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by anniedavis2 on Jun 29, 2018 at 4:34 PM 1st Post

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I noticed last week that access to our externally hosted website is intermittent on our internal network. When trying to reach our website, we often receive this message:

We can occasionally access it, even from PCs and browsers that 5 minutes prior could not access it. I'm stumped as to what would cause intermittent time outs for only 1 website (access to all other websites that we try is normal).

Doing a tracert for www.worknetdupage.org traces the path all the way to the hosting service (a couple of hops time out every time). Pinging the site results in replies, with some intermittent time outs.

Using a proxy server or the Chrome Browsec VPN application for allows us to access the site every time and without issue. Disabling the proxy/VPN app and trying to access the site again results in more timed out errors. I assume that the firewall isn't the issue, because nothing shows up as "blocked" in the logs and we can still access the site intermittently. That being said, the network admin that retired last month hadn't updated the firmware on the firewall for at least 2 years. I've been running updates, and I thought that it had resolved the issue, as we had consistent access to the website for several hours yesterday. Then the intermittent access started up again.

I tried flushing the DNS cache on the router/firewall, the servers, and the workstations. I disabled AV software, windows firewall, made sure that a proxy server wasn't being used. I tried different browsers, different workstations, different DNS servers (OpenDNS, Google DNS). I got rid of the root hints and added Google (8.8.8.8/8.8.4.4) as forwarders.

I found some forums that talked about similar issues when your domain and website have the same name (our domain is worknetdupage.org, our hosted website is www.worknetdupage.org ), so I made sure that I had an A record for both www and (same as parent folder) with the correct IP.My assumption, based upon all of the forums that I've read, is that this is a DNS issue, but I don't know what else to try.

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We also attempted to identify nucleotide polymorphisms, but found only one polymorphism in a total of 3920 bp sequenced nucleotides, both upstream and downstream of the insertion site, in 23 flies of the Okayama population. These observations are consistent with previous work indicating that Hsp83 coding and regulatory regions show very little sequence polymorphisms [ 50 ].

The Hsp83 gene is located close to the breakpoint of the cosmopolitan inversion Inv(3L)P [ 51 ]. We wished to determine if any phenotypic effects of the Hsp83 mutation may have been indirect, caused by the inversion and its association (linkage disequilibrium) with the Hsp83 mutation. To this end, we scored the inversion Inv(3L)P polymorphisms in the two natural populations Okayama and Ivory Coast. We found no evidence of linkage disequilibrium with the Hsp83 alleles (Fisher's exact test, p > 0.05), and only rare inversion arrangements in flies with either Hsp83 genotype (See additional file 4 ). Thus, the phenotypic effects of Hsp83 mutations cannot be attributed to effects of the cosmopolitan inversion Inv(3L)P .

Figure 3

Mutant flies have lower relative fitness in a co-culture competition assay . We determined the relative fitness of mutant and wild-type individuals through a co-culture competition test lasting five generations at 25°C. For this test, we had obtained flies isogenic and homozygous for and from the Okayama, Tokyo, and Ivory Coast populations, as described in the main text. We seeded three co-cultures with an equal number of flies of the mutant and wild-type genotype in three parallel co-culture competition experiments started with individuals from the Okayama (filled diamonds), Tokyo (filled cycles), and Ivory Coast population (filled triangles). We seeded two further co-culture competition experiments with lower percentages of mutant flies (30 and 10 percent, open diamonds) from the Okayama population. Each seeding population comprised 100 flies. We genotyped fifty flies in each generation. Note the consistent decrease in allele frequencies in most lines as time progresses.

The second variant of the assay that we performed involved only individuals from the Okayama population. It started with different proportions of mutant individuals. Specifically, it involved two starting populations, each consisting of 100 flies, where 30 and 10 flies, respectively, were initially homozygous for the Hsp83 P allele. In the first population Hsp83 P allele frequencies decreased by 89.3 percent. In the second population they fluctuated around the initial 10 percent. Both populations had a low Hsp83 P allele frequency of 3.2 percent at the end of this experiment.

In sum, our observations suggest that in populations where mutant flies constitute a substantial fraction of the population, they are competitively inferior to wild-type flies, and their population frequency declines over several generations. The number of individuals we used, as well as the consistent genotype frequency changes we observe in our replicate competition assays make genetic drift an unlikely sole cause of the changes we see.

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