Good morning and welcome to the insecticide resistance in mosquitoes webinar.
Today's presentation will be hosted by Dr James Burgess at Cornell university and
John Shepard of the Connecticut Agricultural Experiment Station.
Hello everybody thank you for joining us today, I'm going to
start this off by talking about pesticide resistance monitoring techniques in disease vectors.
A few of the things we're going to cover today.
I'll start out with; what is pesticide resistance and how does it emerge. We'll talk about how resistance
bioassays diagnostics are created. How you determine the resistance status of field-collected mosquitoes.
and how you actually use those results to inform your managment
decisions. After that John is going to talk a little bit about collecting mosquitoes for
resistance assays, and also techniques for rearing different mosquito species in the lab.
So to start out why is this substance monitoring important?
So we have a lot of different classifications of the insecticides but
the ones you see here in green and blue are the insecticide clasifications
that are generally used you for mosquito control.
So there aren't that many, the ones in green are adulticides
the ones in blue are larvicides.
And then state regulations can actually further restrict the ability to cycle
through these different pesticides so the actual number that can be used in
a state may be even lower than what you're seeing here for example many states only
have pyrethriods registered for vector control. So this means that
resistant monitoring is necessary to maintain the efficacy of these products because we
just don't have a lot of them so we have to we have to guard the ones that we do have.
I have included some additional information
from the EPA about what active ingredients are used for larval and adult control
in the bottom here.
Looking at the Northeast US, where the north east regional center is located
many mosquito control agencies both use adulticides and larvicides
in the field. This is from a survey that we deployed last year to determine
what mosquitoe control in our region we're doing to control mosquito populaitons.
From that same survey you can see the top adulticides are all pyrethroids
and some states will use organophosphates,
but the overwhelming majority of applications are one classification
using one mode of action.
There are and I will explain what these different mechanisms are and
how they work in a minute, but you should know that we've already identified that
multiple resistance mechanisms can affect pyrethriods,
they can also affect organophosphates they maybe cross resistance but
that's not always the case. If you look at esterases for example,
just because an esterase, they are generalists esterase that affect everything and
there were targeted esterase that my only affec one active ingredient.
So looking at the resistance mechanisms we have
3 basic classifications. 1st we have metabolic resistance.
These involve the metabolic breakwdon of the insecticide itself. These include esterases
monooxygenases, glutathion s-transferace. So for
example I'm going to use the video games to explain this.
Think of an oxygenase as packman, it will attack that permethrin or that
malathion chemical, it will break it down into less harmful compounds and
those less harmful compounds and those compounds
wont necesarrily kill the insect.
Altered site resistance is another mechanism of resistance for
example knock down resistance or modified ACHE.
This so
generally when a pesticide impacts or attacks pill a cellular bonding site there has to be
somewhere where that pesticide can attach to the cell or to the cellular mechanism.
Under
sustained selective pressure
that site can actually physically change configuration.
and change shape so that pesticide can no longer bind to that area making him very sad.
There's also penetration resistance so in order to get
into the body of the insect pesticide has the penetrate the cuticle
over time with selection, thicker
cuticle can be developed that will less of the pesticide to get into the body of the insect.
These are the 3 primary mechanisms for
resistance and then there can be more specific versions of these like an
esterase that only effects permethrin or an esterase that affects a broad range of
pesticide active ingredients.
So when we think about how resistance emerges
first you have to have resistance in the natural population,
this may be a gene that's expressed in a low level.
But when you add pressure
from your pesticide application you kill off the susceptible individuals and
you can allow those resistant individuals to start competing
in the field. Over time, those resistant individuals will
start to take over the population and you will have fever susceptible individuals.
The time scale can vary here, this may be your 2nd applications or it
could be your 20th applicantion and we will talk about some of the factors that will affect
how long it takes for application to allow resistance to emerge at the end.
Also I want to define the difference between resistance vs
susceptibility. Susceptibility is usually determined by comparing mortality curves from
susceptible colony in a field collected colony or a field collected strain rather. So
susceptibility curve is if you look at the axis here you have a mortality and
pesticide concentrations. So as pesticide concentrations is going up,
mortality increases until it flatlines at this threshold, 100% mortality.
S shaped curve and
you're going to compare that curve with another one built with field specimens.
So we would say that this field colony here is less susceptible because
fewer are dying at the same concentration compared againt the mortality.
Resistance is defined by a threshold diagnostic concentration
dose, time, it can be a resistance ratio, they're all based on the susceptibility curve.
In our case for our program we've been using LC 99, this is also what the WHO uses for
many of their bioassays.
This is the point at which 99 percent, a concentration at which 99 percent of the individuals
from the susceptible colony would die. So let's say you have 50 percent mortality at that
point for a field specimen or for a field population
that would be resistant population and we'll talk about scaling resistance in a minute aswell.
Resistance ratios are also an option so
this will be a comparison of LC99 and the error around them, because there is always error
involved in these measurements, but in order to do that
you need to collect enough specimens to construct an entire curve which
requires quite a few more specimens than you would normally collect to test
LC 99 or a single value and in field populations
there can varaibility in those curves requiring more time.
Looking at the susceptibility curve this is our bti susceptibility curve,
for Culex pipiens, you can see I've ploted all the individual points here, each of those
points represents a cup with 15 larvae in it so
you can see it takes thousands of larva to create one of these curves and
that's because you get a fair bit of variability so you need to
continue to sample increase replication to overcome that variability and
get a good estimate of your probit curve. Probit analysis is the kind of
regression used to analyze dose response curves this can be used to
determine the pesticide concentration at which point agiven percentage of mosquitoes would die.
There's also always errors associated with this estimate, again
that is why you need to need to increase replication.
If you're experimental design is sound, as you increase replication error should decrease.
We are actually developing some
freeware guidelines that would allow people to do these calculations easily
we hope to have those on our website within the next few months.
Using this probit curve you're going to define your threshold, your LC 99 you can use
LC95 although they are going to detect a lower level of resistance.
When we talk about defining resistance.
For larvicidal bioassays or any assay that is using an LC 99
you can kind of scale this way for a program WHO
uses a similar technique as well.
So we test the population at a
single concentration the LC99 if less than 90 percent of the
individuals die in that sample we call that a low level resistant.
population. Then we scale it so you have to follow up
if you do detect low level resistance with a moderate level resistance test, five times the LC99 and if needed
a hihg level resistance test, about ten times the LC99.
Some of you will be familiar with the bottle bioassay, its the CDC
method that is most commonly used in states.
This is a diagnostic time rather than a diagniostic dose or concentration so
instead of the threshold being
pesticide concentration itss a time threshold. So
in this case let's say the threshold is 30 minutes.
If you have less than 90 percent mortality at that diagnostic time we call it low-leve resistance and
then again you can scale with time this time which is convenient because you can
do it all in one test you don't have to run multiple tests so 2 times
that threshold whould be moderate resistance and 3 times a threshold would be high level resistance.
Some of you may have heard of the who tube test.
This is a contact assay the CDC bottle bioassay.
Two tubes divided by a divider
there is filter paper in here its a exposure tuber where hte mosquitoes
are allowed to come into contact with a treated piece of paper for an hour and then
they are transferred to this holding tube in top and held for 24 hours after which
mortality is calculated. These are based on LC 99
and again they scal much like ours 5 times 10 times the concentrations for
higher levels of reistance. I've also included a website where you can
find information on ordering kits if you want to try it out.
With this and the CDC bottle bioasssay its very important you use the most
current guidelines specially if you are ordering a kit because they do update them and sometimes
the old guideline that you're using will match with the supplies that you have been sent.
Usually they will send you a upadated guidline in the kit.
CDC bottle bioassay I just wanted to show you
many of you are going to be familiar with this already.
This uses treated bottles, 250 mml bottles.
And its a contact assay so mosquitoes are allowed to fly in the bottle unitl they die over time,
so you are checking at given time points, start with 5 minutes and
after 15 minutes it's every 15 minutes. I just wanted to point out so
let's look at permitherin you can see that the concentration is the same for
all species but the is what changes for each species.
CDC bottle bioassay is fairly easy to run the procedure is simple.
You only need one test to scale the level of resistance which is excellent but
if you do detect resistance it takes a lot of time and effort to collect those data and
also a lot of time and effort to clean the bottles after you're done.
And sometimes the level of resistance you detect is difficult to
connect to fiedl efficacy, although some people are working on
connecting those values better in the future.
WHO test its only checked once every 24 hours it's a little faster in that way.
The diagnostic doses are based on susceptibility curves that are published.
The kits are a little difficult to obtain in the states sometimes.
if they're low on supplies they will
preferentially send kits to more impoverished countries than the US.
You can try to build them yourself as well that takes tools.
You do also have to run multiple tests to detect where to scale
different levels of resistance.
Larvicide bioassays are a little more involved.
So I will run you through our procedure again an adaptation of the WHO prodecudre so we
used to wax-lined paper cups and you can see some pictures on the bottom here is the bioassay setup.
One of the cups is filled with 74 ml of DI water and 50 mg of fish food.
If you dont have a scale that goes to milligrams its
the quarter of a fish food pellet. You want to make sure density of larvae and the amount of
food that they are fed is consistent becasue that will affect mortality and survival.
We add 15 fourth instar larvae to the cup. place the cup small cup into
the larger cup, add the larvicide or active ingredient you're interested in.
And then place a fine piece of fabric over the top of the larger container.
This allows adults to merge in the cup so you can count them, special with methoprene.
you can count the number of adults emerged rather than mortality.
In order to build a susceptibility curve you need generally between 4 and
6 replicates and 5 to 6 concentration.
Finding these concentrations can take a little bit of extra time, its
actually what we are working on right now.
You have to do what I call range finding usually start
at a very very high concentration and do one to ten dilutions
until you find those points where all the larvae are dead or
all the larvae are alive and then if you do 1:2 or 1:5 dilutions between those points.
This does take a little time but it is it is necessary to
figure out your concentration range. You can also look in the literature and
sometines find that there, althought with biopesticides that does vary stock to stock so going to have to play wiht it a little bit.
When running these trials you also need a negative control, this is the control with just
acetone or just DI water or whatever you use as a solute.
Sorry, to make your solute
added so that you can make sure especially with again methoprene can be
a 7 to 9 day trial you want to
make sure that there isn't just natural mortality over that time so that you can correct for it.
Once you have those data you can conduct the probit analysis and determin your diagnostic dose,
again we should have guidlines for that to help people to figure out how to do that
in the next few months.
And once you have that concentration you can test field population
at that one concentration, you dont have to build the curve every time, this allows you to use
far less and test much more broadly than you would if you had to build
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
natural mortality over the time period of the trial.
For collecting mosquitoes for assays you can collect at different life stages.
Adult collection can be advantageous if you are testing for adulticides because
you don't have to hold them for very long but unfortunately you don't know the age
with body condition you can't test larvae without rearing them
and you need to identify each individual which takes a lot of time.
Larval collection is another option.
If you allow them to emerge as adults you know the age of the adults which is good
but you don't know if you're testing for larvicides, you don't know the age of larvae, and
each individual has to be identified again and it requires space for rearing.
The best option for most species is egg collection,
if you're collecting a species like Culex pipiens that lays an egg raft you can allow
you can isolate each egg raft and then just identify one or 2 larvae from the egg raft and you
know the species of the entire egg raft which saves you a lot of time in species identification.
You also know the age of the mosquitoes because you watch the hatch.
And it does require some space for rearing.
As you'll see when John speaks it's not a lot of space and the materials can be fairly
simple which is excellent.
So just a few factors that can affect
the selection of pesticide resistance in your
specific population you want to think about genetic factors. So these are you
know the dominance in frequency of the ressitance gene in the population of whether
those genes are actually expressed and very importantly whether the individuals
with the resistance genes can compete against those without them.
That will determine whether or not when removed the pressure
the susceptible popoulation starts outcompete the resistant population again.
They're also operational considerations these are a lot of things you can
actually control, so the chemical nature of the pesticide and
what has been used historicaly. If you always used pyrethriods
that increases the chance that you're going to see resistance in that population.
Persistence of residues
How strong the application is in the 1st place the life stages that are targeted.
The different modes of application, just how effective they are and how effective they are to treat
and reach the mosquitoes themselves and whether you're actually rotating chemicals or
modes of action. You also have biological factors.
Generation time and reproductive output, so how quickly they reproduce and how many
babies they have essentially, whether they mate once or
multiple times, this is related to reproductive output, distance travelled or
how connected different populations are so if you have an isolated population.
and you carpet bomb it and you see resistance emerge
that resistance may spread in that population but it may not affect aneighboring area.
And then you have refugee or
shelter places behavior mosquitoes are hiding from your pesticide aplication.
And on the environment you're spraying and the species of mosquitoe target.
So when you think about managing pesticide reistnace kinda the 1st thing to do is
start monitoring for it in the field. The best way to do this without
spending all of your time doing this is to target the subset
a subset of sites that you know experience high levels of control activity. So
you know where you're targeting your control activities, go collect mosquitoes from
those sites and test those sites for products you're using and
if possible the products that you could also use so alternative products.
If you start to see developing resistance this would be 96 to 90 percent mortality
in your tests you want to go out and re-test old sites include and include a few new
locations so you want to make sure that your test was accurate.
That resistance that you detected is still maintained in those sites that you already
tested and then you want to test new sites to see if that resistance is
more popular
localized population effect or
if it's actually covering a large portion of your county or state.
If you start to detect resistant mosquitoes
less than 90 percent mortality this is where you want to
look at it intensity testings so figure out is it just low level resistance that
you're seeing kind of cross ranged that may not affect efficacy and
efficacy trial could be helpful here.
You also might if you are starting to detect moderate or high level resistance
you may want to consider alternate options let me let me talk a little bit about those.
So a few general options you have management by moderation using lower dosage
a number of aplications, limiting the amount of pesticide you are putting out into the environment
using chemicals that are short lived so that it can degrade quickly so again lest contact.
In trying to treat a targeted area so that you're not broadcasting in
places where mosquitoes are not high at high densities.
So another option is kind of the opposite managment by saturation.
So the idea here is that you're you're basically carpet bombing.
Eliminating mosquitoes that have resistance genes at low levels before it can emerge
in the populaiton, this can be an issue if
if resistance is already starting to emerge put fuel in the fire and cause it to explode.
So this is something I don't generally suggest you can also
use synergists in the field to
increase the effectivity of your pesticide aplication.
Some of the better options involve management by multiple attackss a mixture of
chemicals, rotation chemicals, mosaics.
I will explain what I mean by that.
So rotation would be switching between different
products with different modes of action.This could be two adulticides an
organophosphate and a pyrithriod it coul also be a pyrithriod and a organopesticide.
You just want to make sure you're not deploying the same products
all the time there are limitations whats registered in what state so
it's important to figure out what the rules are and work within those constraints.
You want to if you use the strategy want to pair with a resistance monitoring for
all products to detect emerging resistance so
that you know that your alternative product will be effective if you have to switch to it.
The timeframe here will kind of vary depending on your own capabilities
operational. You may want to try switching monthly, you may want to switch
season to season, that will again depend on what's viable for your operation.
Mosaic is another option this does require some more resistance testing and
rotation so these black bars that you're seeing here would be like
geographic isolation if you're in Vermont that could be topography
limited movement of mosquitoes. In a city
that could be cement structure that are dividing mosquiote populaitons.
But identify where these populations are and then applying different pesticides
in different areas and switching them over time so you're not always applying the same one.
This requires that you test pockets these these pocket locations for
resistance or multiple products but it can be an excellent option for managing resistance
in a larger area or in a city especially.
And then you have a mixture of insecticide,
this is deployin two products at the same time at the same time.
You do want to test for resistance of those products.
It is possible if you're using 2 pyrithriods, especially that
you need to see if resistance emerged for both of them so if you're going to use 2
products you want to try and use 2 products with different modes of action.
Organophosphates or pyrithriods and if you are using larvivides mehtoprene and bti, a biopesticide and a hormone regulator.
To decrease the chances that you are going to see resistance
to both products at the same time.
That was a lot.
But we're going to hold questions until the end of the webinar and
I'm going to give it over to John to kind of take it away and talk about rearing
a little bit. I will be covering how to establish and maintain mosquito colonies.
What do we need to do in terms of establishing mosquito colonies, what do you
need, kind of need 3 basic things you need space
to have all of these colonies going on, dedicated space often,
you need to have the right equipment and materials to be able to rear mosquitoes
from larvae through adult and you need effort and
I think what some people underestimate is the amount of effort that stablishing and
maintaining mosquito colonies will take of the 3 effort is the most important.
Because you can overcome space and
equipment limitations given the right amount of effort.
Going over this presentation I came across some resources that are going to be
really valuable to you if you haven't worked with colonies in the past
this 1st manuscript is open access
it covers the rearing of Culex speicies and Aedes he's mosquitoes.
It includes a very detailed list of supplies many of
the supplies that I will cover here are also listed in this publication and
many of the techniques are then covered again in detail.
Another resource is the manual for mosquito rearing and
experimental techniques they will basically cover just about everything you
need to know that compiles information from almost every reference.
Up until the early 1990 s from other groups that have
stablished populations. If you want to know for example how people have
attempted to colonize Aedes vexans or
to perturb ns that's the place to go to but it is only currently available for
purchase directly through the American mosquito control Association website.
I'm going to talk a little bit about B.e.i. resources it's a repository
and a service that was established by the National Institute of Allergy and
Infectious Diseases in order to access materials from this
you need to have registration certain eligibility
requirements. The registration and eligibility requirements are mostly nonprofit
public, private or academic research there are permits that will need to be
in place for you to to receive material from this
organization. These are all going to be noninfectious material so
most organizations that would be viewing this webinar would be able to receive
some of the
materials given. Going through the paperwork, there are four
species of mosquitoes available.
For use: Aedes aegypti, Culex quinquefasciatus,
Culex tarsalis and Aedes albopictus. The Aedes albopictus if you look at it
it's a pesticide susceptible colony
that is available Bailable for use. They also have two really
great publications as p.d.f. on their website covering
methods in Aedes research and again these are going to be how to colonize new
species and methods in an Anopheles research which is a really big document covers
everything you'd want to know and more but certainly really good information on
how to establish colonies and how to get
your protocols
in order
o establish colonies and maintain them.
I'm going to talk a little bit about the facilities we have here to house our
colonies.
If you need short term colony
use just you're setting up some larvae to rear through to adulthood to do some pesticide
resistance monitoring all you really need is dedicated bench space this can all be
done in season if you do that you don't need to control for temperature or
photo period you just need an area for trays of larvae in cages of adults.
And you may need to increase the humidity in the cages for adult survival.
And you can do that with damp paper toweling and plastic sheeting.
If you're going to stablish mosquitoes long term you do need dedicated space it's
a much bigger operation you need an incubator or
a growth chamber, you can also use a walk in incubator or climat control room.
Again you'll need controls for temperature and photo period to
help these mosquitoes survive optimally and reproduce optimally.
This is an example of two of the growth chambers we have here.
These are essentially large freezer carcasses
that are adapted to have light and temperature control
their free standing you can maintain one or 2 colonies in each
of these units that we have them control for temperature we maintain them
at 25.5 degrees Celsius or 70 degrees Fahrenheit and
we have a light and dark cycle of 16 hours of light and 8 hours of dark.
Use a fluorescent light source
for the light timing. We also have a dedicated insectary
it provides a lot more space in humidity control and
we will go over our set up down next.
Again we maintain this at about 22 to 25 degrees centigrade
we have
a split system a heat pump cooling pump system in
there we maintain it at about 70 percent relative humidity we have a
humidifier mounted employed in indirectly, we have 16 hours of light and
we have overhead lights that are on a cycle that will start to mimic
dusk and dawn and we also have lights on each shelf unit.
There are 2 shelf units in each room each has light control and
then to simulate dawn and dusk we use a clip on light with a 10 watt
incandescent bulb and that goes on the half hour
before the main light start to turn on and will stay on for
about half an hour or an hour until after the lights are totally out.
This allows us to have things like space in a insectary like an 8 foot cage
the cubic foot cage which we used to house our Culiseta melanura colony.
Excuse me.
For species that are available to use in colony or
really convenient to use in colony I would recommend multivoltine species
in Culex pipiens or quinqs, albopicuts, Aedes aegypti, Aedes triseriatus,
Culex pipiens form molestus and Aeides atroplus are autogenous
mosquitos meaning that if they get enough larval nutrition
you can establish them in colony without needing to have a blood source or
blood meal to have progeny more difficult mosquitoes
Culesita melanura, Culex restuans, Culex salinarues, Culex tarsalis
Aedes japonicus and Anopheles quadirmaculatus.
Again the recommended mosquitoes are all
species that were larvae will readily develop in containers.
Univoltine species can be reared more often from larvae to adult but
are very difficult
to stablish as colonies based on limitations of mating or having them
blood feed or then oviposit
viable eggs for collection and then subsequent
hatch.
Specimens can be collected from the field in a variety of ways.
Ovi-trap is one way
we use a 32 ounce casino Cup lined with a 38 pouts in germination paper.
This will collect a number of Aedes or Ochlerotatus species.
You will have to hatch them out and then Id larvae 2nd 3rd or
4th instars with ovi-traps you can get
depending on where what part of the country you're in the Northeast
we would routinely collect Albopictus in areas where it's present.
Ochlerotatus or Aedes triseratus and Japonicus.
You can also use a container for
collecting Culex egg rafts, we use a grvis trap infusion as our bait and
as James had mentioned earlier you can segregate individual eggs rafts and
then ID larvae a couple larvae from each egg graft as 1st or 2nd instars.
We can also
collect larvae with the traditional dipper you can collect from a variety of
of habitats and then I gear id larvae or
rear them to adulthood and
identify them at that stage. I recently did this
with collecting of some Ochlerotatus stimulans from a rental pool.
I was able to collect them as 2nd instars and rear them up to fourth instart and
then through adult
with just some of the techniques we use in the laboratory here.
You can also use a mechanical aspirator we've had success with a backpack
aspirator and collecting adults of Aedes ablopictus to establish our
colony. You collect them similar to the way you would conduct lading bite
counts in that way you're collecting mated adult females.
You then identify the females after chilling them to immobilize them.
Then you put them into a cage offer them a blood meal and
then collect those eggs hatch and rear the larvae for certain species.
when you're ready to rear a larvae some of the equipment you need are large
pans to rear them in ,larger pans will work best, if you're working with a white
pan that's great if not you want to have a white background. And in for these pans
you want to have a surface area that is greater than the volume that it can hold.
You can also use some inexpensive options such as these 24 ounce takeout containers
or a 16 ounce deli container and with the deli container you're going to want
to only fill that up about an inch or an inch in depth.
Because as the water depth increases sometimes have issues
with some of the species surviving as larvae. Now if you need to source these
you get these smaller white plastic pans from bioquip they are Rubbermaid type
storage containers that are available from discount stores and
if you have white enamel pans you probably have to go back to the 1960 s
to get those. This inexpensive containers are often available
online from webstaruant supply stores.
And you once you establish these Larvae you're going to
need a certain particular diet.
There are multiple options many labs will use Tetramin
tropical fish flake that's finely ground these are relatively inexpensive and
readily available. We use a mixture of liver powder and brewers yeast for
most of our colonies here certainly all of our Aedes and Culex colonies.
You can also use finely ground and sifted pet food and
examples are Koi food, dog food, cat food, rabbit food pellets, and
rat chow you can use various combinations of the above and
a lot of those are going to be in that AMCA manual.
What's worked and what hasn't worked useful to make them in a suspension
rather than applying them dry to the surface of the water and we use it
relatively fresh storing it one to 2 days in the refrigerator between uses.
Now what we'll do is we'll discuss how to hatch eggs once you
once you have them.
Water is important.
You want to use distilled reverse osmosis or
condition tap water that's water that has been left out for about 24 hours so
any of the chlorine that's in a municipal water system
eould dissipate out and not affect larval development.
You want to add the water to the pan.
To a depth of about 4 centimeters or
a little bit more than an inch deep.
4 centimeters of the maximum, eggs in the larval food the general
rule you want to have enough for larval density is one larva for every
cubic centimeter or milliliter and up to 5 larva per square
centimeter of surface area. This is a probably about
the maximum, we try to keep it less than that and
I'll show you some examples of what our densities are. So you have eggs
And on the left there's a close up picture of the eggs on the filter paper that we
collect from our colonies these were eggs that were flooded on Friday and
by Monday we had 2nd or 3rd instars with this zoom close up in
the lower left hand corner of the zoom you can see the filter paper in the water and
multiple 2nd and 3rd instar larvae in the pan and
that would be about the density of larvae that's probably about.
500-700 larvae per pan
as a maximum. This one's probably in the 300-400 range.
For Culex egg rafts figure that each raft is going to be 50 to 200
eggs or more per raft you want to make sure that they're oriented
in the head down position Culex egg rafts will have a natural
concave shape to them you want to make sure that
is maintained as down.
You add water and larval food and the eggs will hatch in 24 to 48 hours.
This is the egg rafts are in the oviposition dish for
our colony then pick them out with a spatula or scoop or
sometimes a wooden applicator stick and then add them to the pan.
And then in a sometimes in as few as 12 to 24 hours
you'll have 1st larvae that will hatch from those eggs and then you.
Want to make sure that in each case that you maintain these and
we will feed our larvae Monday, Wednesday and Friday we increase feeding or
the amount of food that we add as larvae develop from 1st to 3rd instar
often with certain diets especially ones that have a higher fat content
you neeed to skim the surface of the water with a paper towel if a scum layer develops.
If the water is cloudy
you tend to feel less and
you also decrease feeding when pupae 1st develop but you still want to maintain
lower levels of feeding as those 4th instars need to develop into pupae.
Transferring pupae, you pick up puape
as they developed usually 5 to 7 days after hatching in our labs. Use
a pipette either with a tip cut off either a glass or plastic pipette.
In the bottom of the screen we have the pupae that are in the pan.
Then transfer using that plastic pipette.
Hold the paupae out and transfer them into either the bottom of a mosquito breeder or
into a 16 ounce deli cup and then we place those
into a cage and if you look at the larger
cup on the left on the upper
picture you will see that I have included some forth instar larvae in that
this was done on Friday and what we tend to do is use a fish net for
straining out larvae in fourth instars
just because it's time consuming to pick all those larvae and pupae
in order to get them into the cage for the weekend. The last thing you
want to do is come in on Monday morning and have a bunch of adults flying off
from a larval rearing pan.
For the cages for adults we have 2 types that will use readily.
The 30x30x30 centimeter or one cubic foot cage, we use the metal cage
with aluminum screening there is also then a hammock towards the front of that
that you can add either sugar or blood meal sources to it if you desire.
The plastic cage is a bugdorm with a little bit as you
can see the opening to access the inside the cage is a little smaller.
So it for certain things.
Each one has its own advantages and disadvantages.
Inside the cage you're going to need a sugar source.
And we use table sugar we make it up as a ten percenct solution and
stored in refrigerator for up to a week we'll use that sugar syrup
solution from a squirt bottle and will soak cotton balls into one ounce cup or
we will use a cotton wick in a 50 ml Earl Meyer flas. As the sugar
source you can also use things like raisins or apple slices or other fruits.
As sugar or carbohydrate sources for maintaining males and
females you'll also need to put in an open oviposition dish for
Culex we use a cup with water with a little bit of food and for Aedes
we use a cup and we add a fluted filter paper to that.
The mosquitos will oviposit on that damp filter paper.
For adults you can still use either the you can also use the large
mosquito breeder if you need if you have lots of space options and
what we do is we simply take a sugar soaked cotton ball and
put it on the top of mosquito breeder and cover it with a
one ounce plastic cup just to limit evaporation of that sugar water.
Again this is the setup we have in the cage we have ovipositio, dishes.
Pupae that are merging into the cage and then sure Wicks for our Culex colonies.
If you need to remove adults from the cage for the pesticide resistance
monitoring testing you can use a battery powered aspirator
those are one sourced for our lab through quark mosquito control products or
you can use a long powered aspirator So depending on how many
you're how many you're trying to pull out the mechanical aspirator might be
to your benefit certainly if you can afford to purchase them they're about
65-70 dolars at this point. And then if
you want to maintain mosquitoes long term you will need to consider blood feeding.
If you're using a live animal.
Institutional Animal Care and use committee approval is required this
required detail protocols and you need to maintain animals appropriately here at
the experiment Station we maintain guinea pigs and Button quail for blood feeding.
If you're going to use a human subject it is regulated by the us Department of
Health and Human Services.
An institutional review board approval is required.
Even as it may be an occupational activity.
Just to use a human subject to feed to provide a blood meal for
a colony. The paper by Achee
that was published vector borne and zoonotic
diseases will cover a lot of the
operations and considerations that need to be taken into account
for using human subjects for for any purpose in mosquitoe
and vector biology research is really good resource.
If you need to use an artificial blood feeding source you
need to order blood from a supplier we ordered defibrinated sheep blood
although a variety of blood sources are available including cow and chicken.
You need to use an artificial membrane
we use a sausage casing a sugar or pig intestine and
you need to warm the blood to 37 degrees or up to 40 degrees in the picture
on the bottom is a sausage casing filled with blood that's from the the Kauffman
paper that I referenced earlier you have the unfilled
sausage casing on the left and filled the sausage casing on the right and
then that sausage casing can either be put into the cage or into that hammock.
For the aluminum cages that I show.
You can also consider an artificial membrane feeding system
one is a Hemotek and you can use
parafilm or
sausage casing as the membrane and as highlighted there
is an aluminum disc that screws into the bottom that can be filled with blood and
then you stretch a pair of film or the sausage casing over the top and
secure it with a rubber band or an O ring. And then
that gray cylinder maintains the blood at the proper temperature and then the
cylinder and blood meal can either be offered through the screening of the cage
or can be put internal into the cage
in a horizontal fashion.
I can also use a glass feeder these again use parafilm or
sausage casing as a membrane requires a circulating water bath. These
glass feeders are jacketed so
warm water will circulate around an inner chamber that can be filled with blood and
then a again the membrane goes against the screen or
part of the cage and mosquitoes will readily feed or can readily feed through the screen
onto the membrane system. And then once you have
successfully
had mosquitos in
in a cage and fed on an artificial or
natural blood meal you will have to consider storing Aedes eggs
oviposition will occur 3 to 5 days after blood feeding.
And here you can see the eggs on the filter paper again as I described earlier.
You take that filter paper out you remove any adults that might be on the filter
paper and we dry them on a paper towel for 15 to 30 minutes.
Then we fold the paper with the eggs on the inside and
place it into a labeled either whirl pack bag or
you can use a Ziploc bag and then we can store those
in the rearing incubator or the insectary by species
for 1 to 6 months.
Will open then things now to questions and
if you any of you have any questions for me that are not covered
or if you want to
check in with me
as you're trying to establish things just feel free to shoot me an e-mail
at John.Shepard@ct.gov.
All right I'd like to say thank you to our 2 presenters and we will at this
point open up for the Q&A session. I also wanted to highlight just for
those who may not have been aware of our presenters affiliations
Dr James Burtis is our post-doctoral associate working for
The Northeast Regional Center for Excellence in Vector-Borne Diseases
at Dr Laura Harrington's Lab at Cornell University and he is the manager of our
pesticide resistance monitoring program, you can contact him at the information on your
screen or by visiting our Northeast Regional Center website and
John Shepherd has been working with the Connecticut agricultural experiment station
since 1997 where he coordinates the trapping and identification of mosquitoes for
the Connecticut Mosquitoe trapping and arvi-virus surveillance program his contact
information and the ag station website are also on your screen. So with that
we will jump in to our questions the 1st question is what information is there in
regards to whether mosquitoe adulticides affect resistance if pyrethroid
are ubiquitous, in other words if we stop spraying pyrethroids the public and
other industries may continue using them as well.
I can try to answer this one.
So there is some information about the effect of like agricultural pesticides
on resistance mosquito populations and
from early studies it does appear that yes,
when pesticides are deployed broadly by agriculture
or by people who are not mosquitoe control
its possible that resistance can emerge in
mosquito populations even if they're not targeting them.
This is one of the reasons it's really important to get
an idea of basline susceptibility of your population
because it's not always under your control to manage if you have to kind of work around
what resistance if any is already in the population and the of course
the work around is available in our state and
this is why our guidelines are broad you have to kind of have figure out
what is going to work in your specific region or your state or county.
Thank you and our next question is actually for John the question is do you
have any advice in avoiding pathogens in the insectary, an example is epivians.
We haven't had too many issues in terms of having any types of
pathogens you know any fungal contaminants or things of that nature.
The only thing you want to make sure you're doing is
we do clean things down wipe things down on a weekly basis
we don't use any types of disinfectants in our labs we just usually tend to
clean things with with water and scrubbing and let them dry out.
Again you know sometimes with the you may have issues.
With getting some things if you have
some pathogens if you're if you're using food for a long period of time.
You know.
Those we don't have we will change those sugar Wicks or
sugar sources on a weekly basis so we don't have any mold issues.
In the in the insectary
and I guess you could have issues with the food if it's not fresh but
that's one of the things we do is we make it up fresh we go through a lot of
material on a weekly basis so we always have
relatively fresh material going in so I think that's all often helpful for us.
Thank you and I believe your next question is going to be for Dr Brutis so
the question is what life's stage in species sent to you most frequently for
resistance testing.
So last season let's say the bulk of our samples were Culex pipiens testing for
larvicides bti and methoprene.
That being said we're trying to encourage people to test Aedes albopictus
as well and we know that adulticide testing don't it's not just.
You know we want to push people to push.
Request more tests for organophosphates especially.
So that we can know what the general separate bill of the.
Illusion in the northeast looks like considering how widely spread I mean since
I read from it use is you know region our next
question is when you're trying to eighty's eggs are there any other precautions we
need to take to make sure the extract correctly such as temperature humidity.
Sure yeah with eighty's eggs we make it we dry them down so that the filter paper is
damp to the barely damp to the touch and then stored in those sealed bags
if you had it where the where they were where the eggs were to dry or
the paper started to get to dry you can make a humidity chamber by having either.
A wet cotton ball in the corner of a closed container away from
the filter paper and you can kind of you can kind of segregate them that way.
But basically in an airtight container with a little bit of humidity you can
you can bring up the humidity on those eggs so
they don't dry out too much and cause mortality to the eggs.
The next question in our Q After like the heating makes a p.p.s.
unless it squeaks and hybrid you need to get in for creating a fine
i can answer that question like.
So there are good molecular diagnostics for the species and
they're also really good taxonomic features for the larvae at least for the.
Some of the species so.
And of course where you are in the country or what you're going to get x..
So.
There are some in some ways that you can identify those are usually check
our companies to confirm that they are indeed what they are but understandably
it can be difficult for some folks whom you know have access to the p.c. are.
Making a comparison and the next question is is there less so
if they publish the resistance testing results to you and
is that let's say to be able to access to the public.
So we in our initial survey we have asked of no cheering
the resistance results publicly in him most of our collaborators
expressed that they wished us not to we are working on a publication.
Right now that will.
That's based on our survey from last year you know we are offering
co-authorship to people sent us citizens so they.
Are not how we report the results as well
we have to be careful how we how we report them in the.
Community so we don't overstep to make them uncomfortable
with releasing results or share.
I mean in the next question is regarding the mosaic method of pesticide
application you have to factor in the flight range of different populations
how much does flight range very biased you know species.
Sorry I was mean again.
So.
Yes So I'm actually going to ask Lauren to answer the flight range questions.
Answered.
But.
You you do have to account for
flight range also if they count who are geographic isolation like physically how
connected skeeters be those 2 populations already might answering the question
I'm not a doll so typically flavorings for mosquitoes is is very short
typically a couple of 100 meters Max and the one exception are some of
the salt marsh mosquitoes which are known to travel up to a mile or more but for
most of the skiers that you'd be focusing on the plate ranges can be pretty small.
All right well if there's no more questions for
today again we have the contact information of our presenters and
the Northeast Regional Center on your screen so please feel free to reach out
to us as a follow up to today's presentation we are going to be making
the video of today's presentation available on our website as well as.
Print material summarizing our q.
and a session as well as the slides that are presenters today for
you all to access in the future and with that I would like to say thank you again
to our presenters and thank you to everybody who joined us today.