1 00:00:02,220 --> 00:00:06,150 Good morning and welcome to the insecticide resistance in mosquitoes webinar. 2 00:00:06,150 --> 00:00:11,950 Today's presentation will be hosted by Dr James Burgess at Cornell university and 3 00:00:11,950 --> 00:00:15,190 John Shepard of the Connecticut Agricultural Experiment Station. 4 00:00:17,170 --> 00:00:20,380 Hello everybody thank you for joining us today, I'm going to 5 00:00:20,380 --> 00:00:24,280 start this off by talking about pesticide resistance monitoring techniques in disease vectors. 6 00:00:25,970 --> 00:00:27,320 A few of the things we're going to cover today. 7 00:00:28,390 --> 00:00:35,110 I'll start out with; what is pesticide resistance and how does it emerge. We'll talk about how resistance 8 00:00:35,110 --> 00:00:40,060 bioassays diagnostics are created. How you determine the resistance status of field-collected mosquitoes. 9 00:00:40,060 --> 00:00:43,700 and how you actually use those results to inform your managment 10 00:00:43,700 --> 00:00:47,750 decisions. After that John is going to talk a little bit about collecting mosquitoes for 11 00:00:47,750 --> 00:00:53,170 resistance assays, and also techniques for rearing different mosquito species in the lab. 12 00:00:55,650 --> 00:00:59,100 So to start out why is this substance monitoring important? 13 00:00:59,100 --> 00:01:03,370 So we have a lot of different classifications of the insecticides but 14 00:01:03,370 --> 00:01:09,400 the ones you see here in green and blue are the insecticide clasifications 15 00:01:10,980 --> 00:01:14,920 that are generally used you for mosquito control. 16 00:01:14,920 --> 00:01:17,360 So there aren't that many, the ones in green are adulticides 17 00:01:18,400 --> 00:01:18,900 the ones in blue are larvicides. 18 00:01:21,380 --> 00:01:24,970 And then state regulations can actually further restrict the ability to cycle 19 00:01:24,970 --> 00:01:30,050 through these different pesticides so the actual number that can be used in 20 00:01:30,050 --> 00:01:34,880 a state may be even lower than what you're seeing here for example many states only 21 00:01:34,880 --> 00:01:40,640 have pyrethriods registered for vector control. So this means that 22 00:01:40,640 --> 00:01:45,550 resistant monitoring is necessary to maintain the efficacy of these products because we 23 00:01:45,550 --> 00:01:49,580 just don't have a lot of them so we have to we have to guard the ones that we do have. 24 00:01:50,670 --> 00:01:52,470 I have included some additional information 25 00:01:53,720 --> 00:01:56,540 from the EPA about what active ingredients are used for larval and adult control 26 00:01:58,790 --> 00:01:59,830 in the bottom here. 27 00:02:02,710 --> 00:02:07,220 Looking at the Northeast US, where the north east regional center is located 28 00:02:09,210 --> 00:02:12,510 many mosquito control agencies both use adulticides and larvicides 29 00:02:12,510 --> 00:02:18,140 in the field. This is from a survey that we deployed last year to determine 30 00:02:18,140 --> 00:02:22,950 what mosquitoe control in our region we're doing to control mosquito populaitons. 31 00:02:25,870 --> 00:02:30,360 From that same survey you can see the top adulticides are all pyrethroids 32 00:02:30,360 --> 00:02:35,180 and some states will use organophosphates, 33 00:02:36,560 --> 00:02:41,750 but the overwhelming majority of applications are one classification 34 00:02:41,750 --> 00:02:42,880 using one mode of action. 35 00:02:46,650 --> 00:02:52,060 There are and I will explain what these different mechanisms are and 36 00:02:52,060 --> 00:02:56,260 how they work in a minute, but you should know that we've already identified that 37 00:02:57,680 --> 00:03:02,440 multiple resistance mechanisms can affect pyrethriods, 38 00:03:02,440 --> 00:03:05,730 they can also affect organophosphates they maybe cross resistance but 39 00:03:05,730 --> 00:03:09,520 that's not always the case. If you look at esterases for example, 40 00:03:10,680 --> 00:03:14,370 just because an esterase, they are generalists esterase that affect everything and 41 00:03:14,370 --> 00:03:19,430 there were targeted esterase that my only affec one active ingredient. 42 00:03:21,520 --> 00:03:24,380 So looking at the resistance mechanisms we have 43 00:03:25,530 --> 00:03:29,240 3 basic classifications. 1st we have metabolic resistance. 44 00:03:30,770 --> 00:03:36,540 These involve the metabolic breakwdon of the insecticide itself. These include esterases 45 00:03:36,540 --> 00:03:41,431 monooxygenases, glutathion s-transferace. So for 46 00:03:41,431 --> 00:03:43,461 example I'm going to use the video games to explain this. 47 00:03:45,041 --> 00:03:49,181 Think of an oxygenase as packman, it will attack that permethrin or that 48 00:03:51,431 --> 00:03:55,841 malathion chemical, it will break it down into less harmful compounds and 49 00:03:55,841 --> 00:03:57,461 those less harmful compounds and those compounds 50 00:03:58,771 --> 00:03:59,271 wont necesarrily kill the insect. 51 00:04:00,961 --> 00:04:05,151 Altered site resistance is another mechanism of resistance for 52 00:04:05,151 --> 00:04:07,391 example knock down resistance or modified ACHE. 53 00:04:10,132 --> 00:04:10,982 This so 54 00:04:10,982 --> 00:04:16,312 generally when a pesticide impacts or attacks pill a cellular bonding site there has to be 55 00:04:16,312 --> 00:04:21,032 somewhere where that pesticide can attach to the cell or to the cellular mechanism. 56 00:04:22,903 --> 00:04:23,623 Under 57 00:04:25,511 --> 00:04:28,251 sustained selective pressure 58 00:04:28,251 --> 00:04:31,831 that site can actually physically change configuration. 59 00:04:32,901 --> 00:04:40,951 and change shape so that pesticide can no longer bind to that area making him very sad. 60 00:04:42,821 --> 00:04:48,252 There's also penetration resistance so in order to get 61 00:04:48,252 --> 00:04:53,502 into the body of the insect pesticide has the penetrate the cuticle 62 00:04:57,553 --> 00:05:01,373 over time with selection, thicker 63 00:05:01,373 --> 00:05:06,933 cuticle can be developed that will less of the pesticide to get into the body of the insect. 64 00:05:09,034 --> 00:05:12,354 These are the 3 primary mechanisms for 65 00:05:12,354 --> 00:05:16,454 resistance and then there can be more specific versions of these like an 66 00:05:16,454 --> 00:05:21,234 esterase that only effects permethrin or an esterase that affects a broad range of 67 00:05:23,656 --> 00:05:25,516 pesticide active ingredients. 68 00:05:28,898 --> 00:05:31,348 So when we think about how resistance emerges 69 00:05:32,479 --> 00:05:34,319 first you have to have resistance in the natural population, 70 00:05:37,419 --> 00:05:40,479 this may be a gene that's expressed in a low level. 71 00:05:42,010 --> 00:05:44,940 But when you add pressure 72 00:05:44,940 --> 00:05:48,970 from your pesticide application you kill off the susceptible individuals and 73 00:05:48,970 --> 00:05:52,280 you can allow those resistant individuals to start competing 74 00:05:53,310 --> 00:05:58,252 in the field. Over time, those resistant individuals will 75 00:05:58,252 --> 00:06:02,802 start to take over the population and you will have fever susceptible individuals. 76 00:06:02,802 --> 00:06:06,192 The time scale can vary here, this may be your 2nd applications or it 77 00:06:08,242 --> 00:06:10,032 could be your 20th applicantion and we will talk about some of the factors that will affect 78 00:06:11,953 --> 00:06:17,313 how long it takes for application to allow resistance to emerge at the end. 79 00:06:19,891 --> 00:06:22,431 Also I want to define the difference between resistance vs 80 00:06:24,621 --> 00:06:29,911 susceptibility. Susceptibility is usually determined by comparing mortality curves from 81 00:06:31,031 --> 00:06:36,921 susceptible colony in a field collected colony or a field collected strain rather. So 82 00:06:36,921 --> 00:06:42,081 susceptibility curve is if you look at the axis here you have a mortality and 83 00:06:42,081 --> 00:06:45,751 pesticide concentrations. So as pesticide concentrations is going up, 84 00:06:45,751 --> 00:06:50,471 mortality increases until it flatlines at this threshold, 100% mortality. 85 00:06:53,121 --> 00:06:55,741 S shaped curve and 86 00:06:55,741 --> 00:07:00,011 you're going to compare that curve with another one built with field specimens. 87 00:07:01,851 --> 00:07:07,111 So we would say that this field colony here is less susceptible because 88 00:07:07,111 --> 00:07:10,571 fewer are dying at the same concentration compared againt the mortality. 89 00:07:12,971 --> 00:07:17,301 Resistance is defined by a threshold diagnostic concentration 90 00:07:17,301 --> 00:07:21,521 dose, time, it can be a resistance ratio, they're all based on the susceptibility curve. 91 00:07:25,934 --> 00:07:30,664 In our case for our program we've been using LC 99, this is also what the WHO uses for 92 00:07:30,664 --> 00:07:31,944 many of their bioassays. 93 00:07:33,126 --> 00:07:38,146 This is the point at which 99 percent, a concentration at which 99 percent of the individuals 94 00:07:38,146 --> 00:07:43,577 from the susceptible colony would die. So let's say you have 50 percent mortality at that 95 00:07:43,577 --> 00:07:48,998 point for a field specimen or for a field population 96 00:07:48,998 --> 00:07:53,248 that would be resistant population and we'll talk about scaling resistance in a minute aswell. 97 00:07:55,810 --> 00:07:58,140 Resistance ratios are also an option so 98 00:07:58,140 --> 00:08:00,860 this will be a comparison of LC99 and the error around them, because there is always error 99 00:08:02,360 --> 00:08:07,000 involved in these measurements, but in order to do that 100 00:08:07,000 --> 00:08:11,590 you need to collect enough specimens to construct an entire curve which 101 00:08:11,590 --> 00:08:16,510 requires quite a few more specimens than you would normally collect to test 102 00:08:19,400 --> 00:08:21,140 LC 99 or a single value and in field populations 103 00:08:23,080 --> 00:08:25,800 there can varaibility in those curves requiring more time. 104 00:08:29,300 --> 00:08:32,660 Looking at the susceptibility curve this is our bti susceptibility curve, 105 00:08:34,290 --> 00:08:39,150 for Culex pipiens, you can see I've ploted all the individual points here, each of those 106 00:08:39,150 --> 00:08:43,230 points represents a cup with 15 larvae in it so 107 00:08:43,230 --> 00:08:46,410 you can see it takes thousands of larva to create one of these curves and 108 00:08:46,410 --> 00:08:50,740 that's because you get a fair bit of variability so you need to 109 00:08:52,170 --> 00:08:57,762 continue to sample increase replication to overcome that variability and 110 00:08:57,762 --> 00:09:02,322 get a good estimate of your probit curve. Probit analysis is the kind of 111 00:09:02,322 --> 00:09:07,002 regression used to analyze dose response curves this can be used to 112 00:09:07,002 --> 00:09:12,182 determine the pesticide concentration at which point agiven percentage of mosquitoes would die. 113 00:09:15,383 --> 00:09:16,753 There's also always errors associated with this estimate, again 114 00:09:16,753 --> 00:09:20,913 that is why you need to need to increase replication. 115 00:09:20,913 --> 00:09:25,703 If you're experimental design is sound, as you increase replication error should decrease. 116 00:09:27,093 --> 00:09:30,283 We are actually developing some 117 00:09:30,283 --> 00:09:35,713 freeware guidelines that would allow people to do these calculations easily 118 00:09:35,713 --> 00:09:38,673 we hope to have those on our website within the next few months. 119 00:09:41,666 --> 00:09:46,736 Using this probit curve you're going to define your threshold, your LC 99 you can use 120 00:09:46,736 --> 00:09:52,206 LC95 although they are going to detect a lower level of resistance. 121 00:09:55,520 --> 00:09:57,400 When we talk about defining resistance. 122 00:09:58,630 --> 00:10:02,000 For larvicidal bioassays or any assay that is using an LC 99 123 00:10:03,660 --> 00:10:07,360 you can kind of scale this way for a program WHO 124 00:10:07,360 --> 00:10:09,040 uses a similar technique as well. 125 00:10:11,040 --> 00:10:12,670 So we test the population at a 126 00:10:12,670 --> 00:10:17,810 single concentration the LC99 if less than 90 percent of the 127 00:10:18,870 --> 00:10:22,510 individuals die in that sample we call that a low level resistant. 128 00:10:23,911 --> 00:10:28,872 population. Then we scale it so you have to follow up 129 00:10:28,872 --> 00:10:32,882 if you do detect low level resistance with a moderate level resistance test, five times the LC99 and if needed 130 00:10:32,882 --> 00:10:37,522 a hihg level resistance test, about ten times the LC99. 131 00:10:40,184 --> 00:10:42,564 Some of you will be familiar with the bottle bioassay, its the CDC 132 00:10:43,674 --> 00:10:45,664 method that is most commonly used in states. 133 00:10:48,054 --> 00:10:53,134 This is a diagnostic time rather than a diagniostic dose or concentration so 134 00:10:53,134 --> 00:10:55,124 instead of the threshold being 135 00:10:56,444 --> 00:10:59,094 pesticide concentration itss a time threshold. So 136 00:10:59,094 --> 00:11:01,564 in this case let's say the threshold is 30 minutes. 137 00:11:02,965 --> 00:11:07,705 If you have less than 90 percent mortality at that diagnostic time we call it low-leve resistance and 138 00:11:07,705 --> 00:11:11,925 then again you can scale with time this time which is convenient because you can 139 00:11:11,925 --> 00:11:16,845 do it all in one test you don't have to run multiple tests so 2 times 140 00:11:16,845 --> 00:11:20,575 that threshold whould be moderate resistance and 3 times a threshold would be high level resistance. 141 00:11:24,440 --> 00:11:27,000 Some of you may have heard of the who tube test. 142 00:11:28,590 --> 00:11:32,220 This is a contact assay the CDC bottle bioassay. 143 00:11:33,390 --> 00:11:37,950 Two tubes divided by a divider 144 00:11:37,950 --> 00:11:41,690 there is filter paper in here its a exposure tuber where hte mosquitoes 145 00:11:43,640 --> 00:11:49,150 are allowed to come into contact with a treated piece of paper for an hour and then 146 00:11:49,150 --> 00:11:54,600 they are transferred to this holding tube in top and held for 24 hours after which 147 00:11:55,750 --> 00:11:59,060 mortality is calculated. These are based on LC 99 148 00:12:00,300 --> 00:12:06,000 and again they scal much like ours 5 times 10 times the concentrations for 149 00:12:06,000 --> 00:12:10,692 higher levels of reistance. I've also included a website where you can 150 00:12:10,692 --> 00:12:13,182 find information on ordering kits if you want to try it out. 151 00:12:16,512 --> 00:12:21,052 With this and the CDC bottle bioasssay its very important you use the most 152 00:12:21,052 --> 00:12:28,492 current guidelines specially if you are ordering a kit because they do update them and sometimes 153 00:12:28,492 --> 00:12:34,392 the old guideline that you're using will match with the supplies that you have been sent. 154 00:12:35,592 --> 00:12:37,052 Usually they will send you a upadated guidline in the kit. 155 00:12:40,593 --> 00:12:43,843 CDC bottle bioassay I just wanted to show you 156 00:12:43,843 --> 00:12:45,853 many of you are going to be familiar with this already. 157 00:12:47,143 --> 00:12:50,333 This uses treated bottles, 250 mml bottles. 158 00:12:53,094 --> 00:12:57,274 And its a contact assay so mosquitoes are allowed to fly in the bottle unitl they die over time, 159 00:12:57,274 --> 00:13:00,854 so you are checking at given time points, start with 5 minutes and 160 00:13:00,854 --> 00:13:06,375 after 15 minutes it's every 15 minutes. I just wanted to point out so 161 00:13:06,375 --> 00:13:11,355 let's look at permitherin you can see that the concentration is the same for 162 00:13:11,355 --> 00:13:14,535 all species but the is what changes for each species. 163 00:13:18,700 --> 00:13:23,690 CDC bottle bioassay is fairly easy to run the procedure is simple. 164 00:13:24,830 --> 00:13:29,590 You only need one test to scale the level of resistance which is excellent but 165 00:13:29,590 --> 00:13:33,640 if you do detect resistance it takes a lot of time and effort to collect those data and 166 00:13:33,640 --> 00:13:36,110 also a lot of time and effort to clean the bottles after you're done. 167 00:13:38,110 --> 00:13:41,540 And sometimes the level of resistance you detect is difficult to 168 00:13:42,690 --> 00:13:46,850 connect to fiedl efficacy, although some people are working on 169 00:13:46,850 --> 00:13:48,550 connecting those values better in the future. 170 00:13:50,851 --> 00:13:55,961 WHO test its only checked once every 24 hours it's a little faster in that way. 171 00:13:55,961 --> 00:14:00,141 The diagnostic doses are based on susceptibility curves that are published. 172 00:14:01,441 --> 00:14:03,141 The kits are a little difficult to obtain in the states sometimes. 173 00:14:04,221 --> 00:14:05,451 if they're low on supplies they will 174 00:14:07,661 --> 00:14:11,771 preferentially send kits to more impoverished countries than the US. 175 00:14:13,551 --> 00:14:17,001 You can try to build them yourself as well that takes tools. 176 00:14:18,821 --> 00:14:23,061 You do also have to run multiple tests to detect where to scale 177 00:14:23,061 --> 00:14:24,491 different levels of resistance. 178 00:14:27,540 --> 00:14:29,740 Larvicide bioassays are a little more involved. 179 00:14:31,550 --> 00:14:37,590 So I will run you through our procedure again an adaptation of the WHO prodecudre so we 180 00:14:37,590 --> 00:14:43,260 used to wax-lined paper cups and you can see some pictures on the bottom here is the bioassay setup. 181 00:14:45,040 --> 00:14:48,820 One of the cups is filled with 74 ml of DI water and 50 mg of fish food. 182 00:14:48,820 --> 00:14:52,020 If you dont have a scale that goes to milligrams its 183 00:14:52,020 --> 00:14:57,780 the quarter of a fish food pellet. You want to make sure density of larvae and the amount of 184 00:14:57,780 --> 00:15:03,220 food that they are fed is consistent becasue that will affect mortality and survival. 185 00:15:04,300 --> 00:15:08,420 We add 15 fourth instar larvae to the cup. place the cup small cup into 186 00:15:08,420 --> 00:15:12,400 the larger cup, add the larvicide or active ingredient you're interested in. 187 00:15:13,580 --> 00:15:18,230 And then place a fine piece of fabric over the top of the larger container. 188 00:15:19,400 --> 00:15:23,080 This allows adults to merge in the cup so you can count them, special with methoprene. 189 00:15:24,320 --> 00:15:28,140 you can count the number of adults emerged rather than mortality. 190 00:15:32,273 --> 00:15:39,355 In order to build a susceptibility curve you need generally between 4 and 191 00:15:39,355 --> 00:15:45,165 6 replicates and 5 to 6 concentration. 192 00:15:46,995 --> 00:15:50,335 Finding these concentrations can take a little bit of extra time, its 193 00:15:50,335 --> 00:15:51,995 actually what we are working on right now. 194 00:15:53,225 --> 00:15:56,325 You have to do what I call range finding usually start 195 00:15:56,325 --> 00:15:59,515 at a very very high concentration and do one to ten dilutions 196 00:15:59,515 --> 00:16:04,955 until you find those points where all the larvae are dead or 197 00:16:04,955 --> 00:16:10,295 all the larvae are alive and then if you do 1:2 or 1:5 dilutions between those points. 198 00:16:12,676 --> 00:16:16,236 This does take a little time but it is it is necessary to 199 00:16:17,396 --> 00:16:21,356 figure out your concentration range. You can also look in the literature and 200 00:16:21,356 --> 00:16:26,516 sometines find that there, althought with biopesticides that does vary stock to stock so going to have to play wiht it a little bit. 201 00:16:28,550 --> 00:16:33,110 When running these trials you also need a negative control, this is the control with just 202 00:16:33,110 --> 00:16:39,040 acetone or just DI water or whatever you use as a solute. 203 00:16:41,090 --> 00:16:42,280 Sorry, to make your solute 204 00:16:43,520 --> 00:16:47,070 added so that you can make sure especially with again methoprene can be 205 00:16:47,070 --> 00:16:50,140 a 7 to 9 day trial you want to 206 00:16:50,140 --> 00:16:53,460 make sure that there isn't just natural mortality over that time so that you can correct for it. 207 00:16:55,041 --> 00:16:59,591 Once you have those data you can conduct the probit analysis and determin your diagnostic dose, 208 00:16:59,591 --> 00:17:03,991 again we should have guidlines for that to help people to figure out how to do that 209 00:17:03,991 --> 00:17:05,071 in the next few months. 210 00:17:07,722 --> 00:17:11,852 And once you have that concentration you can test field population 211 00:17:11,852 --> 00:17:16,472 at that one concentration, you dont have to build the curve every time, this allows you to use 212 00:17:16,472 --> 00:17:21,492 far less and test much more broadly than you would if you had to build 213 00:17:21,492 --> 00:17:26,152 the curve for each population we are testing. You have to run a negative control along with this to make sure that it isnt just 214 00:17:27,762 --> 00:17:30,242 natural mortality over the time period of the trial. 215 00:17:36,093 --> 00:17:40,433 For collecting mosquitoes for assays you can collect at different life stages. 216 00:17:42,323 --> 00:17:45,993 Adult collection can be advantageous if you are testing for adulticides because 217 00:17:45,993 --> 00:17:50,283 you don't have to hold them for very long but unfortunately you don't know the age 218 00:17:50,283 --> 00:17:53,713 with body condition you can't test larvae without rearing them 219 00:17:55,103 --> 00:17:58,123 and you need to identify each individual which takes a lot of time. 220 00:17:59,223 --> 00:18:00,783 Larval collection is another option. 221 00:18:02,225 --> 00:18:06,545 If you allow them to emerge as adults you know the age of the adults which is good 222 00:18:06,545 --> 00:18:11,075 but you don't know if you're testing for larvicides, you don't know the age of larvae, and 223 00:18:11,075 --> 00:18:15,835 each individual has to be identified again and it requires space for rearing. 224 00:18:19,610 --> 00:18:22,900 The best option for most species is egg collection, 225 00:18:23,900 --> 00:18:28,670 if you're collecting a species like Culex pipiens that lays an egg raft you can allow 226 00:18:28,670 --> 00:18:33,200 you can isolate each egg raft and then just identify one or 2 larvae from the egg raft and you 227 00:18:33,200 --> 00:18:37,900 know the species of the entire egg raft which saves you a lot of time in species identification. 228 00:18:38,950 --> 00:18:42,620 You also know the age of the mosquitoes because you watch the hatch. 229 00:18:44,050 --> 00:18:46,290 And it does require some space for rearing. 230 00:18:48,001 --> 00:18:52,441 As you'll see when John speaks it's not a lot of space and the materials can be fairly 231 00:18:52,441 --> 00:18:53,481 simple which is excellent. 232 00:18:57,515 --> 00:18:59,475 So just a few factors that can affect 233 00:19:01,075 --> 00:19:05,667 the selection of pesticide resistance in your 234 00:19:05,667 --> 00:19:10,457 specific population you want to think about genetic factors. So these are you 235 00:19:10,457 --> 00:19:15,477 know the dominance in frequency of the ressitance gene in the population of whether 236 00:19:15,477 --> 00:19:21,307 those genes are actually expressed and very importantly whether the individuals 237 00:19:22,868 --> 00:19:25,378 with the resistance genes can compete against those without them. 238 00:19:27,679 --> 00:19:31,719 That will determine whether or not when removed the pressure 239 00:19:33,869 --> 00:19:35,909 the susceptible popoulation starts outcompete the resistant population again. 240 00:19:40,660 --> 00:19:44,480 They're also operational considerations these are a lot of things you can 241 00:19:44,480 --> 00:19:48,620 actually control, so the chemical nature of the pesticide and 242 00:19:48,620 --> 00:19:52,380 what has been used historicaly. If you always used pyrethriods 243 00:19:53,720 --> 00:19:56,130 that increases the chance that you're going to see resistance in that population. 244 00:19:57,370 --> 00:19:59,170 Persistence of residues 245 00:20:00,230 --> 00:20:05,830 How strong the application is in the 1st place the life stages that are targeted. 246 00:20:07,500 --> 00:20:12,320 The different modes of application, just how effective they are and how effective they are to treat 247 00:20:12,320 --> 00:20:16,690 and reach the mosquitoes themselves and whether you're actually rotating chemicals or 248 00:20:16,690 --> 00:20:20,320 modes of action. You also have biological factors. 249 00:20:21,560 --> 00:20:25,600 Generation time and reproductive output, so how quickly they reproduce and how many 250 00:20:25,600 --> 00:20:28,780 babies they have essentially, whether they mate once or 251 00:20:28,780 --> 00:20:33,590 multiple times, this is related to reproductive output, distance travelled or 252 00:20:33,590 --> 00:20:38,140 how connected different populations are so if you have an isolated population. 253 00:20:40,551 --> 00:20:43,951 and you carpet bomb it and you see resistance emerge 254 00:20:45,621 --> 00:20:49,971 that resistance may spread in that population but it may not affect aneighboring area. 255 00:20:52,484 --> 00:20:53,974 And then you have refugee or 256 00:20:53,974 --> 00:20:59,924 shelter places behavior mosquitoes are hiding from your pesticide aplication. 257 00:21:01,334 --> 00:21:05,034 And on the environment you're spraying and the species of mosquitoe target. 258 00:21:07,254 --> 00:21:10,494 So when you think about managing pesticide reistnace kinda the 1st thing to do is 259 00:21:10,494 --> 00:21:16,536 start monitoring for it in the field. The best way to do this without 260 00:21:16,536 --> 00:21:20,186 spending all of your time doing this is to target the subset 261 00:21:20,186 --> 00:21:24,266 a subset of sites that you know experience high levels of control activity. So 262 00:21:24,266 --> 00:21:28,276 you know where you're targeting your control activities, go collect mosquitoes from 263 00:21:28,276 --> 00:21:32,956 those sites and test those sites for products you're using and 264 00:21:32,956 --> 00:21:38,438 if possible the products that you could also use so alternative products. 265 00:21:41,899 --> 00:21:46,379 If you start to see developing resistance this would be 96 to 90 percent mortality 266 00:21:46,379 --> 00:21:51,889 in your tests you want to go out and re-test old sites include and include a few new 267 00:21:51,889 --> 00:21:57,549 locations so you want to make sure that your test was accurate. 268 00:21:59,840 --> 00:22:03,680 That resistance that you detected is still maintained in those sites that you already 269 00:22:03,680 --> 00:22:07,090 tested and then you want to test new sites to see if that resistance is 270 00:22:08,100 --> 00:22:08,690 more popular 271 00:22:09,720 --> 00:22:11,260 localized population effect or 272 00:22:11,260 --> 00:22:14,640 if it's actually covering a large portion of your county or state. 273 00:22:18,002 --> 00:22:20,182 If you start to detect resistant mosquitoes 274 00:22:21,512 --> 00:22:24,732 less than 90 percent mortality this is where you want to 275 00:22:24,732 --> 00:22:29,422 look at it intensity testings so figure out is it just low level resistance that 276 00:22:29,422 --> 00:22:33,702 you're seeing kind of cross ranged that may not affect efficacy and 277 00:22:33,702 --> 00:22:35,312 efficacy trial could be helpful here. 278 00:22:37,305 --> 00:22:40,815 You also might if you are starting to detect moderate or high level resistance 279 00:22:40,815 --> 00:22:46,775 you may want to consider alternate options let me let me talk a little bit about those. 280 00:22:46,775 --> 00:22:51,255 So a few general options you have management by moderation using lower dosage 281 00:22:51,255 --> 00:22:55,865 a number of aplications, limiting the amount of pesticide you are putting out into the environment 282 00:22:55,865 --> 00:23:00,355 using chemicals that are short lived so that it can degrade quickly so again lest contact. 283 00:23:02,145 --> 00:23:07,095 In trying to treat a targeted area so that you're not broadcasting in 284 00:23:07,095 --> 00:23:11,635 places where mosquitoes are not high at high densities. 285 00:23:12,755 --> 00:23:16,185 So another option is kind of the opposite managment by saturation. 286 00:23:17,445 --> 00:23:21,775 So the idea here is that you're you're basically carpet bombing. 287 00:23:23,405 --> 00:23:27,715 Eliminating mosquitoes that have resistance genes at low levels before it can emerge 288 00:23:27,715 --> 00:23:31,176 in the populaiton, this can be an issue if 289 00:23:31,176 --> 00:23:35,286 if resistance is already starting to emerge put fuel in the fire and cause it to explode. 290 00:23:37,016 --> 00:23:41,958 So this is something I don't generally suggest you can also 291 00:23:41,958 --> 00:23:44,798 use synergists in the field to 292 00:23:45,868 --> 00:23:47,548 increase the effectivity of your pesticide aplication. 293 00:23:50,621 --> 00:23:54,461 Some of the better options involve management by multiple attackss a mixture of 294 00:23:54,461 --> 00:23:56,841 chemicals, rotation chemicals, mosaics. 295 00:23:57,971 --> 00:23:59,091 I will explain what I mean by that. 296 00:24:00,191 --> 00:24:02,781 So rotation would be switching between different 297 00:24:02,781 --> 00:24:06,371 products with different modes of action.This could be two adulticides an 298 00:24:06,371 --> 00:24:11,061 organophosphate and a pyrithriod it coul also be a pyrithriod and a organopesticide. 299 00:24:12,511 --> 00:24:15,431 You just want to make sure you're not deploying the same products 300 00:24:15,431 --> 00:24:19,261 all the time there are limitations whats registered in what state so 301 00:24:19,261 --> 00:24:22,991 it's important to figure out what the rules are and work within those constraints. 302 00:24:24,871 --> 00:24:28,531 You want to if you use the strategy want to pair with a resistance monitoring for 303 00:24:28,531 --> 00:24:31,521 all products to detect emerging resistance so 304 00:24:31,521 --> 00:24:35,381 that you know that your alternative product will be effective if you have to switch to it. 305 00:24:36,572 --> 00:24:41,062 The timeframe here will kind of vary depending on your own capabilities 306 00:24:41,062 --> 00:24:45,622 operational. You may want to try switching monthly, you may want to switch 307 00:24:45,622 --> 00:24:49,572 season to season, that will again depend on what's viable for your operation. 308 00:24:52,252 --> 00:24:56,402 Mosaic is another option this does require some more resistance testing and 309 00:24:56,402 --> 00:25:00,974 rotation so these black bars that you're seeing here would be like 310 00:25:00,974 --> 00:25:05,744 geographic isolation if you're in Vermont that could be topography 311 00:25:05,744 --> 00:25:11,454 limited movement of mosquitoes. In a city 312 00:25:11,454 --> 00:25:15,144 that could be cement structure that are dividing mosquiote populaitons. 313 00:25:15,144 --> 00:25:20,974 But identify where these populations are and then applying different pesticides 314 00:25:20,974 --> 00:25:24,904 in different areas and switching them over time so you're not always applying the same one. 315 00:25:26,724 --> 00:25:31,794 This requires that you test pockets these these pocket locations for 316 00:25:31,794 --> 00:25:37,934 resistance or multiple products but it can be an excellent option for managing resistance 317 00:25:39,154 --> 00:25:42,124 in a larger area or in a city especially. 318 00:25:43,720 --> 00:25:46,280 And then you have a mixture of insecticide, 319 00:25:46,280 --> 00:25:50,480 this is deployin two products at the same time at the same time. 320 00:25:53,612 --> 00:25:56,702 You do want to test for resistance of those products. 321 00:25:57,982 --> 00:26:03,092 It is possible if you're using 2 pyrithriods, especially that 322 00:26:03,092 --> 00:26:07,772 you need to see if resistance emerged for both of them so if you're going to use 2 323 00:26:07,772 --> 00:26:13,052 products you want to try and use 2 products with different modes of action. 324 00:26:17,432 --> 00:26:18,312 Organophosphates or pyrithriods and if you are using larvivides mehtoprene and bti, a biopesticide and a hormone regulator. 325 00:26:19,672 --> 00:26:22,312 To decrease the chances that you are going to see resistance 326 00:26:22,312 --> 00:26:23,842 to both products at the same time. 327 00:26:27,296 --> 00:26:27,906 That was a lot. 328 00:26:29,326 --> 00:26:32,956 But we're going to hold questions until the end of the webinar and 329 00:26:32,956 --> 00:26:37,206 I'm going to give it over to John to kind of take it away and talk about rearing 330 00:26:37,206 --> 00:26:41,796 a little bit. I will be covering how to establish and maintain mosquito colonies. 331 00:26:43,716 --> 00:26:48,266 What do we need to do in terms of establishing mosquito colonies, what do you 332 00:26:48,266 --> 00:26:51,836 need, kind of need 3 basic things you need space 333 00:26:53,657 --> 00:26:58,327 to have all of these colonies going on, dedicated space often, 334 00:26:58,327 --> 00:27:03,497 you need to have the right equipment and materials to be able to rear mosquitoes 335 00:27:03,497 --> 00:27:07,247 from larvae through adult and you need effort and 336 00:27:07,247 --> 00:27:11,987 I think what some people underestimate is the amount of effort that stablishing and 337 00:27:11,987 --> 00:27:17,187 maintaining mosquito colonies will take of the 3 effort is the most important. 338 00:27:18,847 --> 00:27:22,047 Because you can overcome space and 339 00:27:22,047 --> 00:27:26,217 equipment limitations given the right amount of effort. 340 00:27:27,941 --> 00:27:32,901 Going over this presentation I came across some resources that are going to be 341 00:27:32,901 --> 00:27:38,151 really valuable to you if you haven't worked with colonies in the past 342 00:27:38,151 --> 00:27:43,221 this 1st manuscript is open access 343 00:27:43,221 --> 00:27:47,741 it covers the rearing of Culex speicies and Aedes he's mosquitoes. 344 00:27:48,881 --> 00:27:53,891 It includes a very detailed list of supplies many of 345 00:27:53,891 --> 00:27:59,201 the supplies that I will cover here are also listed in this publication and 346 00:27:59,201 --> 00:28:02,321 many of the techniques are then covered again in detail. 347 00:28:04,243 --> 00:28:08,263 Another resource is the manual for mosquito rearing and 348 00:28:08,263 --> 00:28:13,033 experimental techniques they will basically cover just about everything you 349 00:28:13,033 --> 00:28:18,053 need to know that compiles information from almost every reference. 350 00:28:19,273 --> 00:28:25,623 Up until the early 1990 s from other groups that have 351 00:28:25,623 --> 00:28:31,703 stablished populations. If you want to know for example how people have 352 00:28:31,703 --> 00:28:36,573 attempted to colonize Aedes vexans or 353 00:28:36,573 --> 00:28:42,543 to perturb ns that's the place to go to but it is only currently available for 354 00:28:42,543 --> 00:28:47,493 purchase directly through the American mosquito control Association website. 355 00:28:50,083 --> 00:28:55,923 I'm going to talk a little bit about B.e.i. resources it's a repository 356 00:28:55,923 --> 00:29:00,143 and a service that was established by the National Institute of Allergy and 357 00:29:00,143 --> 00:29:05,343 Infectious Diseases in order to access materials from this 358 00:29:06,573 --> 00:29:11,453 you need to have registration certain eligibility 359 00:29:11,453 --> 00:29:16,853 requirements. The registration and eligibility requirements are mostly nonprofit 360 00:29:19,000 --> 00:29:25,630 public, private or academic research there are permits that will need to be 361 00:29:26,760 --> 00:29:32,250 in place for you to to receive material from this 362 00:29:33,821 --> 00:29:38,551 organization. These are all going to be noninfectious material so 363 00:29:38,551 --> 00:29:42,881 most organizations that would be viewing this webinar would be able to receive 364 00:29:42,881 --> 00:29:43,481 some of the 365 00:29:44,731 --> 00:29:50,361 materials given. Going through the paperwork, there are four 366 00:29:50,361 --> 00:29:52,511 species of mosquitoes available. 367 00:29:53,841 --> 00:30:00,311 For use: Aedes aegypti, Culex quinquefasciatus, 368 00:30:00,311 --> 00:30:04,921 Culex tarsalis and Aedes albopictus. The Aedes albopictus if you look at it 369 00:30:06,282 --> 00:30:09,452 it's a pesticide susceptible colony 370 00:30:11,082 --> 00:30:17,452 that is available Bailable for use. They also have two really 371 00:30:17,452 --> 00:30:21,992 great publications as p.d.f. on their website covering 372 00:30:23,202 --> 00:30:27,842 methods in Aedes research and again these are going to be how to colonize new 373 00:30:27,842 --> 00:30:34,162 species and methods in an Anopheles research which is a really big document covers 374 00:30:34,162 --> 00:30:39,932 everything you'd want to know and more but certainly really good information on 375 00:30:41,752 --> 00:30:44,182 how to establish colonies and how to get 376 00:30:45,782 --> 00:30:48,052 your protocols 377 00:30:49,345 --> 00:30:50,005 in order 378 00:30:51,115 --> 00:30:52,785 o establish colonies and maintain them. 379 00:30:54,320 --> 00:30:57,700 I'm going to talk a little bit about the facilities we have here to house our 380 00:30:57,700 --> 00:30:58,500 colonies. 381 00:31:00,260 --> 00:31:02,620 If you need short term colony 382 00:31:04,590 --> 00:31:12,190 use just you're setting up some larvae to rear through to adulthood to do some pesticide 383 00:31:12,190 --> 00:31:17,340 resistance monitoring all you really need is dedicated bench space this can all be 384 00:31:17,340 --> 00:31:22,370 done in season if you do that you don't need to control for temperature or 385 00:31:22,370 --> 00:31:27,710 photo period you just need an area for trays of larvae in cages of adults. 386 00:31:29,150 --> 00:31:32,960 And you may need to increase the humidity in the cages for adult survival. 387 00:31:34,710 --> 00:31:37,870 And you can do that with damp paper toweling and plastic sheeting. 388 00:31:38,932 --> 00:31:43,952 If you're going to stablish mosquitoes long term you do need dedicated space it's 389 00:31:43,952 --> 00:31:47,752 a much bigger operation you need an incubator or 390 00:31:47,752 --> 00:31:52,952 a growth chamber, you can also use a walk in incubator or climat control room. 391 00:31:54,332 --> 00:31:59,252 Again you'll need controls for temperature and photo period to 392 00:31:59,252 --> 00:32:04,352 help these mosquitoes survive optimally and reproduce optimally. 393 00:32:07,013 --> 00:32:10,683 This is an example of two of the growth chambers we have here. 394 00:32:11,753 --> 00:32:15,503 These are essentially large freezer carcasses 395 00:32:15,503 --> 00:32:20,443 that are adapted to have light and temperature control 396 00:32:20,443 --> 00:32:24,483 their free standing you can maintain one or 2 colonies in each 397 00:32:26,874 --> 00:32:32,024 of these units that we have them control for temperature we maintain them 398 00:32:32,024 --> 00:32:38,774 at 25.5 degrees Celsius or 70 degrees Fahrenheit and 399 00:32:38,774 --> 00:32:43,914 we have a light and dark cycle of 16 hours of light and 8 hours of dark. 400 00:32:45,024 --> 00:32:46,854 Use a fluorescent light source 401 00:32:48,761 --> 00:32:54,111 for the light timing. We also have a dedicated insectary 402 00:32:55,311 --> 00:32:59,521 it provides a lot more space in humidity control and 403 00:32:59,521 --> 00:33:02,751 we will go over our set up down next. 404 00:33:04,361 --> 00:33:08,651 Again we maintain this at about 22 to 25 degrees centigrade 405 00:33:09,791 --> 00:33:10,291 we have 406 00:33:11,361 --> 00:33:14,801 a split system a heat pump cooling pump system in 407 00:33:14,801 --> 00:33:20,671 there we maintain it at about 70 percent relative humidity we have a 408 00:33:20,671 --> 00:33:26,251 humidifier mounted employed in indirectly, we have 16 hours of light and 409 00:33:26,251 --> 00:33:31,121 we have overhead lights that are on a cycle that will start to mimic 410 00:33:31,121 --> 00:33:35,861 dusk and dawn and we also have lights on each shelf unit. 411 00:33:37,281 --> 00:33:42,293 There are 2 shelf units in each room each has light control and 412 00:33:42,293 --> 00:33:47,363 then to simulate dawn and dusk we use a clip on light with a 10 watt 413 00:33:47,363 --> 00:33:52,273 incandescent bulb and that goes on the half hour 414 00:33:52,273 --> 00:33:57,203 before the main light start to turn on and will stay on for 415 00:33:57,203 --> 00:34:02,033 about half an hour or an hour until after the lights are totally out. 416 00:34:02,033 --> 00:34:07,433 This allows us to have things like space in a insectary like an 8 foot cage 417 00:34:07,433 --> 00:34:12,533 the cubic foot cage which we used to house our Culiseta melanura colony. 418 00:34:16,810 --> 00:34:17,370 Excuse me. 419 00:34:18,490 --> 00:34:22,210 For species that are available to use in colony or 420 00:34:22,210 --> 00:34:27,390 really convenient to use in colony I would recommend multivoltine species 421 00:34:27,390 --> 00:34:32,550 in Culex pipiens or quinqs, albopicuts, Aedes aegypti, Aedes triseriatus, 422 00:34:32,550 --> 00:34:37,970 Culex pipiens form molestus and Aeides atroplus are autogenous 423 00:34:37,970 --> 00:34:42,730 mosquitos meaning that if they get enough larval nutrition 424 00:34:43,810 --> 00:34:49,350 you can establish them in colony without needing to have a blood source or 425 00:34:49,350 --> 00:34:54,240 blood meal to have progeny more difficult mosquitoes 426 00:34:54,240 --> 00:34:59,080 Culesita melanura, Culex restuans, Culex salinarues, Culex tarsalis 427 00:34:59,080 --> 00:35:04,310 Aedes japonicus and Anopheles quadirmaculatus. 428 00:35:05,712 --> 00:35:08,632 Again the recommended mosquitoes are all 429 00:35:08,632 --> 00:35:12,102 species that were larvae will readily develop in containers. 430 00:35:13,192 --> 00:35:19,222 Univoltine species can be reared more often from larvae to adult but 431 00:35:19,222 --> 00:35:20,382 are very difficult 432 00:35:21,592 --> 00:35:29,942 to stablish as colonies based on limitations of mating or having them 433 00:35:31,252 --> 00:35:33,012 blood feed or then oviposit 434 00:35:34,122 --> 00:35:37,832 viable eggs for collection and then subsequent 435 00:35:38,972 --> 00:35:39,512 hatch. 436 00:35:41,880 --> 00:35:45,630 Specimens can be collected from the field in a variety of ways. 437 00:35:46,700 --> 00:35:49,290 Ovi-trap is one way 438 00:35:49,290 --> 00:35:55,070 we use a 32 ounce casino Cup lined with a 38 pouts in germination paper. 439 00:35:55,070 --> 00:35:59,610 This will collect a number of Aedes or Ochlerotatus species. 440 00:36:00,760 --> 00:36:04,900 You will have to hatch them out and then Id larvae 2nd 3rd or 441 00:36:04,900 --> 00:36:08,400 4th instars with ovi-traps you can get 442 00:36:08,400 --> 00:36:13,490 depending on where what part of the country you're in the Northeast 443 00:36:13,490 --> 00:36:18,040 we would routinely collect Albopictus in areas where it's present. 444 00:36:19,130 --> 00:36:23,420 Ochlerotatus or Aedes triseratus and Japonicus. 445 00:36:24,640 --> 00:36:26,740 You can also use a container for 446 00:36:26,740 --> 00:36:32,590 collecting Culex egg rafts, we use a grvis trap infusion as our bait and 447 00:36:32,590 --> 00:36:38,500 as James had mentioned earlier you can segregate individual eggs rafts and 448 00:36:38,500 --> 00:36:44,520 then ID larvae a couple larvae from each egg graft as 1st or 2nd instars. 449 00:36:47,572 --> 00:36:48,502 We can also 450 00:36:50,333 --> 00:36:56,893 collect larvae with the traditional dipper you can collect from a variety of 451 00:36:56,893 --> 00:37:02,413 of habitats and then I gear id larvae or 452 00:37:04,676 --> 00:37:06,576 rear them to adulthood and 453 00:37:06,576 --> 00:37:10,566 identify them at that stage. I recently did this 454 00:37:11,886 --> 00:37:17,706 with collecting of some Ochlerotatus stimulans from a rental pool. 455 00:37:17,706 --> 00:37:21,756 I was able to collect them as 2nd instars and rear them up to fourth instart and 456 00:37:21,756 --> 00:37:22,496 then through adult 457 00:37:23,556 --> 00:37:26,796 with just some of the techniques we use in the laboratory here. 458 00:37:28,170 --> 00:37:32,670 You can also use a mechanical aspirator we've had success with a backpack 459 00:37:32,670 --> 00:37:37,170 aspirator and collecting adults of Aedes ablopictus to establish our 460 00:37:37,170 --> 00:37:41,840 colony. You collect them similar to the way you would conduct lading bite 461 00:37:41,840 --> 00:37:46,200 counts in that way you're collecting mated adult females. 462 00:37:47,230 --> 00:37:51,210 You then identify the females after chilling them to immobilize them. 463 00:37:52,310 --> 00:37:55,350 Then you put them into a cage offer them a blood meal and 464 00:37:55,350 --> 00:38:00,450 then collect those eggs hatch and rear the larvae for certain species. 465 00:38:02,891 --> 00:38:07,961 when you're ready to rear a larvae some of the equipment you need are large 466 00:38:07,961 --> 00:38:13,361 pans to rear them in ,larger pans will work best, if you're working with a white 467 00:38:13,361 --> 00:38:18,341 pan that's great if not you want to have a white background. And in for these pans 468 00:38:18,341 --> 00:38:23,281 you want to have a surface area that is greater than the volume that it can hold. 469 00:38:24,631 --> 00:38:30,411 You can also use some inexpensive options such as these 24 ounce takeout containers 470 00:38:31,511 --> 00:38:36,291 or a 16 ounce deli container and with the deli container you're going to want 471 00:38:36,291 --> 00:38:40,071 to only fill that up about an inch or an inch in depth. 472 00:38:42,232 --> 00:38:47,352 Because as the water depth increases sometimes have issues 473 00:38:47,352 --> 00:38:53,623 with some of the species surviving as larvae. Now if you need to source these 474 00:38:53,623 --> 00:38:59,486 you get these smaller white plastic pans from bioquip they are Rubbermaid type 475 00:39:00,726 --> 00:39:04,216 storage containers that are available from discount stores and 476 00:39:04,216 --> 00:39:07,836 if you have white enamel pans you probably have to go back to the 1960 s 477 00:39:07,836 --> 00:39:12,366 to get those. This inexpensive containers are often available 478 00:39:12,366 --> 00:39:16,886 online from webstaruant supply stores. 479 00:39:19,280 --> 00:39:22,130 And you once you establish these Larvae you're going to 480 00:39:22,130 --> 00:39:24,180 need a certain particular diet. 481 00:39:25,390 --> 00:39:30,080 There are multiple options many labs will use Tetramin 482 00:39:30,080 --> 00:39:36,070 tropical fish flake that's finely ground these are relatively inexpensive and 483 00:39:36,070 --> 00:39:41,940 readily available. We use a mixture of liver powder and brewers yeast for 484 00:39:41,940 --> 00:39:47,470 most of our colonies here certainly all of our Aedes and Culex colonies. 485 00:39:48,692 --> 00:39:51,682 You can also use finely ground and sifted pet food and 486 00:39:51,682 --> 00:39:56,892 examples are Koi food, dog food, cat food, rabbit food pellets, and 487 00:39:56,892 --> 00:40:02,084 rat chow you can use various combinations of the above and 488 00:40:02,084 --> 00:40:05,664 a lot of those are going to be in that AMCA manual. 489 00:40:06,784 --> 00:40:12,034 What's worked and what hasn't worked useful to make them in a suspension 490 00:40:12,034 --> 00:40:17,414 rather than applying them dry to the surface of the water and we use it 491 00:40:18,584 --> 00:40:23,844 relatively fresh storing it one to 2 days in the refrigerator between uses. 492 00:40:26,394 --> 00:40:29,924 Now what we'll do is we'll discuss how to hatch eggs once you 493 00:40:29,924 --> 00:40:31,054 once you have them. 494 00:40:32,494 --> 00:40:33,754 Water is important. 495 00:40:34,984 --> 00:40:38,154 You want to use distilled reverse osmosis or 496 00:40:38,154 --> 00:40:42,864 condition tap water that's water that has been left out for about 24 hours so 497 00:40:42,864 --> 00:40:45,684 any of the chlorine that's in a municipal water system 498 00:40:46,824 --> 00:40:50,124 eould dissipate out and not affect larval development. 499 00:40:51,694 --> 00:40:53,924 You want to add the water to the pan. 500 00:40:55,294 --> 00:40:57,424 To a depth of about 4 centimeters or 501 00:40:57,424 --> 00:40:59,614 a little bit more than an inch deep. 502 00:41:02,460 --> 00:41:08,020 4 centimeters of the maximum, eggs in the larval food the general 503 00:41:08,020 --> 00:41:13,410 rule you want to have enough for larval density is one larva for every 504 00:41:14,650 --> 00:41:19,620 cubic centimeter or milliliter and up to 5 larva per square 505 00:41:19,620 --> 00:41:24,640 centimeter of surface area. This is a probably about 506 00:41:24,640 --> 00:41:29,620 the maximum, we try to keep it less than that and 507 00:41:29,620 --> 00:41:35,231 I'll show you some examples of what our densities are. So you have eggs 508 00:41:36,931 --> 00:41:41,601 And on the left there's a close up picture of the eggs on the filter paper that we 509 00:41:41,601 --> 00:41:46,921 collect from our colonies these were eggs that were flooded on Friday and 510 00:41:46,921 --> 00:41:53,532 by Monday we had 2nd or 3rd instars with this zoom close up in 511 00:41:53,532 --> 00:41:58,352 the lower left hand corner of the zoom you can see the filter paper in the water and 512 00:41:58,352 --> 00:42:03,232 multiple 2nd and 3rd instar larvae in the pan and 513 00:42:03,232 --> 00:42:06,662 that would be about the density of larvae that's probably about. 514 00:42:07,722 --> 00:42:10,692 500-700 larvae per pan 515 00:42:12,852 --> 00:42:17,352 as a maximum. This one's probably in the 300-400 range. 516 00:42:19,983 --> 00:42:25,313 For Culex egg rafts figure that each raft is going to be 50 to 200 517 00:42:25,313 --> 00:42:30,843 eggs or more per raft you want to make sure that they're oriented 518 00:42:30,843 --> 00:42:35,093 in the head down position Culex egg rafts will have a natural 519 00:42:36,193 --> 00:42:38,473 concave shape to them you want to make sure that 520 00:42:39,703 --> 00:42:41,383 is maintained as down. 521 00:42:43,223 --> 00:42:50,133 You add water and larval food and the eggs will hatch in 24 to 48 hours. 522 00:42:51,441 --> 00:42:56,061 This is the egg rafts are in the oviposition dish for 523 00:42:56,061 --> 00:43:00,411 our colony then pick them out with a spatula or scoop or 524 00:43:00,411 --> 00:43:04,981 sometimes a wooden applicator stick and then add them to the pan. 525 00:43:07,522 --> 00:43:13,152 And then in a sometimes in as few as 12 to 24 hours 526 00:43:13,152 --> 00:43:18,492 you'll have 1st larvae that will hatch from those eggs and then you. 527 00:43:19,592 --> 00:43:23,382 Want to make sure that in each case that you maintain these and 528 00:43:23,382 --> 00:43:29,733 we will feed our larvae Monday, Wednesday and Friday we increase feeding or 529 00:43:29,733 --> 00:43:33,653 the amount of food that we add as larvae develop from 1st to 3rd instar 530 00:43:33,653 --> 00:43:39,203 often with certain diets especially ones that have a higher fat content 531 00:43:40,363 --> 00:43:44,933 you neeed to skim the surface of the water with a paper towel if a scum layer develops. 532 00:43:46,054 --> 00:43:47,854 If the water is cloudy 533 00:43:49,524 --> 00:43:52,234 you tend to feel less and 534 00:43:52,234 --> 00:43:58,934 you also decrease feeding when pupae 1st develop but you still want to maintain 535 00:43:58,934 --> 00:44:03,624 lower levels of feeding as those 4th instars need to develop into pupae. 536 00:44:05,975 --> 00:44:08,415 Transferring pupae, you pick up puape 537 00:44:08,415 --> 00:44:13,445 as they developed usually 5 to 7 days after hatching in our labs. Use 538 00:44:13,445 --> 00:44:18,935 a pipette either with a tip cut off either a glass or plastic pipette. 539 00:44:21,217 --> 00:44:25,547 In the bottom of the screen we have the pupae that are in the pan. 540 00:44:27,121 --> 00:44:30,511 Then transfer using that plastic pipette. 541 00:44:32,511 --> 00:44:39,171 Hold the paupae out and transfer them into either the bottom of a mosquito breeder or 542 00:44:39,171 --> 00:44:43,101 into a 16 ounce deli cup and then we place those 543 00:44:44,121 --> 00:44:48,401 into a cage and if you look at the larger 544 00:44:48,401 --> 00:44:51,941 cup on the left on the upper 545 00:44:54,383 --> 00:44:59,443 picture you will see that I have included some forth instar larvae in that 546 00:44:59,443 --> 00:45:04,403 this was done on Friday and what we tend to do is use a fish net for 547 00:45:04,403 --> 00:45:06,753 straining out larvae in fourth instars 548 00:45:07,763 --> 00:45:12,493 just because it's time consuming to pick all those larvae and pupae 549 00:45:15,234 --> 00:45:19,954 in order to get them into the cage for the weekend. The last thing you 550 00:45:19,954 --> 00:45:25,274 want to do is come in on Monday morning and have a bunch of adults flying off 551 00:45:27,624 --> 00:45:29,754 from a larval rearing pan. 552 00:45:31,527 --> 00:45:35,757 For the cages for adults we have 2 types that will use readily. 553 00:45:36,827 --> 00:45:44,357 The 30x30x30 centimeter or one cubic foot cage, we use the metal cage 554 00:45:44,357 --> 00:45:49,867 with aluminum screening there is also then a hammock towards the front of that 555 00:45:52,767 --> 00:45:57,547 that you can add either sugar or blood meal sources to it if you desire. 556 00:45:57,547 --> 00:46:02,477 The plastic cage is a bugdorm with a little bit as you 557 00:46:02,477 --> 00:46:06,547 can see the opening to access the inside the cage is a little smaller. 558 00:46:08,037 --> 00:46:09,337 So it for certain things. 559 00:46:10,477 --> 00:46:13,327 Each one has its own advantages and disadvantages. 560 00:46:15,201 --> 00:46:17,771 Inside the cage you're going to need a sugar source. 561 00:46:19,071 --> 00:46:23,181 And we use table sugar we make it up as a ten percenct solution and 562 00:46:23,181 --> 00:46:27,951 stored in refrigerator for up to a week we'll use that sugar syrup 563 00:46:27,951 --> 00:46:32,801 solution from a squirt bottle and will soak cotton balls into one ounce cup or 564 00:46:32,801 --> 00:46:39,271 we will use a cotton wick in a 50 ml Earl Meyer flas. As the sugar 565 00:46:39,271 --> 00:46:43,341 source you can also use things like raisins or apple slices or other fruits. 566 00:46:44,461 --> 00:46:49,981 As sugar or carbohydrate sources for maintaining males and 567 00:46:49,981 --> 00:46:55,102 females you'll also need to put in an open oviposition dish for 568 00:46:55,102 --> 00:47:00,362 Culex we use a cup with water with a little bit of food and for Aedes 569 00:47:00,362 --> 00:47:06,002 we use a cup and we add a fluted filter paper to that. 570 00:47:07,202 --> 00:47:11,942 The mosquitos will oviposit on that damp filter paper. 571 00:47:14,483 --> 00:47:18,633 For adults you can still use either the you can also use the large 572 00:47:18,633 --> 00:47:23,053 mosquito breeder if you need if you have lots of space options and 573 00:47:23,053 --> 00:47:27,413 what we do is we simply take a sugar soaked cotton ball and 574 00:47:27,413 --> 00:47:31,603 put it on the top of mosquito breeder and cover it with a 575 00:47:33,664 --> 00:47:39,394 one ounce plastic cup just to limit evaporation of that sugar water. 576 00:47:40,584 --> 00:47:45,324 Again this is the setup we have in the cage we have ovipositio, dishes. 577 00:47:48,986 --> 00:47:54,816 Pupae that are merging into the cage and then sure Wicks for our Culex colonies. 578 00:47:56,100 --> 00:48:01,260 If you need to remove adults from the cage for the pesticide resistance 579 00:48:01,260 --> 00:48:05,970 monitoring testing you can use a battery powered aspirator 580 00:48:05,970 --> 00:48:10,870 those are one sourced for our lab through quark mosquito control products or 581 00:48:10,870 --> 00:48:15,730 you can use a long powered aspirator So depending on how many 582 00:48:15,730 --> 00:48:20,300 you're how many you're trying to pull out the mechanical aspirator might be 583 00:48:21,380 --> 00:48:25,940 to your benefit certainly if you can afford to purchase them they're about 584 00:48:25,940 --> 00:48:32,172 65-70 dolars at this point. And then if 585 00:48:32,172 --> 00:48:37,102 you want to maintain mosquitoes long term you will need to consider blood feeding. 586 00:48:38,122 --> 00:48:40,202 If you're using a live animal. 587 00:48:41,272 --> 00:48:46,112 Institutional Animal Care and use committee approval is required this 588 00:48:46,112 --> 00:48:52,512 required detail protocols and you need to maintain animals appropriately here at 589 00:48:52,512 --> 00:48:58,002 the experiment Station we maintain guinea pigs and Button quail for blood feeding. 590 00:48:59,152 --> 00:49:03,452 If you're going to use a human subject it is regulated by the us Department of 591 00:49:03,452 --> 00:49:05,022 Health and Human Services. 592 00:49:06,642 --> 00:49:10,972 An institutional review board approval is required. 593 00:49:12,042 --> 00:49:14,902 Even as it may be an occupational activity. 594 00:49:17,143 --> 00:49:21,223 Just to use a human subject to feed to provide a blood meal for 595 00:49:21,223 --> 00:49:23,593 a colony. The paper by Achee 596 00:49:25,333 --> 00:49:28,533 that was published vector borne and zoonotic 597 00:49:28,533 --> 00:49:32,223 diseases will cover a lot of the 598 00:49:33,423 --> 00:49:38,873 operations and considerations that need to be taken into account 599 00:49:38,873 --> 00:49:43,433 for using human subjects for for any purpose in mosquitoe 600 00:49:44,683 --> 00:49:47,293 and vector biology research is really good resource. 601 00:49:48,871 --> 00:49:53,781 If you need to use an artificial blood feeding source you 602 00:49:53,781 --> 00:49:58,801 need to order blood from a supplier we ordered defibrinated sheep blood 603 00:49:58,801 --> 00:50:04,021 although a variety of blood sources are available including cow and chicken. 604 00:50:06,214 --> 00:50:08,124 You need to use an artificial membrane 605 00:50:09,636 --> 00:50:13,936 we use a sausage casing a sugar or pig intestine and 606 00:50:13,936 --> 00:50:19,726 you need to warm the blood to 37 degrees or up to 40 degrees in the picture 607 00:50:19,726 --> 00:50:24,466 on the bottom is a sausage casing filled with blood that's from the the Kauffman 608 00:50:24,466 --> 00:50:30,156 paper that I referenced earlier you have the unfilled 609 00:50:30,156 --> 00:50:35,406 sausage casing on the left and filled the sausage casing on the right and 610 00:50:35,406 --> 00:50:39,796 then that sausage casing can either be put into the cage or into that hammock. 611 00:50:40,926 --> 00:50:43,736 For the aluminum cages that I show. 612 00:50:46,277 --> 00:50:50,407 You can also consider an artificial membrane feeding system 613 00:50:50,407 --> 00:50:52,767 one is a Hemotek and you can use 614 00:50:53,837 --> 00:50:55,547 parafilm or 615 00:50:56,757 --> 00:51:02,058 sausage casing as the membrane and as highlighted there 616 00:51:02,058 --> 00:51:06,158 is an aluminum disc that screws into the bottom that can be filled with blood and 617 00:51:06,158 --> 00:51:10,518 then you stretch a pair of film or the sausage casing over the top and 618 00:51:10,518 --> 00:51:14,488 secure it with a rubber band or an O ring. And then 619 00:51:16,540 --> 00:51:22,010 that gray cylinder maintains the blood at the proper temperature and then the 620 00:51:23,460 --> 00:51:28,470 cylinder and blood meal can either be offered through the screening of the cage 621 00:51:28,470 --> 00:51:30,900 or can be put internal into the cage 622 00:51:32,590 --> 00:51:34,440 in a horizontal fashion. 623 00:51:36,211 --> 00:51:40,981 I can also use a glass feeder these again use parafilm or 624 00:51:40,981 --> 00:51:45,851 sausage casing as a membrane requires a circulating water bath. These 625 00:51:47,281 --> 00:51:50,311 glass feeders are jacketed so 626 00:51:50,311 --> 00:51:55,301 warm water will circulate around an inner chamber that can be filled with blood and 627 00:51:55,301 --> 00:52:00,341 then a again the membrane goes against the screen or 628 00:52:00,341 --> 00:52:07,451 part of the cage and mosquitoes will readily feed or can readily feed through the screen 629 00:52:07,451 --> 00:52:12,642 onto the membrane system. And then once you have 630 00:52:13,652 --> 00:52:14,572 successfully 631 00:52:16,022 --> 00:52:18,272 had mosquitos in 632 00:52:19,482 --> 00:52:22,172 in a cage and fed on an artificial or 633 00:52:22,172 --> 00:52:26,792 natural blood meal you will have to consider storing Aedes eggs 634 00:52:26,792 --> 00:52:31,492 oviposition will occur 3 to 5 days after blood feeding. 635 00:52:33,094 --> 00:52:37,054 And here you can see the eggs on the filter paper again as I described earlier. 636 00:52:38,096 --> 00:52:41,776 You take that filter paper out you remove any adults that might be on the filter 637 00:52:41,776 --> 00:52:48,176 paper and we dry them on a paper towel for 15 to 30 minutes. 638 00:52:49,396 --> 00:52:52,556 Then we fold the paper with the eggs on the inside and 639 00:52:52,556 --> 00:52:58,116 place it into a labeled either whirl pack bag or 640 00:52:58,116 --> 00:53:02,286 you can use a Ziploc bag and then we can store those 641 00:53:03,656 --> 00:53:08,586 in the rearing incubator or the insectary by species 642 00:53:10,228 --> 00:53:12,628 for 1 to 6 months. 643 00:53:17,009 --> 00:53:19,799 Will open then things now to questions and 644 00:53:19,799 --> 00:53:23,539 if you any of you have any questions for me that are not covered 645 00:53:24,829 --> 00:53:25,639 or if you want to 646 00:53:26,849 --> 00:53:27,869 check in with me 647 00:53:29,079 --> 00:53:32,869 as you're trying to establish things just feel free to shoot me an e-mail 648 00:53:32,869 --> 00:53:34,249 at John.Shepard@ct.gov. 649 00:53:39,291 --> 00:53:43,771 All right I'd like to say thank you to our 2 presenters and we will at this 650 00:53:43,771 --> 00:53:48,291 point open up for the Q&A session. I also wanted to highlight just for 651 00:53:48,291 --> 00:53:52,561 those who may not have been aware of our presenters affiliations 652 00:53:52,561 --> 00:53:55,321 Dr James Burtis is our post-doctoral associate working for 653 00:53:55,321 --> 00:53:58,431 The Northeast Regional Center for Excellence in Vector-Borne Diseases 654 00:53:58,431 --> 00:54:02,941 at Dr Laura Harrington's Lab at Cornell University and he is the manager of our 655 00:54:02,941 --> 00:54:07,081 pesticide resistance monitoring program, you can contact him at the information on your 656 00:54:07,081 --> 00:54:11,441 screen or by visiting our Northeast Regional Center website and 657 00:54:11,441 --> 00:54:14,821 John Shepherd has been working with the Connecticut agricultural experiment station 658 00:54:14,821 --> 00:54:19,861 since 1997 where he coordinates the trapping and identification of mosquitoes for 659 00:54:19,861 --> 00:54:24,431 the Connecticut Mosquitoe trapping and arvi-virus surveillance program his contact 660 00:54:24,431 --> 00:54:30,162 information and the ag station website are also on your screen. So with that 661 00:54:30,162 --> 00:54:35,292 we will jump in to our questions the 1st question is what information is there in 662 00:54:35,292 --> 00:54:40,092 regards to whether mosquitoe adulticides affect resistance if pyrethroid 663 00:54:40,092 --> 00:54:44,532 are ubiquitous, in other words if we stop spraying pyrethroids the public and 664 00:54:44,532 --> 00:54:47,212 other industries may continue using them as well. 665 00:54:50,753 --> 00:54:51,953 I can try to answer this one. 666 00:54:53,093 --> 00:54:58,643 So there is some information about the effect of like agricultural pesticides 667 00:55:00,123 --> 00:55:01,953 on resistance mosquito populations and 668 00:55:03,320 --> 00:55:06,530 from early studies it does appear that yes, 669 00:55:06,530 --> 00:55:10,580 when pesticides are deployed broadly by agriculture 670 00:55:11,730 --> 00:55:14,060 or by people who are not mosquitoe control 671 00:55:15,280 --> 00:55:19,000 its possible that resistance can emerge in 672 00:55:19,000 --> 00:55:21,090 mosquito populations even if they're not targeting them. 673 00:55:23,030 --> 00:55:25,900 This is one of the reasons it's really important to get 674 00:55:25,900 --> 00:55:28,710 an idea of basline susceptibility of your population 675 00:55:28,710 --> 00:55:32,950 because it's not always under your control to manage if you have to kind of work around 676 00:55:34,610 --> 00:55:40,020 what resistance if any is already in the population and the of course 677 00:55:40,020 --> 00:55:42,590 the work around is available in our state and 678 00:55:42,590 --> 00:55:46,020 this is why our guidelines are broad you have to kind of have figure out 679 00:55:47,190 --> 00:55:52,550 what is going to work in your specific region or your state or county. 680 00:55:55,203 --> 00:55:59,163 Thank you and our next question is actually for John the question is do you 681 00:55:59,163 --> 00:56:04,183 have any advice in avoiding pathogens in the insectary, an example is epivians. 682 00:56:05,933 --> 00:56:11,573 We haven't had too many issues in terms of having any types of 683 00:56:11,573 --> 00:56:16,963 pathogens you know any fungal contaminants or things of that nature. 684 00:56:19,064 --> 00:56:22,844 The only thing you want to make sure you're doing is 685 00:56:22,844 --> 00:56:27,914 we do clean things down wipe things down on a weekly basis 686 00:56:27,914 --> 00:56:32,874 we don't use any types of disinfectants in our labs we just usually tend to 687 00:56:32,874 --> 00:56:36,984 clean things with with water and scrubbing and let them dry out. 688 00:56:38,144 --> 00:56:41,604 Again you know sometimes with the you may have issues. 689 00:56:42,950 --> 00:56:46,430 With getting some things if you have 690 00:56:46,430 --> 00:56:50,880 some pathogens if you're if you're using food for a long period of time. 691 00:56:52,120 --> 00:56:53,610 You know. 692 00:56:53,610 --> 00:56:58,270 Those we don't have we will change those sugar Wicks or 693 00:56:58,270 --> 00:57:02,500 sugar sources on a weekly basis so we don't have any mold issues. 694 00:57:03,700 --> 00:57:05,220 In the in the insectary 695 00:57:06,530 --> 00:57:10,640 and I guess you could have issues with the food if it's not fresh but 696 00:57:10,640 --> 00:57:13,670 that's one of the things we do is we make it up fresh we go through a lot of 697 00:57:13,670 --> 00:57:18,400 material on a weekly basis so we always have 698 00:57:18,400 --> 00:57:23,030 relatively fresh material going in so I think that's all often helpful for us. 699 00:57:24,792 --> 00:57:29,942 Thank you and I believe your next question is going to be for Dr Brutis so 700 00:57:29,942 --> 00:57:33,642 the question is what life's stage in species sent to you most frequently for 701 00:57:33,642 --> 00:57:34,582 resistance testing. 702 00:57:36,164 --> 00:57:42,184 So last season let's say the bulk of our samples were Culex pipiens testing for 703 00:57:42,184 --> 00:57:43,524 larvicides bti and methoprene. 704 00:57:45,324 --> 00:57:48,684 That being said we're trying to encourage people to test Aedes albopictus 705 00:57:50,234 --> 00:57:53,684 as well and we know that adulticide testing don't it's not just. 706 00:57:55,994 --> 00:57:58,264 You know we want to push people to push. 707 00:57:59,584 --> 00:58:02,904 Request more tests for organophosphates especially. 708 00:58:04,224 --> 00:58:06,904 So that we can know what the general separate bill of the. 709 00:58:08,544 --> 00:58:14,384 Illusion in the northeast looks like considering how widely spread I mean since 710 00:58:14,384 --> 00:58:20,254 I read from it use is you know region our next 711 00:58:20,254 --> 00:58:24,434 question is when you're trying to eighty's eggs are there any other precautions we 712 00:58:24,434 --> 00:58:28,834 need to take to make sure the extract correctly such as temperature humidity. 713 00:58:30,451 --> 00:58:38,311 Sure yeah with eighty's eggs we make it we dry them down so that the filter paper is 714 00:58:38,311 --> 00:58:43,151 damp to the barely damp to the touch and then stored in those sealed bags 715 00:58:43,151 --> 00:58:47,281 if you had it where the where they were where the eggs were to dry or 716 00:58:47,281 --> 00:58:51,971 the paper started to get to dry you can make a humidity chamber by having either. 717 00:58:53,081 --> 00:58:59,121 A wet cotton ball in the corner of a closed container away from 718 00:58:59,121 --> 00:59:03,761 the filter paper and you can kind of you can kind of segregate them that way. 719 00:59:06,263 --> 00:59:10,613 But basically in an airtight container with a little bit of humidity you can 720 00:59:10,613 --> 00:59:13,533 you can bring up the humidity on those eggs so 721 00:59:13,533 --> 00:59:17,883 they don't dry out too much and cause mortality to the eggs. 722 00:59:21,264 --> 00:59:25,474 The next question in our Q After like the heating makes a p.p.s. 723 00:59:25,474 --> 00:59:29,464 unless it squeaks and hybrid you need to get in for creating a fine 724 00:59:35,426 --> 00:59:37,386 i can answer that question like. 725 00:59:38,466 --> 00:59:43,786 So there are good molecular diagnostics for the species and 726 00:59:43,786 --> 00:59:48,526 they're also really good taxonomic features for the larvae at least for the. 727 00:59:50,956 --> 00:59:52,326 Some of the species so. 728 00:59:53,526 --> 00:59:58,386 And of course where you are in the country or what you're going to get x.. 729 00:59:59,436 --> 00:59:59,936 So. 730 01:00:01,546 --> 01:00:06,636 There are some in some ways that you can identify those are usually check 731 01:00:06,636 --> 01:00:11,856 our companies to confirm that they are indeed what they are but understandably 732 01:00:11,856 --> 01:00:15,706 it can be difficult for some folks whom you know have access to the p.c. are. 733 01:00:21,219 --> 01:00:25,269 Making a comparison and the next question is is there less so 734 01:00:25,269 --> 01:00:28,909 if they publish the resistance testing results to you and 735 01:00:28,909 --> 01:00:31,869 is that let's say to be able to access to the public. 736 01:00:33,331 --> 01:00:38,531 So we in our initial survey we have asked of no cheering 737 01:00:38,531 --> 01:00:42,401 the resistance results publicly in him most of our collaborators 738 01:00:42,401 --> 01:00:47,061 expressed that they wished us not to we are working on a publication. 739 01:00:48,261 --> 01:00:50,251 Right now that will. 740 01:00:52,494 --> 01:00:56,834 That's based on our survey from last year you know we are offering 741 01:00:56,834 --> 01:01:00,504 co-authorship to people sent us citizens so they. 742 01:01:02,114 --> 01:01:04,564 Are not how we report the results as well 743 01:01:04,564 --> 01:01:07,114 we have to be careful how we how we report them in the. 744 01:01:08,914 --> 01:01:13,284 Community so we don't overstep to make them uncomfortable 745 01:01:13,284 --> 01:01:15,944 with releasing results or share. 746 01:01:19,487 --> 01:01:23,787 I mean in the next question is regarding the mosaic method of pesticide 747 01:01:23,787 --> 01:01:28,687 application you have to factor in the flight range of different populations 748 01:01:28,687 --> 01:01:31,737 how much does flight range very biased you know species. 749 01:01:36,967 --> 01:01:38,037 Sorry I was mean again. 750 01:01:39,697 --> 01:01:40,367 So. 751 01:01:42,077 --> 01:01:45,497 Yes So I'm actually going to ask Lauren to answer the flight range questions. 752 01:01:46,547 --> 01:01:47,047 Answered. 753 01:01:48,197 --> 01:01:48,697 But. 754 01:01:50,629 --> 01:01:52,429 You you do have to account for 755 01:01:52,429 --> 01:01:57,799 flight range also if they count who are geographic isolation like physically how 756 01:01:57,799 --> 01:02:03,979 connected skeeters be those 2 populations already might answering the question 757 01:02:03,979 --> 01:02:09,059 I'm not a doll so typically flavorings for mosquitoes is is very short 758 01:02:09,059 --> 01:02:14,119 typically a couple of 100 meters Max and the one exception are some of 759 01:02:14,119 --> 01:02:19,259 the salt marsh mosquitoes which are known to travel up to a mile or more but for 760 01:02:19,259 --> 01:02:23,819 most of the skiers that you'd be focusing on the plate ranges can be pretty small. 761 01:02:28,610 --> 01:02:30,960 All right well if there's no more questions for 762 01:02:30,960 --> 01:02:35,500 today again we have the contact information of our presenters and 763 01:02:35,500 --> 01:02:39,340 the Northeast Regional Center on your screen so please feel free to reach out 764 01:02:39,340 --> 01:02:44,120 to us as a follow up to today's presentation we are going to be making 765 01:02:44,120 --> 01:02:48,650 the video of today's presentation available on our website as well as. 766 01:02:49,680 --> 01:02:51,530 Print material summarizing our q. 767 01:02:51,530 --> 01:02:55,650 and a session as well as the slides that are presenters today for 768 01:02:55,650 --> 01:03:00,171 you all to access in the future and with that I would like to say thank you again 769 01:03:00,171 --> 01:03:03,141 to our presenters and thank you to everybody who joined us today.