The Effect of Synthetic Pyrethroid Insecticides on Honey Bees in Indiana: Laboratory Studies and a Survey of Beekeepers and Pesticide Applicators

The Effect of Synthetic Pyrethroid Insecticides on Honey Bees in Indiana: Laboratory Studies and a Survey of Beekeepers and Pesticide Applicators

The Effect of Synthetic Pyrethroid Insecticides on Honey Bees in Indiana: Laboratory Studies and a Survey of Beekeepers and Pesticide Applicators

A Thesis Submitted to the Faculty of Purdue University by

William Eugene Chaney

In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy.

August 1988

ACKNOWLEDGMENTS

I wish to express my appreciation to my major professor, and friend, Dr. Rich Edwards, for his support in my graduate work, my professional career, and my personal life. I would also like to thank Dr. Eldon Ortman for his support as head of the Department of Entomology. A special thanks to Dr. Marlin Bergman for his encouragement and to Dr. Harry Potter for his help in the survey portion of this work. My gratitude also to Dr. Virgil Anderson who made a statistical difference in my graduate education and to Dr. Tom Jordan for stepping in at the last minute.

Most importantly, I would like to thank several outstanding individuals without whom this work could have never been done. They are, in no particular order: Carl Geiger, Terry McCain, Suzie Appel, Mark Johnson, Mike Gale, Steve Mroczkiewicz and Connie Holderfield.

Abstract

CHANEY, WILLIAM EUGENE. PhD., Purdue University, August 1988. The Effect of Synthetic Pyrethroid Insecticides on Honey Bees in Indiana: Laboratory Studies and a Survey of Beekeepers and Pesticide Applicators.

Major Professor: C. Richard Edwards.

Insecticides are an important component of the row crop production system in Indiana. Concern for the safe use of these products has lead to a system of regulating the application of pesticides that is designed to protect the public, the environment and the applicator. One non-target organism that is affected by some pesticide applications is the honey bee. Because of its social nature, the impact of pesticides on bees is sometimes expressed as detrimental effects on the colony to which the exposed bee delivers her contaminated nectar or honey.

This study looked at several aspects of the honey bee/pesticide problem, including one class of insecticides about which there is controversy concerning their impact on bees. This class is the synthetic pyrethroids. These studies found that the relative toxicity to adult bees of the four products examined was: permethrin> flucythrinate > fenvalerate > fluvalinate, in decreasing toxicity. The toxicity of these products was also shown to increase at 18 degrees C and 12 degrees C as compared to their toxicity at 25 degrees C. These are temperatures in a range which might be experienced by bees in a colony in Indiana during the winter.

This study also demonstrated that no synergism or antagonism was seen when permethrin and fluvalinate were fed to adult bees together with carbaryl, paraquat or mancozeb. This study did demonstrate that some colonies were more resistant to permethrin and carbaryl than others and that this resistance was related to the race of the queen heading the colony.

Beekeepers, public pesticide applicators and private pesticide applicators were surveyed to examine their knowledge of and attitudes toward the poisoning of honey bee colonies by pesticides. The response rate was not significantly different among the groups. The mean response rate was 75%. Less than 10% of the beekeepers and none of the applicators reported any knowledge of specific incidents in which bees were poisoned by pesticides in 1986. Both the beekeepers and the applicators were concerned about this issue and both groups indicated a willingness to take specific actions to attempt to prevent future poisonings. Each of the three groups showed a poor level of knowledge about pesticides as they relate to bees and about integrated pest management.

Introduction

CHANEY, WILLIAM EUGENE. PhD., Purdue University, August 1988. The Effect of Synthetic Pyrethroid Insecticides on Honey Bees in Indiana: Laboratory Studies and a Survey of Beekeepers and Pesticide Applicators.



Insecticides are applied annually to a high percentage of the cultivated acres in Indiana. Some of these insecticides are applied in a manner and at a time as to expose non-target organisms, including the honey bee, to direct sprays or residues (Atkins 1979, E.H.Erickson 1983a). Concern over the impact of pesticides on the beekeeping industry has been expressed by leaders of that industry (Ambrose 1983; Atkins 1980; Crane 1983; E.H.Erickson 1983a, B.J. Erickson 1984a, 1984b; Knol 1983; Stevenson 1978, and others). The public is also becoming more concerned about the impact of pesticides on the environment as evidenced by increased regulation pesticide applicators face arid the removal of products from the marketplace (Adkinsson 1971, Pimentel 1980). As many older, more persistent, chlorinated hydrocarbons were removed from use, farmers turned to newer shorter lived insecticides and often found more applications were necessary to achieve acceptable control.

One class of insecticides that contains many of the newly registered insecticides is the synthetic pyrethroids. As a group, the synthetic pyrethroids are loosely related by their chemistry and mode of action on the target pests. Within the group are a wide range of products that have diverse target pests. Most of these products are characterized by their relatively low mammalian toxicity and their effectiveness against invertebrate pests at low doses (Sine 1988). In Indiana, these products are being utilized in the pest management programs of a growing number of corn, soybean and alfalfa farmers.

Some synthetic pyrethroids are reported to be quite safe to honeybees in some areas of the United States (Atkins 1979, Johansen 1983, Moffet 1982, Stoner 1984). Early evidence indicates that the toxicity of some synthetic pyrethroids to honeybees may be greater in Midwestern areas than in warmer more arid parts of the country (B.J.Erickson 1983; E.H.Erickson 1983b,1984; Smart 1982). The fact that the toxicity of some synthetic pyrethroids is inversely related to temperature (Georghiou 1964, Morton 1979) and may be important in a contaminated honey bee colony’s ability to overwinter in the midwest. It was also evident from early studies that the toxicity of this group to any particular species was very diverse (Atkins 1981, Moffet 1982, Smart 1982). For example, some of these products are highly toxic to mites and are used as miticides (Herbert 1988, Witherell 1988), while others are so safe to mites as to actually increase their population (Flaherty 1981, Flint 1985).

As farmers have become more specialized producers of a declining range of crops, the direct importance of bees as pollinators on the farm has also been declining. While some studies indicated that soybean yields might benefit from insect pollination (Abrams 1978, Erickson 1978, Mason 1979), other crops such as corn, alfalfa grown for hay and wheat require no insect pollination. As these crops captured an increasing proportion of the acreage, the number of nectar producing plants plummeted. The decrease in forested acres, the intensive planting of non-nectar producing plants such as fescue and crown vetch on roadsides and in pastures, the intensive use of herbicides in cropland and the increasing urban demand for land, seriously reduced the nectar resources available to bees. This combined with the low world honey price over the past several years, has driven nearly all commercial beekeepers from many parts of Indiana.

Most of the remaining beekeepers in Indiana are hobbyist or part-time beekeepers who keep bees for pleasure as well as profit. For these beekeepers, the impact of pesticides on their bees is a highly-charged, emotional issue. Much misunderstanding exists between beekeepers and applicators and there is considerable misinformation on both sides. Since discontinuation of the federal governments Beekeeper Indemnification Program, there has been very little effort to evaluate, document, or record reports of pesticide poisonings of honey bee colonies, unless litigation was instituted or threatened (Coleman 1979, Pimentel 1981). It is generally believed, but undocumented, that beekeepers have overestimated the severity of the problem, while pesticide applicatiors have underestimated the extent of the problem.

In Indiana, application of those products which have been classed by the United States Environmental Protection Agency (EPA) as “Restricted Use Pesticides is regulated by the State Chemist’s Office. This office has the responsibility for enforcing the laws and regulations relating to pesticides including pesticide applicator testing and certification.

Because many individuals who apply pesticides may never use a restricted product, many private applicators are not required to be trained or show competency in pesticide use. The Indiana Cooperative Extension Service and the Office of the State Chemist have worked together to train pesticides applicators in the safe handling and application of pesticides, as well as safety to the environment. Pesticide applicators are further divided into those who apply pesticides for hire and those who use the products only in conjunction with their own crop production operation. These groups are referred to as Public Pesticide Applicators and Private Pesticide Applicators, respectively.

Among the information which is required for certification is knowledge of the safe handling and use of pesticides, including their toxicity to non-target organisms such as the honey bee. The issue of honey bee poisoning is a complicated one and can not be covered in depth during applicator training due to time constraints.

The factors which determine the extent to which a given colony of honey bees will be affected by the application of a pesticide to a given field are complex (Atkins 1981, Johansen 1977, Lieberman 1964, Quatitlebaum 1983). The single most important factor is the number of bees from a particular hive that are foraging in the treated area (Nowakawski 1982). This is influenced by many factors such as attractiveness of the crop treated, presence or absence of blooming weeds in the area, distance from the treated field to the colony, strength of the colony, weather, needs of the particular colony, genetic make-up of the colony, etc (Atkins 1977, B.J. Erickson 1983a, Mayer 1983, Mayland 1970, Smirle 1987, Ross 1981, Wailer 1984).

In addition to factors relating to the honey bee colony, factors relating to the pesticide such as the active ingredient, the formulation, the time of application, the method of application, the weather conditions during and following application, etc., will all influence the extent to which a given colony will be affected. There is the additional complicating factor that some pesticide products may cause no observable damage at the time of application, but may cause delayed mortality of the overwintering colony during a period of greater stress.

The synthetic pyrethroid insecticides are of particular concern in this regard due to the inverse relationship between their toxicity and temperature (Yu 1984). Lehner (in press) has shown that the toxicity of permethrin to bees dramatically increases at 20 degrees C over the toxicity at 26 degrees C. Delayed mortality may often not be detected or identified as a result of earlier pesticide exposure. The insecticide stored in the hive may not singularly cause colony mortality, but may act in conjunction with other factors to increase the stress on the hive and cause a decline of the population. This decline may or may not be reversed by the colony as weather and other conditions improve, depending on their reserve strength and size of the initial population.

An understanding by both beekeepers and pesticide applicators of the factors that influence poisoning of colonies of honey bees by pesticides is critical to establishing a situation in which the two groups can operate without conflict. Because this is such an emotional issue, it is often difficult to separate emotion from fact when discussing this subject with either side. Given the complexity of the problem and the limited resources available to try to deal with the situation, a multi-faceted approach to the problem was undertaken. This included laboratory work to examine some the the most critical questions relating to synthetic pyrethroids and bees as well as an examination of the groups of people directly involved; that is beekeepers, farmers and public pesticide applicators in Indiana.

Chapter One: Relative Toxicities and Temperature

CHANEY, WILLIAM EUGENE. PhD., Purdue University, August 1988. The Effect of Synthetic Pyrethroid Insecticides on Honey Bees in Indiana: Laboratory Studies and a Survey of Beekeepers and Pesticide Applicators.


This part of the study was designed to answer two basic questions important to further studies. These were: 1) what are the relative effects of four common synthetic pyrethroid insecticides on adult honey bees and 2) what impact does temperature have on each of these products. The synthetic pyrethroids chosen to represent this insecticide class were: fenvalerate (Pydrin 2.4EC), permethrin (Ambush 2E), flucythrinate (Pay-Off 2.5EC) and fluvalinate (Spur 2E). Formulated product at five dosages was fed in 50% sucrose syrup to caged adult bees held in different growth chambers at three temperatures (25 degrees C, 18 degrees C and 12 degrees C). Toxicity was measured by monitoring the number of dead bees daily for five days.Background and Objectives

Since the introduction of synthetic pyrethroid insecticides, there has been controversy concerning their impact on honeybees. Early reports indicated that these products were safe to bees because they repelled the foraging workers (Atkins 1977, Bos 1983, Moffet 1982, Stoner 1984). Later evidence indicated that the reduced number of foragers observed in treated areas was not due to repellency, but to a disruption of the normal system of communication among foragers or to direct mortality of foraging bees (Rieth 1986, Cox 1987). Because honey bees tend to work the same nectar source until it is depleted, the gradual return of foragers to the treated area could be due to a new set of foraging bees being recruited to the area or due to a loss of repellency as the product degraded.

While this question was still unresolved, reports of serious mortality of bees and the eventual death of colonies of bees exposed to synthetic pyrethroid insecticides began to surface. Laboratory tests indicated that some of these products were toxic to bees when applied topically or ingested, yet field studies did not show the expected damaging effects (Atkins 1981, Johansen 1983, Moffett 1982, Smart 1982, Stevenson 1978). Many of the reports of damage from synthetic pyrethroid insecticides were undocumented because the beekeepers involved did not feel that there was a reasonable chance of reimbursement. Since the USDA Beekeeper Indemnification Program had been terminated, few beekeepers sought restitution for pesticide damage through other legal means (Happ 1971).

The greatest number of reported poisonings came from the midwestern and eastern states. Geographical differences in toxicity to insecticides have been documented in relation to honeybees. As an example, methomyl was found to be safe to honeybees in western states (Atkins, 1979) but highly toxic when used in Wisconsin in sunflower fields leading to massive bee kills (Krause 1983). The differences in toxicity were attributed to the higher humidity of Wisconsin and increased incidences of heavy dew on treated plants which made the pesticide more available to the foraging bees.

In addition to immediate bees kills of greater magnitude, reports by northern beekeepers indicated a greater incidence of winter mortality in colonies exposed to synthetic pyrethroid insecticides during the previous season. If indeed the foraging bees were returning to the hive with nectar or pollen contaminated with sub-lethal doses of synthetic pyrethroid insecticides, perhaps the lower colony temperature in winter was resulting in these doses becoming lethal due to the inverse relationship between the toxicity of synthetic pyrethrbid insecticides and temperature.

In winter, bees control their temperature by forming a cluster. This cluster is roughly a sphere which expands or contracts to regulate the temperature on the surface at about 8 degrees C (45 degrees F) (Owens 1971, Szabo 1985). The center of the cluster may be considerably warmer, depending on the rate of heat loss. In honeybee colonies in the Midwest, the rearing of immatures, or brood, normally begins in January or early February. There is commonly no brood present from October to that time. After the initiation of brood rearing, the temperature at the center of the cluster is maintained at 33 degrees C to 35 degrees C (92-94 degrees F) (Owens 1971, Szabo 1985). Bees move from the inner areas of the cluster to the outer surface in a slow but constant rotation.

The method of storage of food by honeybees, both honey – the carbohydrate source, and pollen – the protein source, is important in this situation. Nectar is collected from flowers and contains 15-35% sugars. The bees evaporate the excess water from the nectar to increase this sugar concentration to about 80%. During this process, the conversion of most of the 12-carbon sugars to 6-carbon sugars is accomplished by a bee supplied esterase. The resulting product, now correctly called honey, is very stable chemically and biologically. The sugar concentration makes it unsuitable for yeast, fungal or bacterial growth. If pesticides contaminated nectar, they are concentrated in the process of producing honey and end up in a quite stable environment (Barker 1980, Winterlin 1973).

Pollen is also collected from the flowers and stored in the cells. It has been shown to be carried to the hive contaminated with pesticides (Johansen 1972, Mayer 1983, Rhodes 1980, Winterlin 1973). The bees add nectar to this pollen to make it more workable and often seal it under a cap of honey. This situation is also very stable chemically and biologically. Unlike the honey, pollen is not concentrated and is usually consumed only by very young bees and those involved in caring for larvae. Under normal conditions in this highly social colony, most bees spend a period of their early lives caring for larvae. This period coincides with the greatest activity of the hypopharyngeal gland in the head. It is also the period when the most pollen is consumed. In winter when brood rearing is initiated, older bees are often needed to care for larvae and they again begin to consume large amounts of pollen. These older bees are also more likely to have previously been exposed to pesticides than young bees reared in the spring.

In the winter cluster, bees are able to maintain temperature by consuming honey stored from the previous season. If this honey is contaminated with pesticides, it is possible that bees moving from the center where they consumed contaminated honey to the outside of the cluster, could experience a temperature drop from 35 degrees C to 7 degrees C (95 degrees F to 45 degrees F). One of the objectives of this study was to look at the toxicity of the selected products at temperatures within this range.

There were also conflicting reports as to the relative toxicity to bees of the two most commonly available synthetic pyrethroid insecticides of the time, permethrin and fenvalerate (Atkins 1979, B.J.Erickson 1983b, Smart 1982, Stoner 1985). Manufacturers were also seeking registrations for new products for agricultural use. Two of these newer products were chosen because they represented both ends of the spectrum as to bee toxicity. These products were fluvalinate and flucythrinate.

The objectives of this first study were:

  1. To devise an easy method of testing the toxicity of synthetic pyrethroid insecticides to adult honeybees in the laboratory
  2. To determine the relative toxicity of the four selected synthetic pyrethroid insecticides
  3. To determine the effect of three temperature ranges on the toxicity of the selected synthetic pyrethroid insecticides

Materials and Methods

Adult honey bee workers were collected from a single colony headed by an Italian queen purchased from a commercial queen breeder. The queen was introduced into the colony approximately 120 days before the start of the experiments. Adult bees were collected from the honey supers during mid-morning on days when the colonies were in active flight. Collected bees were held without food in one gallon paper Fonda Cups (cardboard ice cream cartons) with screen tops for ventilation until needed.

The bees were anesthetized with CO2 and then counted out directly into one half pint Fonda cups containing a vial of 50% sucrose syrup with the assigned concentration of formulated pesticide. The bees were handled by a leg with larval forceps. Any worker bees appearing damaged or exhibited unusual behavior in any way or any drone bees were discarded.

The cups were filled in numerical order, each cup having been randomly assigned a treatment after numbering. The cups were provided with a seven-dram glass vial of sucrose solution with a perforated plastic cap to serve as a feeder when inverted. The inverted vials were held in place on the side of the cup above the floor by a piece of rubber band stapled on either end to the walls of the cup. The top was replaced by a piece of netting to provide ventilation and to facilitate observation of the bees.

The assigned pesticide treatment was prepared just prior to the introduction of the bees by mixing formulated pesticide with 50% sucrose solution at room temperature with a magnetic stirrer. Dilutions of a stock 1000 PPM solution were used to make concentrations of 100, 33, 10, 3 and 1 PPM. The formulated product was obtained from the manufacturing company without the knowledge of the company as to the nature or purpose of the experiment. Product manufactured for the current year was obtained and the concentration was assumed to be as stated on the label.

Once the cups were filled with the 25 adult bees, the bees were allowed to recover from the anesthesia and recounted. The very occasional bee which did not appear to be behaving normally was replaced. As soon as all the bees had recovered, the cups were moved to one of three environmental chambers that held at 12 degrees C, 18 degrees C or 25 degrees C (± 1,25 degrees C). Since only one temperature could be maintained in any one chamber, the design was a split plot randomized complete block design with randomization restricted by temperature. The three replications were made with each of the three chambers held at each of the temperatures.

Within cup variation was estimated by replication of a selected portion of the treatments in any given replication. These were chosen so that when combined they would represent an additional complete set of treatments. These replicates were randomized within the assigned temperatures.

The bees were held in darkness within the chambers and at 60-70% relative humidity. Mortality was determined at 12 and 24 hours the first day and each 24 hours thereafter for 5 days, for a total of 6 determinations. Bees were considered to be dead if they did not respond to a gentle puff of breath. The CO2 in human breath normally produces a fanning response in worker honeybees.

Preliminary studies had shown that the test insecticides, even at concentrations of 1000 PPM in 50% sucrose solution, did not have any fumigant effect within the growth chambers. The toxicity of the insecticide-tainted sucrose solution did change the bees willingness to consume the solution. The toxicity of the insecticide held in solution for seven days was not different from that of solution made fresh the day of introduction. New solutions were prepared for each replication, however.

Results and Discussion

The relative toxicity of the four synthetic pyrethroid insecticides was found to be constant in nearly all situations after the first observation. Regardless of the temperature, concentration or day of observation, the relative order of toxicity from most toxic to least toxic was: permethrin, flucythrinate, fenvalerate and fluvalinate. The product which showed the greatest actual difference in toxicity between the temperature ranges examined was fenvalerate. It was found that the test procedure was acceptable at 25 degrees C and 18 degrees C. The bees were not observed to cluster at 12 degrees C as they would in the hive and normal behavior was not observed. At 12 degrees C the bees became so inactive that feeding was reduced significantly, resulting in lower mortalities than those observed at 18 degrees C. Even under these conditions, however, the relative order of the toxicities of the test insecticides was unchanged. An accurate test at this temperature would require the use of at least three frame broodless colonies. Normal clustering behavior would be initiated and would have to be controlled so as to not raise the temperature of the bees in the cluster.

Data Handling

The ANOVA, GLM (General Linear Model) and TTEST procedures of SAS (Statistical Analysis System) were used for data analysis as appropriate. The GLM procedure was used when unequal cell sizes were present. The TTEST procedure was used to examine the variance between cups within a treatment. The ANOVA procedure was used for all other tests. Duncan’s Multiple Range Test (DMRT) at the 5% level was used to indicate significance of differences between means tested together.

Because the number of dead bees tended to converge on 100% from the higher concentrations of the more toxic products, the mean number of dead bees observed over the five days presented a clearer picture of the relative toxicities. Since the bees were exposed to the test insecticide over the entire time, this mean percentage of dead bees better represented the threat these products present to the bees in an overwintering colony. The mean number of dead bees per observation was used for all calculations unless otherwise noted and results are reported as the mean percentage of dead bees per observation.

Duplicates of one-third of each of the treatments were used to evaluate the within-treatment, between-cup variance. Analysis showed this to be such a minor contribution to total variance (0.96%) that these observations were used as a fourth replicate in further analysis, even though one-third had been run simultaneously with each of the three other replications. Analysis using only the first three replicates did not give different results than using all four.

Overall Results.

Figure 1 The mean percentage of dead bees per observation by product.Figure 1 The mean percentage of dead bees per observation by product.

Figure 1 shows the mean percentage of test dead bees at each observation period and the standard deviation of this measurement. This represents the mortality for each product at all five concentrations and at all three temperature ranges. The relative order of the toxicity of the products was the same in nearly all of the situations examined. Permethrin was most toxic to the bees followed by flucythrinate, fenvalerate and fluvalinate, in that order. Each of the insecticides was significantly different from the others at the 5% level using Duncans Multiple Range test. Fluvalinate was not significantly different from the control, however.

Figure 2 The mean percentage of dead bees per observation for each treatment-concentration combinationFigure 2 The mean percentage of dead bees per observation for each treatment-concentration combination

Figure 2 shows the mean percentage of dead bees per observation for each treatment by concentration. Cups receiving pure 50% sucrose solution as a control were previously assigned to a concentration for analysis. In an analysis of variance by product, concentration was a significant source of variation at a 0.01 level for all insecticides, including fluvalinate, which was not significantly different from the control in an overall analysis.

An analysis of variance by concentration (Table 1) showed significant differences between flucythrinate and fenvalerate only at a concentration of 33 PPM, even though overall means were significantly different. The only concentration at which fluvalinate and the control were significantly different was at 100 PPM. At all concentrations permethrin was significantly more toxic than the other products. Analysis using a pooled estimate of all observations of the value for the control did not change the significance of any differences. The LC50 values calculated using a log-probit analysis were: permethrin – 2.6 PPM, flucythrinate – 8.4 PPM, fenvalerate – 14.7 PPM, and fluvalinate – 799.6 PPM. Each mean represents 6 observations over 5 days for 12 cups, 3 for each of 4 replications, for a total of 120 observations.

Table 1. The mean percentage of dead bees per observation for each treatment by concentration.
Mean percentage of dead bees*
Treatment 1PPM 3PPM 10PPM 33PPM 100PPM
Permethrin 36.0a 49.6a 80.1a 84.7a 91.7a
Flucythrinate 26.6b 34.3b 49.5b 74.2b 77.4b
Fenvalerate 24.4b 31.3b 41.9b 60.5c 71.0b
Fluvalinate 23.5b 21.8c 24.0c 34.3d 40.1c
Control 18.9b 19.3c 20.5c 20.1d 23.5d
*Means within a column followed by the same letter are not significantly different at the 5% level by the Duncan’s Multiple Range Test

Temperature Effects.

Figure 3

 

The effect of reduced temperature was to increase the toxicity of all of the materials tested over the toxicity observed at 25 degrees C (Figure 3) . The only exception was the mean number of dead bees per observation for permethrin at 12 degrees C, which was lower than for the same product at 25 degrees C. As previously explained, the results at 12 degrees C were affected by the extremely reduced bee activity and food consumption at this temperature. Permethrin caused such a high mortality so quickly that even the relatively high mortality at 12 degrees C was lower than that observed at 25 degrees C or 18 degrees C. The insecticide which showed the greatest increase in the mean percentage of dead bees was fluvalinate, the least toxic of the four synthetic pyrethroid insecticides tested. Fenvalerate showed nearly as large an increase.

The values for the LC50 of the three most toxic products at 25 and 18 degrees C are shown in Figure 4. This figure points out the increasing numerical differences in toxicity at 18 degrees C compared to 25 degrees C for the less toxic materials. The values for fluvalinate are not shown because of the great difference in magnitude. The values for fluvalinate dropped from an LC50 at 25 degrees C of 800 PPM to 615 PPM at 18 degrees C, however, consistent with the previous observation. This decrease in LC50 for fluvalinate is a 23% drop in LC50 compared to drops in LC5O’s of 83.4%, 86.1%and 96.2% for fenvalerate, flucythrinate and permethrin, respectively.

Figure 4

Figure 4

Permethrin was significantly more toxic than any of the other treatments at all temperatures (Table 2). Flucythrinate was significantly more toxic than fenvalerate and fluvalinate was significantly more toxic than the control at 18 degrees C only. The relative order of toxicities was the same as had been seen at each concentration and overall for all of the temperatures examined. Each mean represents 6 observations over 5 days of 20 cups, 5 in each of 4 replications, for a total of 120 observations.

Table 2. The mean percentage of dead bees per observation for each treatment at three temperatures.
Mean percentage of dead bees*
Treatment 25 Degrees C 18 Degrees C 12 Degrees C
Permethrin 65.3a 80.8a 59.2a
Flucythrinate 43.8b 66.3b 47.1b
Fenvalerate 32.3c 59.3c 45.8b
Fluvalinate 13.7d 41.3d 31.2c
Control 13.6d 27.4e 25.7c
*Means within a column followed by the same letter are not significantly different at the 5% level by the Duncan’s Multiple Range Test

Discussion.

Permethrin was found to be the most toxic of the four synthetic pyrethroid insecticides tested at all temperatures and at all concentrations. It was also found to have the largest percentage increase in toxicity with a temperature change from 25 degrees C to 18 degrees C. The extremely low LC50 at 18 degrees C of 0.202 PPM for permethrin certainly suggests that even very small concentrations of permethrin in nectar, collected at temperatures often 25 degrees C or higher, then concentrated by the bees in the process of ripening of nectar to honey, could produce significant mortality in overwintering colonies of bees. If one considers the relative importance of an individual member of a colony of bees in December or January compared to the value of an individual worker in May, June or July, the implications for a significant impact of even the smallest contamination of nectar by permethrin become even more staggering.

Even using optimistic estimates of the rate of degradation of the insecticide while in storage in a solution that is nearly 80% sugars, naturally antibiotic, and sealed within a dark wax cell, the concentration of permethrin in nectar which would result in a 0.202 PPM concentration in the resulting honey, is very small. If we assume a best case scenario of 30% sugar in nectar and a degradation of 80% of the permethrin over the period from collection to consumption, a concentration of only 0.38 PPM permethrin in nectar will result in honey containing the LC50 of permethrin at 18 degrees C.

Flucythrinate was found to be significantly more toxic than fenvalerate overall and at a concentration of 33 PPM, while at other tested concentrations the differences were not significant. Flucythrinate was found to result in a numerically greater mean percentage of dead bees per observation than fenvalerate at all concentrations. The LC50 of the fenvalerate was approximately double that of flucythrinate at both 25 degrees C and 18 degrees C, however. The percentage of change between the two temperature ranges were approximately equal. Using the same best case scenario as outlined for permethrin, a concentration of 8.8 PPM of fenvalerate or 3.45 PPM of flucythrinate in nectar would result in the LC50 of each being reached at 18 degrees C. These concentrations are in the range of the concentrations of fenvalerate found by Erickson (personal commun.) in nectar being brought back to hives in Wisconsin by bees foraging on sunflowers treated with Pydrin (fenvalerate).

Fluvalinate was significantly less toxic than any of the other synthetic pyrethroid insecticides tested at any concentration or at any temperature It was not found to be significantly different from the control of 50% sucrose except at a concentration of 100 PPM. The LC50 of 615 PPM at 18 degrees C should indicate that even in a worst case scenario considering no degradation of the product after being collected by the bees in nectar and concentrated into honey, the likelihood of a bee receiving a lethal dose is very small. The fact that the product did exhibit some increased toxicity to bees at 18 degrees C should be noted however, especially in light of the recent registration of fluvalinate as a miticide for use in live colonies of bees for control of the varroa mite, Varroa jacobosoni (Oudemans) (Herbert 1987).

The threat to colonies of honey bees in Indiana from synthetic pyrethroid insecticides as suggested by reports by beekeepers was supported by the results of these tests. The LC50′s calculated from the data are certainly not difficult to visualize as a possibility in honey made by colonies foraging on plants treated intentionally or unintentionally with synthetic pyrethroid insecticides. Despite the failure of this methodology to adequately measure the toxicity at temperatures below 18 degrees C, it is reasonable to assume that the same products would show even lower LC50′s at the lower extreme of the temperature range a bee might encounter in a hive over the winter in Indiana.

 

 

Chapter Two: Pesticide Interactions

As an oversimplification, it may be stated that all pesticides work by interrupting some vital life system in the pest. This same action may also impact a non-target organism such as a honey bee, a bird or the pesticide applicator. Although different pesticides may have different modes of action, the combined effects of seemingly unrelated pesticides can sometimes give surprising results. Because the numerical reality is that no one person, group or research effort could examine all the possible combinations of pesticides on any one organism, this study chose to look at the effect of three common pesticides on bees simultaneously exposed to synthetic pyrethroid insecticides at a sublethal dose.

Background and Objectives

Because honey bee colonies are often exposed to several different pesticides during the year, the question of the interaction of synthetic pyrethroid insecticides and other pesticides becomes important in assessing the total impact on bees. Some of these other pesticides may be other synthetic pyrethroid insecticides, some may be other types of insecticides and some may be fungicides or herbicides which by themselves may not be particularly harmful to honey bees (Moffett 1972, Morton 1974, Stevenson 1978, Stoner 1985). Synergism of the toxicity of insecticides to dipterous pests by herbicides was documented by Lichtenstein (1973) but was not seen by Sonnet (1978) with carbamate and organophosphate insecticides fed in combination with various herbicides to honey bees.

In general, honeybees keep nectar from various sources largely segregated in the combs of the hive. That is, within a given cell which makes up the comb, usually only honey from a single floral source is stored. Further, usually the cells of a given area of the hive are filled at the same time and will represent those honeys made from nectar collected in a rather narrow time range. Most of the honey consumed by the colony over the winter is stored in an area of the hive referred to as the brood nest. This is the area of the hive in which brood is reared through the spring and summer.

In the fall as brood production declines, the bees begin to fill cells no longer needed for brood production with the honey that is being made at the time. This generally leads to honey from various sources being stored in concentric circular areas in the brood nest. Later in the season, the bees will begin to move honey stored in other parts of the hive into the brood nest in preparation for winter. These honeys come from a variety of nectar sources, and may be contaminated with a variety of pesticides. The possibility of pesticide interactions in the honey bee colony is increased by their unique method of storing and utilizing food. In the winter, the cluster may be exposed to any of these products simultaneously.

The importance of interactions between unrelated compounds is well documented. The natural pyrethrins, from which the synthetic pyrethroid insecticides take their name, are isolated from the flowers of certain chrysanthemum plants. They are known for their very quick knockdown of many pest species. They are often formulated in combination with piperonyl butoxide, a compound which by itself has little toxicity to insects. Together, the piperonyl butoxide acts as a synergist increasing the toxicity of the pyrethrins by a factor greater than the additive toxicities would suggest.

The behavior of bees as a unit is highly dependent on chemical cues being passed from individual to individual in the colony. Sharing food among worker bees as they pass in the colony facilitates this form of communication. Because of this food exchange behavior and the pattern of food storage in the brood nest, it is likely that any individual worker may be consuming honey from a number of sources on any one day. In order to simulate this, a study of the effect of exposure to synthetic pyrethroid insecticides simultaneously with one of three types of pesticides was devised.

The fungicide chosen was mancozeb, the active ingredient in the product Dithane M-45, among others. This product is frequently used in vegetable and fruit production (Sine 1988). Bees are attracted to blooming orchards in the spring at a time when they are replenishing honey stores depleted by the winter. Honey produced at this time may be stored for long periods. Because stored honey must be diluted with water prior to consumption by adult bees or for feeding larvae, nectar is preferred as a food over stored honey. If subsequent conditions are good, honey reserves are not needed to sustain the colony through poor nectar flows later in the season. Surpluses that are produced in good nectar years in the spring and stored in empty cells in the brood nest are likely to remain there until winter.

The herbicide chosen was paraquat, the active ingredient in the product Gramoxone 1.5EC. This product was chosen because it is a nonselective contact herbicide used to kill growing vegetation and because it has a higher toxicity to animals than most herbicides (Sine 1988). The areas in which it is applied are more likely to contain vegetation in bloom which is attractive to bees than preplant, preemergence or early postemergence herbicides. It is used throughout the growing season and has seen increased use as the practice of no-till, or reduced tillage farming has become popular in Indiana. In this practice weeds are killed with a herbicide, such as paraquat, in preparation for planting into ground that is not tilled. This may occur early in the season in the case of full season planting or later in the year in preparation for a second or double crop on land from which one crop has already been harvested. In either case, the possibility of some weeds being in bloom and attractive to the bees is high.

The insecticide chosen was Sevin 50W, a formulation of carbaryl, which is a common, general purpose insecticide that is especially toxic to bees. This product is used in nearly all crops, but is of special interest in some parts of Indiana because of the frequency with which it is used in soybeans for control of leaf feeding insects. Soybeans are often treated while in bloom and are often attractive to honeybees as nectar producing plants (Erickson 1979, Kettle 1979, Robacker 1983). Carbaryl is also used in other attractive crops such as sweetcorn, melons, cucumbers and in home gardens and orchards.

Because of the number of pesticide combinations to be examined, only two synthetic pyrethroid insecticides, permethrin and fluvalinate, were used. These products represented the most and least toxic of the insecticides examined in the previous study. The lower temperature range of 12 degrees C was also dropped because of the results of the previous work and to reduce the number of treatments. All of the pesticides were examined at two concentrations, 1 and 10 PPM.

The objective of this study was to determine if there was any synergism or antagonism between the products. This was determined by examining the mortality of bees fed a synthetic pyrethroid insecticide in combination with another pesticide in comparison to the mortality of the two products alone.

Materials and Methods

Bees were collected from the same colony as used in the previous study. The study was conducted one year later, but was headed by the same queen as evidenced by markings placed on her thorax at the time of her introduction to the colony. Bees were collected and handled as previously described. Twenty five bees were again used in cups constructed as before. Each treatment was tested at 18 degrees C and 25 degrees C.

Table 3

Table 3

The experimental design was again a split plot randomized complete block design with the restriction on randomization being the two temperature ranges. Treatments (Table 3) consisted of the assigned pesticides mixed in 50% sucrose solution and prepared to give concentrations of 1 or 10 PPM as assigned when mixed with equal volumes of the paired pesticide. All solutions were prepared fresh before the trial with dilutions of 1000 PPM solutions. The study was replicated four times, with each temperature repeated twice in each of two environmental chambers.

Formulated product was used for all solutions assuming the concentration stated on the packaging to be correct. Fresh formulated product was obtained from the manufacturer before the start of the experiment. Tests for possible fumigant action of the products in 50% sucrose indicated no effect from a concentration of 1000 PPM. Concentrations of 1 PPM and 10 PPM were tested for stability in 50% sucrose solution for seven days and had no significant decrease in toxicity.

Bees were checked for mortality at 12 and 24 hours following introduction to the cups and each 24 hours thereafter for 5 days. As previously described, bees not responding to gentle stimuli were considered dead. Two environmental chambers were used for the trial and each chamber was kept at 18 degrees C for two replications and 25 degrees C for the other two replications. The bees were kept in the dark throughout the experiment and at 50% to 70% relative humidity.

Results and Discussion

No evidence of synergism or antagonism was shown in any of the combinations examined. The toxicities of the fungicide and herbicide were not found to be significantly different from the control of 50% sucrose under the conditions examined. Bee mortality for the treatments containing permethrin showed higher mortality at 18 degrees C than at 25 degrees C, as expected from the previous tests.

Figure 6

Figure 6

Figure 5

Figure 5

Data Handling.

The ANOVA and GLM procedures of SAS were used for data analysis. As in the previous study, mortality is indicated by a mean of the percentage of bees dead at each of the six observations made over five days. Means of three or more groups were tested for significance of differences with the Duncan’s Multiple Range Test with Type I error rate set at 5%.

Overall Results.

Permethrin and carbaryl were the most toxic (Figure 5) and were not significantly different from each other at the 5% level under the conditions tested. There were also no significant differences between fluvalinate, paraquat, mancozeb or the control of 50% sucrose. A control was grouped with both the synthetic pyrethroid insecticides and the three other pesticides and assigned to both concentrations for completeness of balance in design.

As expected from the previous work, mortality was higher at 18 degrees C than 25 degrees C for all treatments containing permethrin. Figure 6 shows the mean percentage of dead bees per observation for all the treatments by pesticide and temperature. That is, all observations of bees exposed to permethrin, in any combination, at 25 degrees C are represented by the first bar of the graph.

Figure 7

Figure 7

Because the value for each pesticide is a mean of all occurrences of that product in combination with all others in a balanced design, much of the increased mortality at lower temperature is due to the combination of that product with premethrin. The mean percentage of dead bees per observation for each of the 48 treatments is given in Figure 7.

Pesticide Interactions.

Comparing the expected mortality of permethrin or fluvalinate with the other pesticides did not indicate that any synergism or interaction was present. The expected mortality was determined by adding the increased mortality over the control for the non-pyrethroid to the increased mortality of the pyrethroid over the control. This calculated, expected value was compared to the observed mortality of the combination minus the control mortality estimated by the control/control mean.

For example, the permethrin/control mean mortality was 43.13%, a 32.17% increase attributable to permethrin over the control/control combination value of 10.96%. The carbaryl/control combination gave a 41.65% mean mortality indicating that 41.65% – 10.96%, or 30.69% was due to carbaryl.

Therefore, the expected mean percent mortality of the permethrin/carbaryl combination was 32.17% + 30.69% or 62.86%. The observed mean percentage of dead bees per observation for the permethrin/carbaryl combination was 70.63%. This value minus the control mortality (70.63% – 10.96%) was 59.67% is the observed combination mortality.

Discussion.

The results of this study indicate that two of the more toxic insecticides to bees, permethrin and carbaryl, have an additive and not a multiplicative toxicity to adult bees. It was also shown that any effects of paraquat and mancozeb are very small in terms of adult mortality and these products do not significantly change the impact of permethrin or fluvalinate on bees. This is especially interesting in light of the perception of many beekeepers and applicators about the impact of herbicides and fungicides on honeybees (see Chapter 4).

Chapter Three: Interhive Variability

CHANEY, WILLIAM EUGENE. PhD., Purdue University, August 1988. The Effect of Synthetic Pyrethroid Insecticides on Honey Bees in Indiana: Laboratory Studies and a Survey of Beekeepers and Pesticide Applicators.


Since the 1960′s, resistance of insects to a number of insecticides has been documented. Resistance of houseflies to DDT was the first in a long and continuing series of pest species which have become resistant to chemical controls. Only recently has this phenomenon been exploited to select for pesticide resistant beneficial insects and mites. It might be expected that honey bees could also be selected for resistance to pesticides. This study concentrated on examining various populations of honey bees for resistance to synthetic pyrethroid insecticides in all three races of bees commonly sold in North America.

Background and Objectives

The development of resistance in insect populations is influenced by a number of factors. These include the generation time of the species, the amount of insecticide pressure on the population, the mobility of the species, the amount of outbreeding with populations not resistant and whether or not the females mate with more than one male (Georghiou 1980). In a population of leafminers in a greenhouse environment for example, it can be seen how each of these factors favors the development of resistance. A population of honey bees presents a very different picture, however.

Genetic changes in honey bees colonies only occur when the queen is replaced (Collins 1980). This may happen under several conditions, but, it most commonly occurs when the colony swarms or when the old queen is superseded. In managed colonies, the intercession of the beekeeper in replacing the queen must also be considered. The most frequently this might be expected to occur would be once every year or two. This is a very long time considering the development of insecticide resistance.

It is possible that colonies exposed to pesticides will be more likely to supersede their queen. This does provide the opportunity for the selection of a larva that is surviving at a time when the colony is challenged by an insecticide. Neither swarming or beekeeper replacement of queens is likely to provide any selection for insecticide resistance. Only strong colonies are likely to swarm and it may be assumed that colonies exposed to substantial levels of insecticide would not be strong enough to swarm. Beekeepers do not have a commercial source of pesticide resistant queens available. Queens are not likely to come from colonies which have been selected for any trait other than honey production. Beekeepers raising their own queens may be able to do some indirect selection for pesticide resistance by choosing colonies that perform well in areas of pesticide use.

The mating behavior of queen honey bees is also not well suited for the development of insecticide resistance. The virgin queen leaves the hive at a few days of age and mates with 5 to 10 drones. From this mating she will store all the sperm necessary for egg laying for her entire life. The drones she mates with are likely to be from colonies other than her own, and may come from colonies several miles away. Because drone bees do not contribute to the welfare of a colony, weaker colonies are likely to produce fewer drones than strong colonies. Therefore colonies challenged by insecticides are not likely to produce as many drones as other colonies.

The development of resistance by selection also requires the assumption that some level of natural resistance exists in the population (Graves 1965). In honey bees a number of traits have been selected for at one time or another (Rothenbuhler 1980). Some of these programs have been quite successful, such as breeding for increased pollen collection (Boelter 1984). Other honey bee queen breeders claim to have strains selected for resistance to various honey bee diseases, honey producing ability, overwintering ability, a preferred color or resistance to some pesticides (Tucker 1980). Most of these traits have been selected for from within one race; others from the offspring of selected crosses. Differential resistance to carbaryl was shown between lines of Apis mellifera ligustica Spin, and A. m. scutellata Lepeltier by Danka (1986). The relative impact of race and previous selection pressure on the two lines was not addressed by Danka.

In the United States, the honey bee genetic pool has been closed since 1912 when Congress closed the United States to the importation of live bees. This was done to prevent the introduction of a mite pest which was having devastating effects on the honey bee population of England at the time (Phillips 1925, Rennie 1921). The border was not closed to Canada. Canada had adopted similar laws, but had excluded New Zealand as well as the U.S. and therefore genetic material could come to the U.S. from New Zealand via Canada. Since 1912, there have been some very limited importations under permit from the USDA as well.

At the time the border was closed in 1912, there were four races of bees known to be present in the United States (Culliney 1983, Severson 1985). These were Apis mellifera ligustica Spin or the Italian, by far the most popular and common; A. m. mellifera L. or the German or Black bee, the first race imported by settlers but an aggressive and unpopular race; A. m. caucasica Gorb. or the Caucasian, a gentle grey bee noted for its ability to overwinter well but not popular for other reasons; and A. m. carnica Pollmann or the Carniolan, also a gentle grey bee but noted for its tendency to swarm. None of these races were genetically pure, with the possible exception of the Italian. The lack of artificial insemination at the time and the sheer number of italian bees, made keeping pure lines of the other races nearly impossible.

Over time A. m. mellifera became nearly impossible to find in a distinguishable form, although its influence on some strains is still detectable. Renewed interest in the grey races (A. m. carnica and A. m. causica) resulted in some breeders selecting strains that showed morphological and behavioral characteristics very close to those of these races as seen in their native areas (Carlisle 1955). Instrumental insemination greatly aided this work because it allowed the use of single drone controlled matings. Today relative pure lines of A. m. ligustica, A. m. laucasica and A. m. carnica are available in the U.S.

A long term selection program for honey bee lines was started in England in 1912 when the native strains were decimated by the Isle of Wright Disease, now believed to be a combination of infection with Nosema apis Zander and infestation with Acarapis woodii Renie (Bullamore 1922). This program was carried out by Brother Adam at Buckfast Abbey. Brother Adam has traveled the world looking for different genetic lines of bees that might contribute to his breeding program and he has imported a number of these races and strains to Buckfast Abbey. His intensive selection and crossing have resulted in a world famous line of bees now known as Buckfast. This line was imported under permit into the United States as eggs which were reared into virgin queens and inseminated with semen also imported under special permit from the USDA. The Buckfast bee is now sold by a single licensed breeder in the U.S. (Sugden 1983). These queens are mated naturally to wild drones, however, so that their progeny are only 50% Buckfast genotype.

The objectives of this study were:

  1. To look for resistance to one or more synthetic pyrethroid insecticides in genetic lines of bees representing all three common races found in North America and a line representing a long term selection program from England.
  2. To determine if any resistance found was related to race of the colony, possible previous selection pressure or simply individual variation between colonies.

Materials and Methods

In the summer of the year previous to the study, colonies of bees were established in a common site near West Lafayette. This was accomplished by removing frames of brood from established colonies and introducing marked queens representing the different lines selected for the study. All of these colonies were established in new hives so that previous pesticide exposure would not be a factor. The old frames removed from other colonies and used to establish the new hives were replaced with new frames as soon as possible.

The queens selected were as follows: two caucasian lines, one each from two breeders; two carniolian lines, one each from two breeders; two buckfast queens from the sole breeder in the U.S.; two Italians from one breeder and two Italians from hives in a high pesticide use area of southern Indiana which had not been requeened for at least seven years. These ten lines allowed comparisons between races, between queens from the same breeder, and between bees which could be reasonably assumed to have been under selection pressure and those not under such pressure within the same race or from other races.

The seven treatments consisted of a control of 50% sucrose and carbaryl as Sevin 50W, permethrin as Ambush 2EC and fluvalinate as Spur 2.4 EC in 50% sucrose at 1 and 10 PPM each. The bees were handled and mortality measured as previously described. All bees were held in the dark in a single environmental chamber at 25 degrees C. The study was conducted as a randomized complete block design and replicated four times. Each treatment was applied to a single test unit (cup of 25 bees) except for one fourth of the treatments which were duplicated to provide an estimate of between cup variance. The duplicated treatments comprised a fifth complete set of insecticide-bee source combinations which were randomly assigned to a replication.

Results and Discussion

Significant differences were found between the different sources of bees examined. These differences were related to the race of the colony. There was surprising little variation between the different sources of the same race. No significant differences in susceptibility were found in the hives which were assumed to have been exposed to insecticides and those which were assumed to not have been exposed.

Data Handling.

The data were analyzed using the ANOVA, GLM and MEANS procedures of SAS. Mortality is presented as the mean percent of bees dead at each of six observations made over five days. Means were tested for significant differences using Duncan’s Multiple Range test with the error rate set at 5%. Two data points of the 350 combinations of 10 hives and 7 treatments for five replications were missing due to the loss of one or more observations used to calculate the mean percentage of bees dead per observation. The significance of differences did not change if the fifth replication was dropped.

Hive to Hive Variation.

There was considerable variation between the hives as to their susceptibility to the products tested (Figure 14). Differences in overall mortality as measured by the mean percentage of bees dead per observation (Table 4) were found to be significant at the 5% level. The ANOVA indicated that variance between duplicate cups within a hive/treatment/replication combination was not a significant component of the total variance (<1%). Analysis by treatment showed that differences between hives were significant for only two treatments, carbaryl and permethrin at 10 PPM (Table 5).

Analysis of Racial Differences.

The influence of race on the hive differences was assessed by grouping the hives into Italian, Caucasian, Carniolan and Buckfast. While this latter group is not a true race, it does represent a different genetic lineage than the other hives and does not fit well into any of the other races, although each of these races was used at some point in the development of the Buckfast bee.

Figure 15 shows the mean percent mortality by race. Each race is represented by two hives except Italian which was represented by four hives. The Italian and Caucasian races were significantly different from the Carniolan and Buckfast races. Further analysis of the four hives grouped as Italian was conducted by splitting the group into Indiana and California strains. Since the Indiana strain was assumed to have been exposed to the materials in question, while the California strain was selected from breeding stock in Canada in an area of no pesticide use, differences representing selection pressure should have been evident between these two groups. The means and standard deviations of the Indiana and California strains were 13.4%(6.1) and 15.9%(7.4), respectively. The difference was not significant the 5% level.

The racial differences were also examined by treatment. Figure 16 shows the mean percent mortality by treatment. The treatments were: Trt. 1 – carbaryl 1PPM, Trt. 2 – carbaryl 10PPM, Trt. 3 – permethrin, Trt. 4 – permethrin 10PPM, Trt. 5 – fluvalinate 1PPM, Trt. 6 – fluvalinate 10PPM, and Trt. 7 – the control of 50% sucrose only. The Italian race was significantly more sensitive to carbaryl than any other race. The Caucasian race was significantly more sensitive to permethrin than the other races and while not significant, showed an interesting sensitivity to fluvalinate at 10 PPM.

Discussion.

Racial differences in susceptibility to the treatments examined were noted. It was especially interesting to note that the Italian race, the most popular in the United States, was the most susceptible to carbaryl and more susceptible to permethrin than the Carniolan and Buckfast races. The similarity between the Buckfast race and the Carniolan is not surprising since the Carniolan was a major line used by Brother Adam in developing the Buckfast bee. It was also very interesting to note the small amount of variation between the different sources of a race compared to racial differences.

Since the so-named Indiana and California strains of the Italian race were not positively known to have represented lines selected under insecticide pressure and a lack of such pressure respectively, the lack of significance of the difference should not be taken to suggest that selection for increased tolerance to carbaryl or the synthetic pyrethroid insecticides would not be effective. The racial differences would suggest that racial hybrids should be considered in any breeding program whose aim was increased resistance to insecticides.

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