities. To support such a rapid growth, it is important for cities to deliver up-to-date information to its residents in a timely manner by adopting modern information and communications technologies. Internet of Things, Cloud Computing, Smart Grids, and Intelligent Transportation Systems are some of the key enabling technologies for city dwellers to interact with their cities seamlessly. This workshop will provide a forum for presenting and discussing the most recent advances in different aspects of smart cities and their enabling technologies. The workshop will gather leading researchers from academia and industry to present their research achievements and share their experiences in developing/deploying those technologies.
Publication: All accepted papers will be published with CyberC 2017 processing (You can find all previous Processing at the IEEE Xplore and EI database).
2017 IWSCET is co-hosted with CyberC 2017. Authors are invited to submit original technical papers covering but not limited to the topics of interest listed below:
• Internet of Things
• Sensor-aided Smart Grids
• Vehicular Sensor Networks
• Video Sensor Networks
• Sensor-aided Social Networks
• Cognitive Radio Sensor Networks
• Robot-aided sensor networks
• Body Sensor Networks
• Sensor-Cloud Applications
• Data Fusion in Smart Sensor Networks
• Big Data in Sensory Systems
• Human-Sensor Interactions
• Bio-Inspired Sensor Networks
• Resource Management
• Cross-Layer Design and Optimization
• Network Security
Important Dates
June 15, 2017 - Conference Paper Submission Deadline
July 15, 2017 - Notification of Acceptance & Registration Starts
August 15, 2017 - Camera-Ready Paper Submission Due & Registration Due
Paper Submission Web: Click here or go to http://edas.info/ by selecting "IWSCET2017".
Electronic submission to firstname.lastname@example.com with title of "IWSCET2017 Submission" is also accepted.
Workshop Chair
Chi-Tsun (Ben) Cheng, The Hong Kong Polytechnic University, Hong Kong
Workshop Co-Chairs
Donglin Wang, New York Institute of Technology, USA
Hongbin Chen, Guilin University of Electronic Technology, China
Qingfeng Zhou, Hefei University of Technology, China
ZhiQiang (Michael) Chen, The University of Hong Kong, Hong Kong
Technical Program Committee Members
Achintha Maddumabandara, Shaw Communications, Canada
Chatura Seneviratne, University of Calgary, Canada
Gayan Attanayake, AnsaldoSTS, Australia
Haruna Matsushita, Kagawa University, Japan
Han Zhang, South China Normal University, China
Homin Kwon, SYSTRAN Software Inc., USA
Jing (Vicky) Wang, The Hong Kong Polytechnic University, Hong Kong
Kai-Yin (Zero) Fok, The Hong Kong Polytechnic University, Hong Kong
Kia Fallahi, Eaton, Canada
Lusheng Wang, Hefei University of Technology, China
Nuwan Ganganath, ASTRI, Hong Kong
Prabhath Ilangakoon, Ceylon Electricity Board, Sri Lanka
Qingquan Sun, Oakland University, USA
Shengli Zhang, Shenzhen University, China
Shintaro Arai, Okayama University of Science, Japan
Sumona Mukhopadhyay, University of Calgary, Canada
Thilina Yapa Bandara, Zone24x7 (pvt) Ltd., Sri Lanka
Wang-Hei Ho, The Hong Kong Polytechnic University, Hong Kong
Wanmai Yuan, Harbin Institute of Technology, China
Xi Chen, State Grid Corporation of China, China
Xiaofan Liu, Southeast University, China
Xinzhi Xu, Global Energy Interconnection Development and Cooperation Organization, China
Yoko Uwate, University of Tokushima, Japan
Zhizhong Ding, Hefei University of Technology, China
Zhuo Chen, ICT Center, CSIRO, Sydney


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Enter cabin through sliding glass door. Large sofa to port with a portion that is a sofa bed to sleep a couple. To starboard is a bar with sink and ice maker. Next starboard is the entertainment center with a 42' flat screen TV and stereo. To port is a a dinette for four.
Down galley is starboard and guest stateroom with two single berths is to port. To starboard is a washer and dryer. To port is the guest stateroom head with a show stall.
All the way forward is the master stateroom with a center line, full sized berth.
Both staterooms have lighted, cedar-lined hanging closets and ample drawer storage.
1225 Island Avenue
San Diego, CA 92101
United States


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Williams II Gin is celebrates the 200th Anniversary of the discovery of the Antarctic Peninsular by Williams Smith of Blyth and the remarkable work of The Blyth Tall Ship Project. In this Special Edition, limited to 100 bottles, you can enjoy the smooth and unique taste of Alnwick Gin in the knowledge that £8.00 will be donated the the Blyth Tall Ship Project. A fantastic gift for anybody interested in Northumberland and Maritime History.
In 1819 Blyth native William Smith, Captain of the Williams, was blown south buy a storm and accidently discovered the land we now call the South Shetland Islands which eventually led them to the discovery of the Antartic Peninsular. 200 years later the Williams II is a 105 year old Oak built gaffed riffed ketch. At 36m in length she is almost exactly the same dimensions as the Williams, built over 100 years earlier.


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Prayer Requests
We have a vibrant prayer ministry comprised of individuals who are passionate about lifting up needs and concerns to our Amazing God. If you would like to submit a prayer request please fill out the form below. All prayer requests are confidential and will only be shared with members of the prayer team.
Do not worry about anything, but in everything by prayer and supplication with thanksgiving let your requests be made known to God. And the peace of God, which surpasses all understanding, will guard your hearts and your minds in Christ Jesus. Philippians 4:6-7


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Free Shipping On All NZ Orders Over $50 (Free to Australia & US for Orders Over $100)
Choose your preferred currency
Buy now online or at our Vape Shops for Mouth To Lung (MTL) Tanks. Vape Crew provides high quality Mouth To Lung (MTL) Tanks. We provide efficient shipping throughout New Zealand and Australia. Check out the rest of our Mouth To Lung (MTL) Tanks !
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Customer Feedback and Profile Of Drivescape Paving
Drivescape Paving are one of the finest driveway and patio contractors in County Dublin. We offer a range of affordable driveway and patio solutions including block paving, tarmac, gravel and concrete driveway in Dublin. We can build a range of patios using garden paving, patio paving and flagstones.
Drivescape Paving are a local family run business that covers all of County Dublin. We have completed hundreds of driveway and patio projects in north and South Dublin. Our team has built its reputation upon delivering quality workmanship at an affordable price.
We have a large team that is capable of tackling any sized driveway or garden area. We give 100% of our attention to each and every project, regardless if its a small or large job.
All our work is done to industry approved standards that is always adhered to. We have a close working relationship with both the main paving suppliers and tarmac suppliers to ensure we always get a quality product.
Drivescape Paving Reviews
Drivescape Paving offer a range of solutions for driveways and patios in Dublin including block paving, tarmac, gravel and natural stone installations. Servicing all of the County Dublin area.
User Review( votes)
- Driveway Installations (Paving, Tarmac, Gravel)
- Patio Installations (Natural Stone, Patio Slabbing)
- Garden Services (New Lawns, Artificial Grass)
- Brick Laying (Walls, Wooden Fencing)
- Driveway Repairs
- Resin Driveways/Patios
Drivescape Paving will always provide written quotations to help you understand the pricing of the project and what the work entails. It has built its reputation upon providing transparency to customers and delivering high quality workmanship.
Every step of the way, they consult their customers to ensure they are 100% satisfied with the development of their driveway and patio.
At Drivescape Paving, they provide a range of driveway solutions. These solutions apply to new driveways and driveway extensions.
Driveway Options:
- Block Paving
- Tarmac
- Gravel
- Concrete
- Resin Bond
Patio Options:
- Natural Stone Patios
- Patio Paving
- Garden Slabbing
- Flagstones
- Lawns
- Wooden Fencing
Get Drivescape Paving in Dublin today to build you a new driveway or patio. Options on laying new driveways, patios and gardens in County Dublin. Top quality workmanship at unbeatable prices.


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Deposits of exposed and embedded silver nanoparticles were grown on Si(100) and silica substrates by laser ablating high-purity silver and SiO2 targets in vacuum using a femtosecond Ti:sapphire laser delivering 45 fs pulses at 804 nm and 1 kHz repetition rate. The effect of the laser fluence and irradiation time on the obtained nanostructures was investigated using several fluences between 650 mJ/cm2 and 3.2 J/cm2 and deposition times in the range of 1-20 minutes. Optical response of the deposits was characterized using optical absorption spectroscopy and the surface morphology was studied by scanning electron microscopy (SEM). Samples with the optimal optical response were obtained by depositing three successive Ag/SiO2 bilayers at the main laser wavelength (804 nm) under vacuum at substrate room temperature. They were composed of silver nanoparticles with an average diameter of 6 nm and a narrow size distribution; each layer of these nanoparticles was then separated by silica layers of approximately 100 nm. The laser fluence and deposition time for Ag (SiO2) were 650 mJ/cm2 (3.2 J/cm2) and 1 min (10 min), respectively.
© 2014 Optical Society of AmericaFull Article | PDF Article
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Executive Summary
The Nigerian army carried out massive attacks over the course of 12 and 13 December 2015 which targeted the Islamic Movement of Nigeria in Zaria.
The operation resulted in the deaths of at least 1000 unarmed civilians, mostly IMN members, killed by gunshots attributed to soldiers. Hundreds more were injured. There were reports that the military blocked access to medical care for the injured and shot at and killed wounded persons. Witnesses and victims reported signs of widespread force and cruelty with allegations of looting, arson, mutilation, mass graves, torture, rape and other inhumane acts levelled at the
Nigerian army.
The supporting materials and eyewitness accounts further indicate that such massive acts of violence were carried out in the context of a widespread and systematic attack against the Nigerian civilian population. The violence unleashed on members of the IMN has a history and it is a history of premeditation.
There is also sufficient evidence to suggest an official plan by Nigerian government officials to cover up these crimes and indeed perpetuate the same.
IHRC is of the view that there are reasonable grounds to believe these crimes fall within the jurisdiction of the court in accordance with the Rome Statute. The crimes of the Nigerian army meet all the relevant criteria to warrant further investigation by the prosecutor of the International Criminal Court.
In view of the above, IHRC calls on the Prosecutor to open a preliminary
investigation proprio motu on the incidents reported.
This IHRC communication aims to provide a solid understanding of the events and crimes committed by the Nigerian Army, and it is based on eyewitness accounts and other supporting materials. The second part focuses on the legal parameters against which crimes against humanity are assessed, in respect of
articles 7, 12, 13, 15, 17 and 53 of the Rome Statute.
Based on the evidence collected so far, IHRC firmly believes that:
1. The crimes committed between 12 and 14 December 2015 in Zaria,
Kaduna State, by the Nigerian army amount to crimes against humanity.
2. The crimes committed by the Nigerian army meet all the necessary legal requirements to warrant a preliminary investigation by the ICC prosecutor.
Based on these grounds, IHRC therefore recommends the following:
1. The ICC prosecutor should open a preliminary examination into the
incidents that occurred between 12 and 14 December 2015 in Zaria, Kaduna State.
2. IHRC demands the Prosecutor expand its monitoring activities on Nigeria and the army's involvement in attacks against the IMN that occurred in the period between 2014 and 2015. Indeed, Nigeria is already under preliminary investigation by the Prosecutor in relation to Boko Haram.
3. IHRC calls on the ICC Prosecutor to issue a preventive statement saying
that he is monitoring the Nigerian crisis and is aware of the commission of crimes and warn the perpetrators about their criminal liability
To be continued


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Bill Woods and his Sodium Distortion motorcycle building buddies are not the run of the mill hobbyists. They want to go as fast or faster than anyone ever has on a 500cc motorcycle and when they and about 175 others gather at the Bonneville Speed Flats they get to find out who really is the fastest. When you want an a bike to go fast and an engine to turn at very high RPMs then you need to very best parts available, so Bill turned to Torque Transmission for L Series timing pulleys.
"We use pulleys to drive a supercharger on the motorcycle. The first nylon pulleys we got from Torque Transmission were used with a speed increaser to jump the RPM's from 7,0000 to about 42,000 RPM an the pulleys worked very well. We have used the nylon L Series pulleys to go to 63,000 RPM but had a catastrophic failure due to timing belt disintegration.
We have used aluminum pulleys but found that they would fail above 40,000 rpm where as the nylon pulley showed no ill effects. The aluminum pulley teeth would begin to "melt" or wear off at those speeds. We found that the nylon pulleys would dissipate the heat very well. The nylon pulleys outlast the belts" said Bill Woods.
These guys have a great story, so great in fact that there is a movie called 'Out of Nothing' coming out soon about their exploits. Check it out below.


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Job Description
A well-established, growing online business, which is investing heavily into its CRM system are looking for a Salesforce CRM Campaign Manager to provide strong, in-depth technical knowledge to push the company forward.
The company has recently introduced Salesforce CRM as its new system and is now looking for a Salesforce CRM Campaign Manager with strong technical Salesforce knowledge to take this forward as part of a growing team. With expert knowledge of the Salesforce Marketing Cloud, you will be responsible for managing and driving the strategy to grow the value of their extensive customer database. Linking customer journeys across the CRM, you will build campaigns in SFMC, including within the communications, and work with internal stakeholders to ensure consistency with customer communications. Performing key analysis and monitoring the campaign performances, you will develop and maintain the strategy to create constant improvements, from developing testing plans to reviewing all targeting. You will be responsible for completing, maintaining and updating a full customer segmentation analysis and managing the quality of data capture across all customer touch points.
A technical expert with deep, broad knowledge of Salesforce Marketing Cloud, you will be highly proficient in the following;
- CRM Systems
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This is a permanent vacancy


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RAMALLAH : Israeli forces rounded up six Palestinians in overnight raids carried out across the occupied West Bank, according to the military on Monday.
In a statement, the army said that the individuals had been detained for "suspected involvement in popular hostile activities".
It did not elaborate on the nature of these alleged "activities". The Israeli army carries out frequent arrest campaigns across the West Bank -- including occupied East Jerusalem -- on the pretext of searching for "wanted" Palestinians.
According to Palestinian figures, roughly 5,700 Palestinians -- including numerous women and children -- are currently languishing in Israeli detention facilities.


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Introgression from Domestic Goat Generated Variation at the Major Histocompatibility Complex of Alpine Ibex
The major histocompatibility complex (MHC), a crucial component of the defense against pathogens, contains the most polymorphic functional genes in vertebrate genomes. The extraordinary genetic variation is generally considered to be ancient. We investigated whether a previously neglected mechanism, introgression from related species, provides an additional source of MHC variation. We show that introgression from domestic goat dramatically increased genetic variation at the MHC of Alpine ibex, a species that had nearly gone extinct during the 18th century, but has been restored to large numbers since. We show that Alpine ibex share one of only two alleles at a generally highly polymorphic MHC locus with domestic goats and that the chromosomal region containing the goat-type allele has a signature of recent introgression. Our finding contradicts the long-standing view that ancient trans-species polymorphism is the sole source of the extraordinary genetic variability at the MHC. Instead, we show that in Alpine ibex introgression generated genetic diversity at a MHC locus. Our study supports the view that loci favoring genetic polymorphism may be susceptible to adaptive introgression from related species and will encourage future research to identify unexpected signatures of introgression.
Published in the journal:
. PLoS Genet 10(6): e32767. doi:10.1371/journal.pgen.1004438
Category: Research Article
doi: 10.1371/journal.pgen.1004438
The major histocompatibility complex (MHC), a crucial component of the defense against pathogens, contains the most polymorphic functional genes in vertebrate genomes. The extraordinary genetic variation is generally considered to be ancient. We investigated whether a previously neglected mechanism, introgression from related species, provides an additional source of MHC variation. We show that introgression from domestic goat dramatically increased genetic variation at the MHC of Alpine ibex, a species that had nearly gone extinct during the 18th century, but has been restored to large numbers since. We show that Alpine ibex share one of only two alleles at a generally highly polymorphic MHC locus with domestic goats and that the chromosomal region containing the goat-type allele has a signature of recent introgression. Our finding contradicts the long-standing view that ancient trans-species polymorphism is the sole source of the extraordinary genetic variability at the MHC. Instead, we show that in Alpine ibex introgression generated genetic diversity at a MHC locus. Our study supports the view that loci favoring genetic polymorphism may be susceptible to adaptive introgression from related species and will encourage future research to identify unexpected signatures of introgression.
The MHC is one of the most gene-dense regions and contains the most polymorphic functional genes in vertebrate genomes [1]–[3]. The major role of MHC gene products is the recognition of foreign peptides and their presentation to specialist immune cells in order to initiate an immune response [4]. Specific MHC haplotypes and MHC heterozygosity were shown to be associated with immunity to diseases [e.g. 5–9]. A higher allelic diversity at MHC loci is expected to be favored because individuals with a broader range of MHC sequences (binding and presenting a broader range of pathogenic peptides) should be able to more successfully fight diseases. However, the mechanisms generating and maintaining the extraordinary MHC diversity are not fully understood [reviewed in 10–12]. Three types of balancing selection, mediated by pathogen-driven or sexual selection, are usually invoked to explain MHC polymorphism: heterozygote advantage, negative frequency-dependent selection and fluctuating selection [11], [13]. Linked recessive deleterious mutations may additionally contribute to a pattern of balancing selection and may explain why MHC alleles are frequently more highly diverged from each other than expected otherwise [12]. While MHC alleles often exhibit high sequence divergence, balancing selection tends to even out allele frequencies among populations and hence such loci show lower population differentiation than neutral loci [14].
Balancing selection at a locus may predate speciation events and maintain a set of highly divergent alleles termed ancient trans-species polymorphism [15]. Adaptive genetic variation at loci under balancing selection is generally assumed to stem from standing genetic variation or mutations. However, adaptive genetic variation may also be generated through introgression, the gene flow between species [reviewed in 16]. Introgression at loci under balancing selection is expected to be favored because of the selective advantage of rare alleles [14], [16]. Therefore, loci under balancing selection are good candidates for adaptive introgression as shown for self-incompatibility genes in plants [17] and a coat pattern gene in animals [18].
Recently, introgression was proposed as an additional mechanism contributing to high levels of genetic diversity at the MHC [19]. Due to the significance of MHC variation for the defense against infectious diseases, introgression at the MHC is likely adaptive. Introgression from archaic humans was suggested to have shaped the human MHC [20]. MHC diversity in domestic mammals may have been augmented by introgression from wild ancestors [21]. Several MHC alleles were found to be shared between two newt species consistent with introgression among species [22]. However, direct evidence for recent introgression events at the MHC in wild species is lacking. Here we present evidence that introgression from domestic goat was an important source of MHC class II variation in Alpine ibex.
The Alpine ibex (Capra ibex ibex) is a species of wild goat occupying high-alpine niches of the European Alps spanning from Northern Italy and France to Slovenia. Several related ibex species are found in mountain ranges of Southern Europe, Central Asia, Northeast Africa, and the Arabian Peninsula (the Mountain goat of North America, Oreamnos americanus, belongs to a different genus). One of these species, the bezoar (Capra aegagrus) is the ancestor of the domestic goat [23]. Following near extinction during the 18th century due to overhunting, Alpine ibex were reintroduced to most parts of the European Alps from the only remaining population in Northern Italy (Gran Paradiso National Park). The reintroduction was very successful, and the species has recovered to more than 40'000 individuals living across the European Alps. Therefore, the Alpine ibex is considered a flagship species of the restoration of large mammals. However, the re-introduction caused several bottlenecks of less than 100 individuals, which substantially depleted genetic variability [24]–[26]. The depletion of genetic variability is particularly striking at the DRB locus of the MHC class II. Only two alleles (Caib-DRB*1 and Caib-DRB*2) were reported at the exon 2 of DRB in at total of 125 individuals [27], [28]. In comparison, both the domestic goat (C. aegagrus hircus) and its wild ancestor the bezoar (C. aegagrus) are highly polymorphic at this exon. We show that introgression from domestic goat is responsible for the fact that Alpine ibex are polymorphic at all at the exon 2 of the DRB locus, suggesting that introgression can be an important evolutionary force shaping the evolution of the MHC.
Results and Discussion
We extended the sequencing by Schaschl et al. [27] and Alasaad et al. [28] from 125 to 203 Alpine ibex from different populations and did not find additional alleles at the exon 2 of the MHC DRB locus. We found that the second allele (Caib-DRB*2) was identical to the Cahi-DRB*16 allele of the domestic goat first reported from the Japanese breed Shiba ([29], Genbank accession AB008361). We will refer to Caib-DRB exon 2 sequence variants (236 bp) as Caib-DRB alleles throughout the manuscript (see Figure 1). We found no recombinant between the two alleles in Alpine ibex, although recombinants at this locus were found in several related ungulate species [27], [30], [31]. This suggests that the two alleles are not both ancient alleles of the Alpine ibex DRB locus.
We investigated the geographic distribution of the Caib-DRB alleles by genotyping the microsatellite marker OLADRB1 (Figure 1A). OLADRB1 is directly adjacent to the exon 2 of DRB in even-toed ungulates [32] and is in complete linkage disequilibrium (i.e. diagnostic) in Alpine ibex [28 and this study]. We found substantial variation in Caib-DRB allele frequencies among 40 Alpine ibex populations (n = 754 individuals). The frequency of the allele Caib-DRB*2 ranged from 0% (most populations of Central and South-Western Swiss Alps) to 31% (North-Western Swiss Alps; Figure 1A and Table S1). Caib-DRB*2 was generally at a high frequency in the Eastern Swiss Alps (Figure 1A). In the founder population for the reintroductions, the Gran Paradiso National Park in the Italian Alps, the frequency of Caib-DRB*2 was 3%. The striking population structure observed at Caib-DRB reflects the complex reintroduction history shown by neutral markers [25]. Individuals were first brought from the Gran Paradiso to Swiss zoos. From these zoos, initial reintroductions established three wild populations across the Swiss Alps, which in turn served as source populations for subsequent reintroductions across Switzerland and elsewhere.
The origin of the Caib-DRB*2 allele in Alpine ibex
The MHC is known to harbor genetically divergent alleles within species [4], [33]. The two Caib-DRB alleles found in Alpine ibex were even more divergent than expected from related species (13.2% nucleotide and 22.6% amino acid difference, Figure 1B). In comparison, the mean pairwise nucleotide difference among MHC DRB exon 2 alleles found in domestic goats and their wild ancestors (bezoar) was substantially lower (8.6%, 95% confidence interval CI 1.8–15.0%; 17.8% amino acid difference, Figure 1B). Domestic sheep (Ovis aries) and Chamois (Rupicapra rupicapra) showed even lower mean allele divergence (Domestic sheep: nucleotide divergence 7.4%; 95% CI: 2.2–15.9% and amino acid divergence 15.0%; Chamois: nucleotide divergence 3.9%; 95% CI: 0.9–6.6% and amino acid divergence 9.5%; Figure 1B, Table S2). The high genetic divergence between the two Alpine ibex Caib-DRB alleles and the identity of Caib-DRB*2 to an allele identified in domestic goats may be indicative of introgression from domestic goats into Alpine ibex. The two species share a common ancestor 2–6 million years ago [34], [35] and hybrids, which can survive and breed have repeatedly been reported in the wild [36]. Alternatively, related species may show similarities at MHC alleles because of ancient trans-species polymorphisms caused by balancing selection [11], [15], [37], [38].
We assessed the likelihood that two species share identical MHC DRB exon 2 sequences by analyzing 112 sequences of length identical to the two Alpine ibex MHC DRB exon 2 sequences (236 bp) from eight species of the Caprinae subfamily comprising domestic goats and Alpine ibex. With the exception of the sequence shared between domestic goats and Alpine ibex, we found no shared alleles among species. However, we found evidence of shared sequences among species by including sequences of shorter minimum length (227 bp; n = 332) representing 11 species. Six pairs of species shared 13 alleles (see Table S3). For all except one of these species pairs, hybridization has either been observed or signatures of past hybridization between the species have been reported (see Table S3 for more details).
Hybrids between Alpine ibex and domestic goats were occasionally reported in the past and microsatellite analyses confirmed the existence of F1 hybrids (see Figures S1, S2 for more details). However, in a survey of Alpine ibex individuals, we found no evidence of recent hybrids based on genetic clustering analyses using the software STRUCTURE [39]. For this, we analyzed 30 neutral microsatellites in 1781 individuals [this study and 25] and 546 SNPs in 95 individuals. Thus, the presence of Caib-DRB*2 in Alpine ibex in extant populations is not solely due to recent hybrids.
Alpine ibex share nearly identical DRB intron and exon sequences with a domestic goat breed
Introgression is expected to generate highly similar sequence tracts between the donor and recipient species [16]. If introgression was the source of Caib-DRB*2, we predict that in addition to the DRB exon 2, intronic and other non-coding regions should be highly similar between Alpine ibex carriers of Caib-DRB*2 and domestic goats. In contrast, if ancient trans-species polymorphism was responsible for maintaining identical DRB exon 2 sequences, we predict that the surrounding non-coding regions should have accumulated significant sequence divergence between Alpine ibex and domestic goats. In order to distinguish between these scenarios, we sequenced four regions of the DRB gene. The MHC region is located on chromosome 23 in domestic goats (Figure 1C) and the 11 kb DRB gene is fully contained on the 136 kb goat reference genome scaffold2167 [40]. The four sequenced regions comprise a total of 2253 bp and covered the complete sequences of exons 3, 5 and 6, the complete intron 5 as well as partial sequences of introns 1–4 and a 3′ UTR sequence (Figure 2, see Supporting Text S1–S4 for full sequence alignments). We sequenced seven Alpine ibex homozygous for Caib-DRB*1, seven Alpine ibex homozygous for Caib-DRB*2 and five domestic goat individuals selected from a screening of diverse breeds. All seven Alpine ibex homozygous for Caib-DRB*1 were homozygous for the same haplotype at all four loci and were strongly differentiated from Alpine ibex homozygous for the Caib-DRB*2 allele (5.1% nucleotide divergence). The haplotype of Caib-DRB*1 Alpine ibex individuals did not show any close similarity to sequences found in domestic goats (Figure 2).
All seven Alpine ibex homozygous for Caib-DRB*2 were homozygous for the same highly distinct haplotype (Figure 2). We found that the domestic goat individual VBN.4 of the breed Valais Blackneck carried a very similar haplotype (99.8% sequence identity across 2253 bp). The haplotypes of VBN.4 and of Alpine ibex homozygous for Caib-DRB*2 differed only by five SNPs at exon 3, intron 3, exon 5, and the region comprising partial introns 4, 5 and 3′ UTR sequences (Figure 2). The domestic goat VBN.4 was homozygous for the DRB exon 2 Cahi-DRB*16, which is identical to Caib-DRB*2. The high similarity in both coding and non-coding regions of the DRB gene between the domestic goat VBN.4 and Alpine ibex homozygous for Caib-DRB*2 strongly suggests domestic goat breeds were the donors of DRB sequences that introgressed into Alpine ibex.
We tested for the presence of recombinant sequences at the MHC DRB. For this, we concatenated sequences of all four loci and used the recombination test based on the Φw-statistic [41]. The five domestic goat sequences showed significant evidence for recombination (64 informative sites; p<0.0001). In contrast, the two haplotypes associated to the Caib-DRB*1 and Caib-DRB*2 allele, respectively, did not show any evidence for recombination (116 informative sites; p = 1). This suggests that Alpine ibex haplotypes associated with Caib-DRB*1 and Caib-DRB*2 alleles, respectively, did not co-exist for a long period in the populations.
High expected heterozygosity extends to the chromosomal region surrounding DRB
A recent introgression event from domestic goats into Alpine is expected to lead to a chromosomal region of high expected heterozygosity in individuals carrying the introgressed allele. To characterize the genomic region surrounding the DRB gene, we performed restriction site associated DNA sequencing (RAD-seq). We genotyped 15 Alpine ibex homozygous for Caib-DRB*1 and 15 Alpine ibex carrying Caib-DRB*2 of which 14 were heterozygous for Caib-DRB*2. The sampling covered individuals from four populations. Additionally, we included nine domestic goat individuals (representing four breeds). The RAD sequences were mapped to the domestic goat genome [40]. We identified 258 polymorphic SNPs located between 22 Mb and 27 Mb on chromosome 23. Eighty-six of these SNPs were polymorphic among Alpine ibex. Individuals carrying Caib-DRB*2 showed high rates of expected heterozygosity in a region of about 750 kb surrounding the second exon of DRB (24.5 to 25.25 Mb) on chromosome 23 (Figure S3). Furthermore, we found high sequence similarities to domestic goat sequences in the same region (for a representative sample of genotypes and SNPs see Figure 3). At 7 out of 10 SNPs, we found an allele that was shared between domestic goats (Figure 3D) and Alpine ibex carrying Caib-DRB*2 (Figure 3C) but not with Alpine ibex homozygous for Caib-DRB*1 (Figure 3A). Similarly, the Alpine ibex GR0201 homozygous for Caib-DRB*2 was homozygous for a SNP allele that was only found in domestic goats and other Alpine ibex carrying Caib-DRB*2 at 6 out of 10 SNPs (Figure 3B). These observations are consistent with introgression at the MHC DRB locus and indicate that introgression has increased genetic diversity of Alpine ibex in this genomic region.
Alpine ibex haplotypes carrying Caib-DRB*2 are highly similar in the region surrounding the MHC region
We extended the SNP genotyping to 95 Alpine ibex individuals (four populations) and 177 domestic goat individuals (six breeds) using the 52 K Illumina Goat SNP Chip [42]. We identified 677 high-quality, polymorphic SNPs among Alpine ibex genome-wide. A total of 35 SNPs were located on chromosome 23 containing the MHC DRB locus. We found that allele SNP16397/G was diagnostic for Caib-DRB*2 in all 91 individuals, which were both sequenced at the exon and SNP genotyped (Table S4). We aimed to identify whether haplotypes carrying Caib-DRB*2 were genetically similar in the chromosomal region surrounding the MHC DRB locus. As the large chromosomal region was likely to contain recombined haplotypes, we constructed NeighborNet networks [43] based on three different sections of chromosome 23. One section covered 6 Mb of the MHC region containing the DRB locus and two sections covered either end of chromosome 23 (Figure 4B). We found that haplotypes containing the diagnostic allele SNP16397/G (associated with Caib-DRB*2) clustered strongly in the section covering the MHC DRB. However, we found no such association of haplotypes containing SNP16397/G at either end of the chromosome (Figure 4B). The tight clustering of haplotypes carrying Caib-DRB*2 in the DRB MHC region indicates a high relatedness among these haplotypes. This suggests that only few recombination events occurred between haplotypes carrying the Caib-DRB*2 and haplotypes carrying the Caib-DRB*1. If the alleles Caib-DRB*1 and Caib-DRB*2 were maintained as an ancient trans-species polymorphism, no or weak clustering of haplotypes associated with either allele would be expected. The clustering of the haplotypes and the increased expected heterozygosity in the individuals carrying the Caib-DRB*2 allele suggest high levels of linkage disequilibria.
Population genetic signature of introgression in the MHC DRB region
Introgression is expected to generate blocks of linkage disequilibrium due to the recent integration of sequences from another species. We used population and species level data for the analysis of linkage disequilibria in order to control for potential alternative sources of linkage disequilibrium [reviewed in 44]. We identified nearly complete linkage disequilibrium between Caib-DRB*2 and two proximal microsatellite markers (OLADRB1 and OLADRB2) covering a physical distance of 107 to 161 kb in the cattle and sheep genome, respectively (Figure 1C). Complete linkage disequilibrium with Caib-DRB*2 was observed for one of four alleles of the diagnostic marker OLADRB1 (allele OLADRB1/184; n = 156, Figure 1D and Table S4). Allele OLADRB1/184 was nearly completely associated with allele OLADRB2/277 (correspondence between the two markers in 705 out of 707 individuals).
Based on data from the 52 K Goat SNP Chip, we calculated linkage disequilibria among SNPs. We found a large block of high linkage disequilibrium containing the MHC DRB in Alpine ibex populations with high Caib-DRB*2 allele frequencies (e.g. population Cape au Moine: r2 = 0.85 across 2.1 Mb, Figure 4A; Table S5). Two smaller linkage disequilibrium blocks (0.2 and 0.4 Mb with r2≥0.85) were observed in the Alpine ibex population with the lowest frequency of the Caib-DRB*2 allele (population Weisshorn, Figure 5A and 6A; Table S5). The smaller blocks of linkage disequilibria in the Weisshorn population may also be explained by a general depletion of genetic diversity at the MHC DRB. Compared to Alpine ibex, blocks of linkage disequilibria were much smaller in domestic goats (e.g. breed Capra Grigia: 0.08 Mb with r2≥0.85) and linkage disequilibria decreased more steeply with pairwise distance between SNPs (Figures 5B and 6B). Domestic goats showed invariably small linkage disequilibrium blocks. Although introgression is expected to lead to increased linkage disequilibria, population subdivisions, genetic drift, and natural selection may also generate linkage disequilibria [5], [6], [44], [45]. Genetic drift is expected to create randomly distributed regions of high linkage disequilibria among chromosomes and populations [reviewed in 44]. Blocks of strong linkage disequilibria (r2>0.8) were generally shorter (0–0.5 Mb) on the chromosomes other than chromosome 23 of Alpine ibex (Figures S4A and S5) and strength of linkage disequilibria was lower for comparable pairwise distances between SNPs (Figure S4B and S4C). Hence, population subdivision and genetic drift are unlikely to explain the large block of high linkage disequilibria at the MHC region in Alpine ibex populations with a high frequency of the Caib-DRB*2 allele.
The MHC Caib-DRB*2 allele increased rapidly in frequency
Successful introgression of the Caib-DRB*2 allele into Alpine ibex populations would require a significant increase in frequency from the time point of the hybridization event to the extant frequency of the Caib-DRB*2 allele. A significant frequency shift of an allele is expected to leave a footprint of selection in the surrounding chromosomal regions. We aimed to test for evidence of positive selection acting on haplotypes containing Caib-DRB*2 using analyses of extended haplotype homozygosity (EHH). The non-recombined segment of haplotypes containing an allele under positive selection is expected to be much longer and less diverse than haplotypes containing alleles not under selection, because the latter have experienced recombination or mutation events. The EHH measures the length of such conserved haplotypes on both sides of a specified core SNP [46] and has been used to show evidence for selection at the human MHC [5]. For EHH = 1 (maximum) at a certain position, all haplotypes containing the SNP allele of interest are identical up to this position. Introgression events followed by a rapid frequency shift due to drift or selection, are expected to lead to long introgressed haplotypes (i.e. a high EHH over a long distance from the core SNP). The non-recombined part of introgressed haplotypes is expected to be much longer than that of non-introgressed haplotypes.
We found that EHH was substantial around SNP16397 (Figures 7A and S6). The related measure iHH [47], integrated on both sides of the core SNP, showed a similar pattern (data not shown). Alpine ibex haplotypes carrying SNP16397/G (the SNP allele diagnostic for Caib-DRB*2) were substantially longer and were less diverse than haplotypes carrying SNP16397/A (Figure 7A, left panel). Similarly, bifurcation diagrams showed that haplotypes carrying SNP16397/G showed fewer bifurcations that were at a greater distance from the core SNP than haplotypes carrying SNP16397/A. For the six goat breeds, measures of EHH and iHH were generally much lower (Figures 7B and S7). Furthermore, the haplotype diversity associated with either of the two alternative SNP16397 alleles was very similar. Thus, these analyses show that the Caib-DRB*2 allele rapidly increased in frequency and that only few recombination events occurred among haplotypes carrying either Caib-DRB*1 or Caib-DRB*2. The increase in frequency of Caib-DRB*2 could have been caused either by genetic drift and/or by positive selection for individuals carrying the introgressed allele. Positive selection is plausible because the introgression increased the genetic diversity at and around the MHC DRB, as evidenced by the fact that individuals lacking Caib-DRB*2 are mainly monomorphic in the region surrounding this locus (Figures 3A and S3).
Introgression as a source of MHC variation
Using a combination of Sanger and RAD sequencing, SNP chip and microsatellite data we found evidence for introgression at the MHC DRB gene in Alpine ibex. The DRB exon 2 comprises only two alleles with one allele being identical to an allele found in domestic goats. We found no recombinants between the two highly divergent alleles, while recombinants at this locus were found in several related ungulate species [27], [30], [31]. Alpine ibex homozygous for the goat-type allele showed nearly identical sequences (99.8%) to a breed of domestic goats over 2253 bp of coding and non-coding sequences surrounding the MHC DRB exon 2. Evidence of long nearly identical non-coding sequences shared between a domestic goat breed and Alpine ibex shows that introgression rather than ancestral trans-species polymorphism accounts for the MHC DRB polymorphism in Alpine ibex.
The chromosomal region in proximity to the MHC DRB locus was genetically highly related for the haplotypes carrying the goat-type Caib-DRB*2 allele. We found nearly complete linkage disequilibrium between the MHC locus and two proximal microsatellites covering more than 100 kb. Linkage disequilibria in proximity of the MHC DRB locus were strongest in Alpine ibex populations with a high frequency of the Caib-DRB*2 allele. On the contrary, linkage disequilibria were lower in a population with a lower frequency of Caib-DRB*2 and in domestic goat breeds. The extended haplotype homozygosity (EHH) was substantially higher in haplotypes carrying the Caib-DRB*2 than in haplotypes carrying the native Alpine ibex allele, suggesting a substantial increase in allele frequency since the original hybridization event that lead to this introgression. High linkage disequilibria, sequence clustering and increased EHH are consistent with introgression and a selective sweep. We suggest that these signals stem from both the initial introgression event and a subsequent Caib-DRB*2 frequency increase in Alpine ibex populations.
We identified a single introgressed goat allele (Caib-DRB*2) among all sampled populations across Switzerland and in the Gran Paradiso National Park, the founder population of all extant Alpine ibex populations. The most parsimonious explanation for the introgression of Caib-DRB*2 is that the introgression originated from a successful hybridization between domestic goats and Alpine ibex in the Gran Paradiso National Park prior to the reintroduction of Alpine ibex across the Alps. The domestic goat breed Valdostana bred in the vicinity of the Gran Paradiso National Park shows striking phenotypic similarities to Alpine ibex (http://eng.agraria.org). This is indicative of efforts to interbreed Alpine ibex with domestic goats in this region. We suggest that the Caib-DRB*2 allele was introduced to Swiss populations through animals in the captive breeding program at a period of historically low Alpine ibex population sizes. The low extant frequency of Caib-DRB*2 in the population of Gran Paradiso may be explained by the fact that the population passed through a bottleneck after animals were reintroduced to Switzerland [24].
As the domestic goat MHC DRB is highly polymorphic, multiple successful introgression events may have introduced different MHC DRB alleles into Alpine ibex populations. However, this is unlikely for several reasons. We found no hybrids in the surveyed populations suggesting that hybridization events were rare over the past decades or that hybrids had a lower fitness compared to Alpine ibex. Furthermore, some domestic goat MHC DRB alleles may not be of adaptive value for Alpine ibex and, hence, genetic drift may have prevented the successful establishment of such alleles.
Alpine ibex are a genetically impoverished species and were subject to considerable species conservation efforts. Introgression is generally considered a threat for species conservation: small populations of endangered species may be substituted with hybrid individuals and introgression may be maladaptive [48]. Introgression from domesticated species into their wild relatives is of particular concern as shown in the case of the American bison [49]. However, introgression from domestic species was shown to be adaptive in wolves [50] and Soay sheep [18]. MHC introgression from domesticated species may contribute to the genetic rescue of wild relatives.
We showed that introgression from domestic goats into Alpine ibex generated variation at the previously monomorphic MHC DRB locus. MHC DRB introgression in Alpine ibex is likely adaptive by broadening the MHC sequence repertoire and thereby conferring an improved immune response. The MHC is a susceptible genomic region for adaptive introgression because balancing selection is expected to favor introgression [17], [19] and alleles are likely to be compatible among species. Introgression may well be an underappreciated mechanism generating the extraordinary genetic diversity at the MHC [16]. Our study supports the view that a broad range of loci under balancing selection may be susceptible to adaptive introgression and will encourage future research to identify unexpected signatures of introgression.
Materials and Methods
We analyzed 754 Alpine ibex samples from 40 populations across Switzerland, six Swiss wildlife parks and a population from the Gran Paradiso National Park in Italy (Figure 1A). The sample size per population varied between 1–61 individuals (average n = 16, Table S1). Allele frequencies reported in the main text and on Figure 1 are based on populations with n>12. 707 individuals were used to study linkage between the two microsatellites OLADRB1 and OLADRB2 (see below). Samples were collected either as tissue, blood or hair. See [25] for detailed information on the populations and sampling procedures.
Sanger sequencing of DRB exon 2
We based our analyses on a total of 98 exon 2 sequences of the MHC DRB class II locus sequenced by Alasaad et al. [28]. In addition to previously published sequences, we sequenced 78 Alpine ibex individuals (Table S4) at the DRB exon 2. This locus is homologous to BoLA-DRB3 and Ovar-DRB1 of cattle and sheep, respectively (Figure 1C). A nested PCR was performed using the primer pair HL030 (located at the boundary of the first intron and second exon), HL031 and HL032 (both located at the boundary of the second exon and second intron, [27]). The 236 bp PCR product was Sanger sequenced on a 3730 DNA Analyzer (Life Technologies, Inc.). Samples of nine individuals were extracted and sequenced two times independently. Sequences were edited and manually corrected in Geneious, version 6.05 (Biomatters, Inc.). The MHC region of cattle and sheep is homologous to goat chromosome 23 and there is strong colinearity between goat and cattle chromosomes [40]. We verified the sequence homology using BlastN to search the NCBI Genbank database. The closest hits to our sequences were previously published DRB exon 2 sequences of Alpine ibex and domestic goats. The MHC class II DRB in domestic goats is located on chromosome 23 at 24.9 Mb and is fully contained on scaffold2167 [40]. Correspondence to BoLA-DRB3 and Ovar-DRB1 was verified by BlastN searching the DRB exon 2 sequence in the cattle and sheep genomes (Figure 1C).
Microsatellite genotyping
We genotyped all 754 Alpine ibex individuals at the microsatellite OLADRB1, known to be associated to the MHC region on chromosome 23 [28], [32]. A subset of 707 Alpine ibex individuals was genotyped at the microsatellite locus OLADRB2 located in the same region. OLADRB1 is directly adjacent to the second exon of MHC DRB [32]. The forward primer of OLADRB1 overlaps with the primers HL031 and HL032. OLADRB2 (also known as OLADRB, Genbank UniSTS: 251420) has been localized to the BoLA-DRB2 gene in cattle and is located at 107 kb from exon 2 of MHC DRB and OLADRB1 (161 kb in sheep, Figure 1C). See Table S6 for primer sequences and references. PCR conditions for OLADRB2 were as described in [25]. For OLADRB1, we used a reaction volume of 6 µl containing 1.5 µl (3–30 ng) of DNA template, 0.4 µM of both forward and reverse primers and 3 µl Qiagen Multiplex PCR Kit. PCR cycling conditions included an initial denaturation step at 95°C for 15 minutes. Microsatellite quality controls and genotyping procedures were followed according to [25].
Sanger sequencing of DRB regions surrounding exon 2
The DRB1 gene of domestic sheep was mapped to scaffold2167 of the reference genome of domestic goat. Primers were designed in introns 1 to 4 and 3′ UTR of exon 6 to amplify four loci including partial sequences of introns 1–4 and the complete intron 5 and the 3′ UTR of exon 6. In addition, the loci comprised complete sequences of exons 3, 5 and 6. For the PCR amplification we used a total reaction volume of 25 µl containing 0.5 µM of both forward and reverse primers, 0.2 mM of each dNTPs, 2.5 µl 10× Buffer and Taq Polymerase. PCR cycling conditions included an initial denaturation step at 94°C for 3 minutes, 35 cycles of 30 sec at 94°C, 30 sec at 54°C, 1 min at 72 °C and a final extension of 7 min at 72 °C. See Table S7 for primer sequences. A total of 2253 bp were sequenced according to the protocol described above.
Seven Alpine ibex homozygous for Caib-DRB*1 and seven Alpine ibex homozygous for Caib-DRB*2 were sequenced. Sequenced individuals were chosen according to their genotype at the microsatellite OLADRB1 as described above. Sixteen domestic goat individuals representing five different breeds were sequenced at the locus containing partial sequences of intron 2. Five individuals covering the sequence diversity found at intron 2 were chosen for sequencing at all four loci. Sequences were edited and manually corrected in Geneious, version 6.05. Heterozygous sites were called if homozygote individuals for each allele were identified.
RAD library preparation
RAD library preparation was performed according to [51] except for the following modifications. A total of 1.35 µg of genomic DNA of each sample was digested with the restriction enzyme Sbf1 (New England Biolabs) in a total volume of 50 µl (one hour at 37°C, heat inactivation at 65°C for 20 min, slowly ramp down {<0.1 °C/s}). For the P1 ligation, adapters containing a unique 6 bp barcode (3.5 µl of 100 nM stock prepared according to [51]), 0.5 µl T4 DNA ligase (New England Biolabs; 2,000,000 Weiss Units/ml) and 4.4 µl H2O were added to each sample and incubated at room temperature overnight. This was followed by heat inactivation at 65°C for 20 min and slow cool down to room temperature. Samples were pooled before shearing in a COVARIS (Duty Factor: 5%; Peak incidence: 105; Cycles per Burst: 200: Time: 75 s). Size selection (300–700 bp) of the purified DNA fragments was performed using a CALIPER. The excised DNA was purified and blunt-ended (New England Biolabs). 1 µl dATP and 3 µl Klenow enzyme (New England Biolabs) were added (30 min at 37°C) and P2 adapters (1 µl of 10 mM stock) were ligated (0.5 µl rATP of 100 mM; 1 µl of 2,000,000 Weiss U/ml T4 DNA Ligase, New England Biolabs). After purification ligation products were PCR amplified using Phusion High-Fidelity DNA polymerase in a total volume of 120 µl: 50.4 µl H2O, 60.0 µl Phusion High Fidelity Master Mix, 2.4 µl primers (10 µM), 4.8 µl library template. Amplification master mixes were divided into 6 separate 20 µl reactions: 98°C 30 s; 14 cycles {98°C 10 s, 65°C 30 s, 72°C 30 s}; final extension for 5′ at 72°C. All purification steps were performed using a MinElute PCR purification kit (Qiagen) according to the manufacturer's recommendations. The library was sequenced on an Illumina HiSeq 2000 platform (100 bp, paired-end).
The FASTX-toolkit was used for P1 barcode splitting (http://hannonlab.cshl.edu/fastx_toolkit/index.html) and Trimmomatic 0.30 (www.usadellab.org/cms/index.php?page=trimmomatic) was used for adapter and quality trimming. Reads were then aligned to the domestic goat reference genome [40] using Bowtie2 2.1.0 [52]. Genotypes were called using UnifiedGenotyper (GATK, version 2.6.5; [53], [54]) and filtered using VariantFiltration and SelectVariants (QD<2.0, MQ<30.0, −12.5>MQRankSum>12.5, FS>40.0, HaplotypeScore>12.0, ReadPosRankSum<−8.000, QUAL>30.0, AN>20).
PGDSpider version 220.127.116.11 [55] was used for data format conversions and to remove genotypes with a genotyping phred-scaled quality score lower than 20. PLINK v. 1.0.7 [56] was used for additional genotype filtering. We required a genotyping rate of SNPs>70% and a minor allele frequency>0.01. PLINK was also used to calculate expected heterozygosity. The R package {ggplot2} was used for data visualization.
SNP Illumina BeadChip genotyping and filtering
We used a recently developed Illumina InfiniumHD BeadChip [42]. The BeadChip comprises 53'347 SNP markers with known physical locations on the goat genome. We genotyped 96 Alpine ibex individuals from four populations (Albris, Cape au Moine, Rheinwald and Weisshorn; n per population 23–24, Table S5). Populations were chosen in order to cover different frequencies of the putatively introgressed MHC DRB allele (Table S1) and the three genetically distinct regions of Alpine ibex [see 25]. In addition, we genotyped 188 domestic goats covering six Swiss breeds: Bündner Strahlenziege, Capra Grigia, Saanenziege, Nera Verzasca, Kupferziege, Gemsfarbene Gebirgsziege (20–49 individuals per breed; see Table S5 for details on sampling).
We used GenomeStudio version 2011.1 for SNP calling and quality filtering. We used PLINK v. 1.0.7 [56] for further locus filtering. Twelve individuals (including one Alpine ibex) were discarded because we required a genotyping rate of at least 90% per individual. Furthermore, SNPs with a genotyping rate lower than 90% (2,817 SNPs) and a minor allele frequency in Alpine ibex below 0.01 (49,832 SNPS) were removed from the data set. Twenty-one additional SNPs were removed because of an observed heterozygosity of one, which may be due to the presence of a duplicated region. A total of 677 SNPs were retained with a genotyping rate of 98% in the remaining individuals. A minor allele frequency of 0.05 (570 SNPs) was applied for the linkage disequilibrium analysis, resulting in a genotyping rate of 99.6%. PGDSpider version 22.214.171.124 [55] was used for data format conversions.
Phylogenetic and population genetic analyses
We constructed haplotype networks of phased SNP data (fastPHASE version 1.4.0, [57]) based on the Neighbor-Net algorithm implemented in Splitstree 4.13.1 [43]. The networks were based on the uncorrected p distance using concatenated SNP loci. Pairwise linkage disequilibrium (LD; r2) estimates among SNPs were obtained using the R package {genetics}. Heatmaps of pairwise LD estimates were produced using the function LDheatmap in R package {LDheatmap}. In order to investigate the decay of LD over distance, a regression of r2 values against pairwise distance was plotted (Figure 6). The expected values of r2 were computed using non linear least squares (nls in R) as shown in the following function by [58].
Where n is the number of haplotypes, C = ρ * distance[bp] and ρ = 4Ner (population recombination rate). All LD analyses were performed for each Alpine ibex population and domestic goat breed separately.
The software STRUCTURE [39] was used in order to search for recent hybrids. See figure legends S1 and S2 for details on parameter values.
Measures of the extended haplotype homozygosity (EHH, [46], Figures 7, S6, S7) were calculated from phased data (fastPHASE version 1.4.0, [57]) using the R package {rehh} [59]. The same package was used for the bifurcation diagrams. We used physical distances for the EHH analyses and refrained from performing statistical tests, as we did not have access to genotyped Alpine ibex families and, hence, estimates of genetic distances.
MHC DRB sequence diversity analyses
For the comparisons of MHC DRB exon 2 sequences among species of the subfamily Caprinae, a BlastN of Caib-DRB*1 was performed in Geneious version 6.05 (Biomatters, Inc.). Sequences were aligned using MAFFT v7.023b [60]. We used the R function dist.dna {ape} (model = "raw", proportion of sites that differ between each pair of sequences, no mutation model) to calculate genetic distances between all pairs of sequences. As most published sequences were shorter than our sequenced DRB exon 2 fragment, we used a universal 227 bp sequence length to calculate percent identities. We used R to identify sequences shared among species. This comparison was done both for 236 bp (112 sequences unique within species) and 227 bp (332 sequences unique within species).
We tested for evidence of recombination at the MHC DRB using the Φw-statistic developed by [41] implemented in Splitstree4 [43], We used the default window size of 100 and k = 5 (Alpine ibex) and k = 3 (domestic goats).
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Seal Support Systems
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To provide maximum service life, a mechanical seal requires four things of the fluid in which it operates:
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Buffer/Barrier Media (Double Seals):
- When the pressure of the medium in the double seal is lower than the pressure in the seal chamber (stuffing box), seal engineers refer to it as a buffer fluid.
- When the pressure is higher in the seal than in the seal chamber, it is called a barrier fluid.
The most common buffer/barrier is water. It is readily available and compatible with most pumped media. Oils often are used where water would not be suitable (see Royal Purple FDA 22), care should be taken to use a double seal with an efficient pumping device or an external circulating pump as oils are more resistant to flow than water. Finally, an inert gas (typically nitrogen) can be used as a medium. The gas must be supplied at a constant pressure and must be clean and free from moisture. A high quality gas panel, such as AESSEAL® Gas 10 Panel, is required to support gas seals.
AESSEAL® manufactures a complete line of seal support systems, download the Catalog.
A Few of the Many API Seal Support Plans:
The API seal support plans shown below were selected to illustrate a few common API Plans. For a complete list including all API Plans, please visit the AESSeal website.