Testing for Resistance In Larval Mosquitos An Overview

Just another WordPress site

Testing for Resistance In Larval Mosquitos An Overview

(soft digital music) – [William] In this video, we will cover the following topics First, the purpose and benefit of carrying out bioassays What information do you acquire about the efficiency and direction of control efforts in your mosquito control program? Second, what supplies do you need to run bioassays that examine the level of resistance in a population of mosquitoes, the various control agents used against the larval stage of the mosquito lifecycle Three, how do you set up a bioassay that has an experimental design that is appropriate for making conclusions about the susceptibility of mosquitoes in your district through larval mosquito control agents Fourth, how do you read the results of the bioassay to determine mortality in the mosquito larvae? Fifth, how do you interpret the bioassay results, including robustness of the results, correcting for mortality in the control treatment, interpreting dose response curves and resistance ratios, and assessing the level of resistance that has evolved in a particular mosquito population Controlling mosquitoes requires that local health departments and vector control organizations have five core capabilities In order to make informed decisions about vector control that reduce the incidence of vector-borne diseases and improve quality of life, agencies carrying out vector control must be able to first, surveil local mosquito populations Second, use surveillance data of mosquitoes and the pathogens that they transmit to inform vector control activities Third, have an action plan to control mosquitoes at all life stages Fourth, use integrated mosquito management that incorporates multiple approaches for vector control And fifth, conduct pesticide resistance testing Practicing informed integrated mosquito management will increase the cost effectiveness of mosquito control activities, assist in choosing the most effective, environmentally friendly method or methods for vector control given the local conditions, and maintain the effectiveness of a comparatively few larvicide and adulticide mosquito control agents in their vector control toolbox The methods used to assess resistance include conventional cup and bottle bioassays, microplate assays, and molecular assays The standard laboratory bioassay, when coupled with biochemical or molecular studies, can help determine the mechanism of resistance In addition to resistance management and susceptibility monitoring, the methods that are described in this video can be used for research investigating the efficacy of new insecticides and new insecticide formulations, product quality control to investigate the consistency of mosquitocidal activity among different batches of the same product, assessing the stability of mosquitocidal activity of product that has been stored for extended periods after purchase, and last, study the biotic and abiotic factors affecting larvicidal activity and efficacy of particular mosquito control agents If we are interested in the mortality of mosquito larvae caused by a particular control agent and we do not know the activity of that control agent against the mosquito population of interest, then to find out the activity range of the control agent, the mosquito larvae are exposed initially to a wide range of test concentrations, together with an untreated control A standard dose response to a larval mosquito control agent should look like this Mortality is plotted on the Y-axis, and a logarithm of dose is plotted on the X-axis Note that there is no mortality of larvae at doses on the left side of the graph On the right side of the graph, mortality reaches a point at which mortality is 100% as dose increases Including doses in both of these regions of the dose response curve in your laboratory bioassay is not informative You want to identify the region in the center of the dose response where mortality increases as a function of dose It is in with this range of doses that you want to target your bioassay You want to use a range of four to five concentrations, yielding between 5% and 95% mortality during the bioassay to determine the LC50 and LC90 values

LC stands for Lethal Concentration The 50 and 90 refer to the concentrations that cause 50% and 90% mortality respectively Typically we want to identify two concentrations above and below the dose that causes 50% mortality in our bioassay test We will linearize the dose response by plotting the number of individuals dying for a known number of larvae exposed at each concentration of the control agent being tested by plotting mortality as probits The percentage of mortality at each of the doses tested in the bioassay is based on a binomial outcome Larvae are either dead or alive The raw dosage mortality data shown in the left panel approximate a cumulative normal curve that is sigmoidal or S-shaped By converting the Y-axis to normal equivalent densities using the probit transformation shown in the middle panel, we will linearize the cumulative normal distribution of mortalities as is shown in the right panel It is much easier to use a line in a curvilinear relationship to carry out inverse prediction, where we are essentially using mortality, the Y variable, to predict dose, the X variable, measured on a logarithmic scale You are interested in the position of the dose response relative to dose, the slope of the line, the fit of the data points to the linear model, among other parameters that we will discuss later The dose response of a population of mosquitoes that is susceptible to the control agent across a range of doses might look like this Mortality increases with an increasing dose of the control agent As the population becomes increasingly resistant to the control agent, the dose response curve will shift to the right The population depicted in the line on the right is comparatively less susceptible to the control agent and is the population whose dose response curve is depicted by the line on the left When a mosquito population becomes highly resistant to a particular control agent, the dose response moves further to the right and may flatten out This indicates that one is unable to obtain meaningful levels of larval mosquito mortality at very high doses of the control agent What agents are used to control mosquitoes in the larval stage? Mosquito larvae occur in standing water The control agents are typically applied to the water’s surface, and active ingredients used to control mosquito larvae fall into three general categories: control agents that must be ingested or eaten by mosquito larvae; control agents that work by contact with mosquito larvae; or control agents that work both through contact and ingestion by mosquito larvae First, there are products derived from bacteria that produce protein precursors that must be ingested by the mosquito larvae and then broken down in the larval digestive tract to form toxins These products are known as bacterial or microbial larvicides Commercial formulations of these biologically derived agents are made from different strains of Bacillus thuringiensis israelensis or Lysinibacillus sphaericus, formally known as Bacillus sphaericus These materials are highly specific in that they are toxic only to mosquitoes and their close relatives at operational application rates Commercial formulations come in a variety of forms: liquids, and different types of granules and briquets The commercial formulations differ in their toxicities The international toxicity units, or ITUs, indicate the toxicity of the formulation to mosquito larvae The higher the international toxicity units, the more toxic is the formulation per unit mass It is important to know the ITU of the formulation that is undergoing bioassay A second category of larval mosquito control agents has a mode of action that requires only contact with the mosquito larvae Juvenile hormone mimics are an example of a control agent that is absorbed through the cuticle Control agents such as monomolecular films that spread across the water’s surface, change the surface tension, and drown the larvae could also be included in this category A third type of control agent works by contact and ingestion Spinosad is derived from a bacterium and consists of neurotoxins for several groups of insects The percentage of Spinosad in formulated products differs For example, Spinosad can range from 2.5 to 20.6% of commercial formulations

In order to calculate the concentration of active ingredient in a bioassay, the investigator needs to know the percentage of the active ingredient in each formulation being assayed How frequently do I need to run bioassays? Mosquito control agents used routinely in mosquito control operations should be evaluated on a regular basis Bioassays are run to establish the baseline level of resistance in local mosquito populations to a new product to be incorporated into a mosquito control program This is more relevant to adulticides than to larvicides because the former are often used to control other insects besides mosquitoes It could be the case that local mosquito populations have been selected for resistance against a widely used control agent, even if that control agent has never been used locally to control mosquitoes Bioassays need to be run more frequently than once or twice each year if you observe that a particular control agent has failed to reduce mosquito populations in the field, or if you are aware that mosquitoes in your area are known to carry genes that confer resistance to a particular control agent For some control agents, resistance to multiple control agents or cross resistance is possible To test the levels of resistance of mosquito larvae, you need the following items: mosquito larvae in the appropriate instar for testing Most bioassays that assess the susceptibility of mosquitoes to comparatively rapid mortality, say within one or two days, to a control agent, use early fourth instar larvae in one day trials, and typically third instars in two day trials For control agents that require ingestion by the larvae, you do not want to use late fourth instars that have ceased feeding prior to pupation Sometimes younger instars, such as second instars, are used in bioassays An important point to keep in mind is that you want to maintain consistency in the instar and husbandry of larvae across all treatments in the bioassay Other supplies that you need to run a bioassay include: plastic, wax-lined, or styrofoam cups containing 100 or 200 milliliters of water When evaluating methoprene or other compounds that are hydrophobic, one needs to take precautions to avoid binding of the control agent to the bioassay cups Silanized glass vessels are recommended Styrene cups also are an alternative bioassay cup Tap water that has been dechlorinated by aging for 24 hours, or distilled water Tap water can be aerated during aging to enhance decholorination Either formulated or technical grade forms of the control agent; 20 milliliter glass vials to mix different concentrations of the active ingredient being tested; pipettes; and screens for transferring the larvae You want to create a range of concentrations of the active ingredient to test against the mosquito larvae, resulting in five to 95% mortality within the exposure period Remember that you also want to include an untreated control in each bioassay As you can see, the range of doses that you will want to target in your bioassay differs among the control agents The range of doses to be tested reflects the inherent toxicity of each formulated control agent Four to five doses should be tested in each bioassay Often concentration of B.t.i and L. Sphaericus products are based on entire formulation with ITU per milligrams specification, while products based on Spinosad and methoprene are typically based on active ingredient levels Most of the time, a stock solution or suspension is made at 1% concentration for a volume of 20 mils then serially diluted at 10 times, taking 2 mils of the stock solution, adding it to 18 mils of distilled water, to appropriate levels depending on treatment concentration ranges needed for a given insecticide As you can see in this table, if you started by mixing 200 milligrams of your control agent and 20 milliliters of water to obtain a 1% solution, the concentration of that initial suspension or solution would be equal to 10 thousand milligrams per liter, or equivalently, 10 thousand parts per million You would carry out 10 full dilutions of each stock solution by adding 2 mils of well-shaken stock solution to 18 mils of distilled water

You continue the dilution process until you obtain a stock solution with the appropriate concentration of control agent for you to obtain the range of concentrations desired in a 100 mil bioassay cup So for example, if you had a .001% stock solution, or 10 milligrams per liter of the control agent, you would add 1000, 500, or 100 microliters of stock solution to cups holding 100 mils of water to obtain final concentrations of 0.1, 0.05, 0.01 milligrams of the control agent per liter If your bioassay cups hold more or less than 100 mils, then you need to adjust the amount of stock solution added to each cup accordingly Let’s run through an example Suppose we want to test four concentrations of a water-dispersible granule formulation of B.t.i against mosquito larvae This formulation is 3000 ITUs per milligram Concentrations used in bioassays of this control agent are typically based on the entire formulated product A 1% stock solution, 200 milligrams of product in 20 mils of diluent, usually distilled water, is equivalent to 10 milligrams per milliliter, or 10 thousand parts per million, is made and then serially diluted as needed 100 to 1000 microliters of diluted stock solution are added to 100 mils of water in replicate bioassay cups Quite often, the treatment concentrations do not go on a 10 times scale, particularly for pesticides with acute toxicity A smaller range of concentrations of control agent is typically required Now let’s suppose the highest concentration that we want to test is equal to 0.1 milligrams per liter of formulated ingredient To obtain 0.1 milligrams per liter of the formulated product in each 100 mil bioassay cup, add 1000 microliters of a 0.001% stock solution to each cup To do this, serially dilute the 1% stock solution three times If we were interested in the amount of B.t.i. in each cup, then we would need to correct for the concentration of B.t.i. in the formulated product Based on the product label, we know that each liter of this formulated control agent contains 37.4% B.t.i Now we also want to have a range of test concentrations that might include the following three concentrations: 0.01 milligrams per liter, 0.025 milligrams per liter, and 0.005 milligrams per liter We would make solutions of these concentrations by adding 100, 250, or 500 microliters of the 0.001% stock solution to each bioassay cup In this example, 50% mortality of the mosquito larvae is expected to occur at about 0.038 milligrams per liter Be careful not to contaminate the water in the cups of the untreated control with your control agent Typically you want to start processing the bioassay cups with the control, then the lowest concentration of the control agent, working your way up to the treatment containing the highest concentration of the control agent in your bioassay Doing so will help to reduce inadvertent contamination among treatments in the bioassay If you plan to compare the level of resistance in a particular field population of mosquitoes to a susceptible population of the same species, then you will need to either maintain a susceptible mosquito colony in your laboratory, or to acquire susceptible individuals from a reputable source To set up the bioassay using the conditions described in this table, add 25 late third instar or early fourth instars to each bioassay cup Mosquito larvae are transferred from the rearing pan to the bioassay cup using a small screen The amount of water transferred with the larvae should be minimized You want to have a minimum of three replicate cups for each concentration in the bioassay, including the untreated control You can use additional replicate cups if you have an adequate supply of larvae which have been raised under the same conditions

The more replicate cups that you use, the more robust are the findings in your bioassay test After you have added the larvae to the bioassay cups, then add the different concentrations of the control agent to the cups In order to avoid contamination, disposable cups are preferred to reusing bioassay containers Remember to follow all required procedures for disposing of materials used in bioassays It is recommended that you repeat the bioassay a minimum of two more times on subsequent days using fresh stock solutions for each test The bioassays are run under a known temperature regime, typically 25 to 28 degrees Celsius Sometimes slightly warmer conditions are maintained in mosquito insectaries A continuously recording temperature sensor can be purchased fairly cheaply Alternatively, a recording maximum-minimum thermometer can be used to keep track of the temperature extremes during the experiment as ambient water temperatures are critical during exposure for ingestion and cuticle absorption of pesticides by mosquito larvae The photoperiod recommended by the WHO for bioassays is 12 hours of light and 12 hours of dark Larvae will need to be fed for long-term bioassays such as required to test insect growth regulators Investigators differ whether or not to feed mosquito larvae during short-term bioassays of 24 hours For bacterial larvicides that must be ingested by larvae, the food levels and organic matter in the environment can influence the outcome of bioassays Whether or not you provide food for the larvae, you want the food supply to be consistent across replicates in each bioassay and among bioassays, especially if you are interested in long-term trends of resistance in your mosquito populations Food level and type need to be consistent and should be quantified For short-term bioassays of 24 hours in length, it is preferable not to add food, but if you do add food, then a couple drops of a 10% rabbit pellet solution can serve as a phagostimulant and avoid starvation of larvae but not cause water quality issues For 48 hour bioassays, larvae could be well-fed for 24 hours before placing larvae in the bioassay cups, and then fed on the morning of the second day of the test For tests of juvenile hormone mimics, a small amount of solid food, such as 100 milligrams of rabbit pellets per bioassay cup is suggested to provide nutrients ’til pupation After a prescribed period of time, mortality of the mosquito larvae is assessed Distinguishing between dead and alive larvae is usually straightforward Alive larvae will maintain their position in the water column or respond to movement Dead larvae usually lie unmoving on the bottom of the bioassay cup Moribund larvae are problematic Is the larva alive or dead? You need to be consistent when dealing with moribund larvae Ones that cannot maintain normal position, do not show the characteristic diving reaction when the water is disturbed, and often exhibit a shrunken body and dark coloration If the larva does not move when touched with a dissection probe or some comparable item at the siphon or cervical region, then consider the larvae to be dead It can be risky to count cadavers as dead larvae can be consumed by survivors during the test period You want to record the number of surviving mosquito larvae on a data sheet Include all relevant information about the conditions of the bioassay on the data sheet Some of the relevant information that should be included is the following: mosquito species, the instar of the larvae used in the bioassay, the location where the mosquitoes were collected, the control agent being tested, the range of concentrations that were tested, the number of mosquito larvae placed initially into each replicate bioassay cup, the duration of the bioassay, or the time since the start of the bioassay that the data are recorded If the control mortality is between 5% and 20%, the mortalities of the treated groups should be corrected according to Abbott’s Formula Tests with control mortality greater than 20% or pupation greater than 10% should be discarded Concentration response data are subject to probit analysis using one of the many computer programs

that offer probit analysis For example, you could use POLO PC or POLO Plus, almost any statistical software to calculate LC50 and LC90 levels, their 95% or 99% confidence intervals, the coefficient of determination, or R-squared to quantify the fit of your bioassay data to the linear probit model, the slope of the dose response line, and population heterogeneity, indicated by the ratio of LC90 over LC50 These parameters vary upon the quality of the data in the bioassay, mosquito species, population genetic diversity, and mode of action of pesticides tested Finally, if you are comparing the dose response of a field population to that of a susceptible laboratory colony, you calculate resistance ratios, which is the LC value for the field population divided by the LC value for the lab population If the resistance ratio is less than five, the field population remains susceptible to the control agent as compared to the laboratory population Resistance ratio can be categorized into three levels: low levels of resistance if the resistance ratio is between five and 20; mid or intermediate levels of resistance occur when the resistance ratio is between 21 and 100; high levels of resistance are indicated by resistance ratios greater than 100 These categories are somewhat arbitrary In practice, one wants to detect the evolution of resistance in mosquito populations early on in the process and implement measures to counteract the further intensification of resistance as soon as possible Let’s look at some bioassay data collected for Qx quinquefasciatus exposed to four different larval mosquito control agents Those response lines for mosquito larvae exposed to B.t.i. with a toxicity of 3000 ITUs per milligram are shown here The LC50 value for the field collected population is 0.075 milligrams per liter The LC50 value of the susceptible laboratory colony is similar but slightly lower at 0.053 milligrams per liter If you compare the 95% confidence intervals for these two values, you can see that the 95% confidence intervals overlap, suggesting that the susceptibilities of the two populations to this control agent do not differ significantly Notice also that the resistance ratio of the two LC50s is only slightly greater than one The coefficient of the termination or R-squared describes how well the bioassay data fit the linear dose response model 0% indicates that the model explains none of the variability of the response data around its need A value of 1.0 indicates that the model explains all, 100% of the variability in the response data In other words, the points fall on the regression line You can see that the fit to the linear model to the data from both bioassays is very good since the R-squared values are near to one The second group of graphs show the response of the same mosquito populations to Lysinibacillus sphaericus Notice that the LC50 values are much smaller than in the previous graphs These mosquito populations are much more susceptible to L. Sphaericus than to B.t.i., even though the potency of the formulation for L. Sphaericus is much lower, only 650 ITUs per milligram as compared to 3000 ITUs per milligram in the B.t.i. formulation The 95% confidence intervals for the LC50s overlap, indicating that the LC50s of the two populations are not statistically significantly different The third group of bioassay results is for the same population and its susceptibility to Spinosad Notice that the LC50s are considerably lower than for the two bacterial larvicides This last group of bioassay results is for the insect growth regulator methoprene Notice that the regression lines are flatter than those shown previously, and the IE50s are in the parts per billion or micrograms per liter range Also notice that the 95% confidence intervals for the field and laboratory populations do not overlap The resistance ratio is 7.6, indicating that the field population shows some resistance to this mosquito control agent as compared

to the susceptible laboratory colony There is a number of factors that influence the outcome of bioassays in mosquito control agents These factors include the material being tested For the same control agent, different formulations have different inherent toxicities The mode of action differs appreciably among bacterial larvicides versus Spinosad versus insect growth regulators Mosquito species is important Mosquito genera and the species can differ appreciably in their inherent susceptibilities to particular larval mosquito control agents Given the factors like the selection pressure from control agents, the extent of genetic exchange among populations, and other factors, different populations of the same mosquito species can differ significantly in their susceptibilities to a particular control agent Age of the larvae and effects of husbandry practices on the quality of larval mosquitoes is also important Water quality, the amount of organic matter in the water and at field-site can influence the efficacy of some control agents Duration of exposure The temperatures that the bioassays are being run Whether food is added to the replicate bioassay cups The type of food, how much food is added, can all affect the outcome of the bioassay In nature, high levels of enrichment in food can reduce the effectiveness of bacterial larvicides that are ingested by mosquito larvae by reducing the amount of bacterially derived proteins ingested per unit time Last, the criteria used for assessing the effect of the control agent on the larval mosquitoes is important Typically mortality is the criterion used However, for IGRs, inhibition of occlosion to the adult stage is one of the criteria used to evaluate the effectiveness of mosquito control Based on the bioassay data results, we can now determine the correct dosage of mosquito control agent for effective control Bioassay, a simple, straightforward process to quantitatively measure the interaction of a lethal agent versus a target organism is an important tool for multiple purposes, ranging from resistance detection and management, active ingredient screening, commercial product evaluation, studies on product stability under harsh conditions, and the efficacy of particular formulations of mosquito control agents and the diversity of larval developmental sites in your district (soft digital music)