Here we explain the general larval bio-assay protocol used throughout the paper

The walnut industry submitted its research results for a heart health claim to the U.S. Food and Drug Administration , and the almond industry submitted its as part of a petition filed by the International Tree Nut Council Nutrition Research and Education Foundation to the FDA for a heart health claim for nuts. The FDA approved a qualified health claim for walnuts, and another for almonds and other selected nuts, on July 15, 2003, which states: “Scientific evidence suggests but does not prove that eating 1.5 ounces per day of as part of a diet low in saturated fat and cholesterol may reduce the risk of heart disease.” Each of these four commodity groups has used news releases and public relations to publicize the nutrition and health benefits of their products. The messages for walnuts and avocados have been communicated almost entirely through third parties such as magazines, newspapers, doctors, nutritionists or other credible sources, rather than paid advertising. The advertising emphasis for walnuts and avocados has been on quality, taste and recipes. The Almond Board of California initially relied on public relations to disseminate its message on the health and nutritional benefits of almonds. Following FDA approval of their qualified health claim, their research results were incorporated into paid advertising and promotion, almost always featuring a health message.

Likewise, in 2003 the California Strawberry Commission introduced a promotion campaign called “Be Well — Get the Red Edge” , which targets health and nutrition professionals,greenhouse ABS snap clamp and consumer and trade media. Commodity groups have found that they can stretch their promotion budgets by partnering with other groups. After FDA approval of the qualified heart health claim for nuts, the Almond Board of California partnered with the American Heart Association and now makes liberal use of the AHA logo in almond advertising. The California Walnut Commission formed a partnership with the Spanish Heart Foundation, and distributed more than 40,000 brochures and samples during “Heart Week” in Spain. The foundation also includes recipes and other information concerning California walnuts on its Web site. The California Avocado Commission is also leveraging its research results by partnering with health organizations, including the American Diabetes Association, UCLA Nutrition Education, American Association of Diabetes Educators, American Dietetic Association and IDEA Health and Fitness Association. Imports can easily create a “free rider” problem for U.S. commodity programs. California avocado producers spent more than $182 million on market development between 1961 and 2003 . They were understandably upset to see producers in other countries taking advantage of state promotional efforts when the national market share of imported avocadoes increased from less than 3% prior to 1990 to about 34% from 1998 through 2002. With the 2002 passage of the Hass Avocado Promotion and Research Order , all Hass avocados sold in the United States, including imports, are assessed 2.5 cents per pound to fund advertising, promotion, research and data dissemination.

Increased avocado demand due to HAPO promotion will offset much of the price impact of increased imports, and importers should enjoy attractive returns from their promotion dollars. Carman estimated that returns for importers’ spending on advertising and promotion ranged from $2.09 to $6.31 per dollar spent, depending on the level of imports and the effectiveness of Hass avocado advertising.An important and often overlooked benefit of mandated marketing programs, in addition to having an organized commodity group, is the value of the information they gather, organize and disseminate. A first-of-its-kind innovation for commodity groups was the establishment, by the Hass Avocado Board , of a Web-based program designed to exchange crop and marketing information among 100 packers and over 20,000 producers from the five HAB members — California, Chile, Dominican Republic, Mexico and New Zealand. The HAB Web site notes: “The program goal is to develop collaborative strategies to achieve an orderly flow of the 750 million pounds of fruit sent annually into the U.S. marketplace.” This Web-based program collects, tracks, analyzes and disseminates information relevant to selling Hass avocados in the U.S. market. It provides all players in the U.S. market with 24-hour access to critical market data that drives decisions about growing, shipping, distribution and marketing. The HAB market information program is an exciting development in produce marketing, made possible by recent worldwide developments in communication networks. It provides a dramatic reversal of trends that have reduced the availability and timeliness of market and price information for produce markets.

The widespread availability of marketing information and data is a theoretical requirement for competitive markets. It will be interesting to see to what degree improvements in marketing efficiency made possible through HAB flow to producers funding the program. If successful, the program template can be extended to other commodities and countries, with benefits flowing to producers and consumers worldwide.Drosophila suzukii , commonly known as the spotted-wing drosophila, is an invasive pest of small fruit and berry production native to Southeast Asia. Starting in 2008, this species underwent a rapid geographic range expansion, spreading across North America, South America, and Europe. Following its arrival to the USA, growers in California, Oregon, and Washington reported yield losses of up to 50% in raspberries and blackberries, 40% in blueberries, and 33% in cherries. Unlike other Drosophila species that typically breed on overripe and rotting fruit, D. suzukii females readily lay eggs into firm, still-ripening fruit using specialized serrated ovipositors. After hatching, larvae continue to feed and develop within the fruit, causing rapid decay and increased susceptibility to subsequent infestation by other drosophilids. Efforts to control D. suzukii and prevent infestation have led to a drastic increase in the use of broad-spectrum insecticide applications in susceptible host crops. Specifically, three main classes of insecticide–organophosphates, pyrethroids, and spinosyns–have shown high efficacy and now form the basis of most conventional D. suzukii management programs. Carbamates and some diamides have also shown moderate efficacy against D. suzukii, but the use of these materials is more limited throughout North America and Europe. The continued reliance on chemical sprays to control this pest, combined with its high fecundity and rapid development time, have elicited concerns that insecticide resistance could quickly develop. For organic growers, the risk of resistance is even greater. Currently, spinosad is the only insecticide approved by the Organic Materials Review Institute with high efficacy against D. suzukii, meaning that growers must rotate spinosad sprays with low-efficacy materials such as pyrethrins and azadirachtin in order to comply with label requirements. Consequently, D. suzukii in organic fields are primarily challenged by only one class of insecticide further increasing the risk of resistance. Indeed, recent work by Gress and Zalom reported low to moderate levels of spinosad resistance in D. suzukii collected from commercial caneberry fields near Watsonville, CA. Specifically, this study utilized dose-response analysis of adult D. suzukii to quantify the degree of resistance and found that Watsonville females exhibited LC50s 5–8 times higher than females from a second, untreated location in California. This finding demonstrates an immediate need to expand our understanding of resistance evolution in D. suzukii and to develop novel tools that aid in the early detection and assessment of resistant populations so that appropriate management actions can be implemented. When decreased susceptibility to an insecticide is first detected in a pest field population,snap clamps ABS pvc pipe clip laboratory selection studies, often referred to as resistance risk assessments, represent a critical first step towards understanding the potential for resistance to develop. These studies are performed by exposing large numbers of individuals from the suspect field population to moderately lethal concentrations of insecticide for multiple generations such that the most susceptible individuals are eliminated and the most tolerant survive. Dose-response analyses are performed both before and after implementing the selection protocol, and the change in LC50 is used to quantify the response to selection and better understand the potential for resistance to develop. This approach, however, is not without its limitations. For example, because laboratory colonies contain limited genetic variation relative to large field populations, these studies can understate the evolutionary potential for resistance to develop in a field setting.

Conversely, selection imposed in laboratory settings may be more intense and consistent than in the field, leading to possible overestimation of resistance development. Nevertheless, an increase in resistance due to laboratory selection provides unambiguous evidence that further loss of susceptibility in the field is possible. Additionally, if laboratory selection is successful and resistant colonies are generated, these strains can assist researchers in the development of novel tools for detecting, monitoring, and managing resistance in the field. For example, resistant colonies can be used to identify the molecular mechanism that produces the resistant phenotype, and these markers can, in some instances, be used to track spatiotemporal patterns of resistance development in the field. Laboratory-generated resistant colonies can also be used to assess whether insecticide resistance in a particular population is associated with fitness costs , and this information can be used to develop programs to manage resistance in the field. The presence of such costs under benign conditions would suggest that efforts to temporarily eliminate the target insecticide from spray programs could successfully maintain susceptibility in the field, at least in the short-term. After decreased spinosad susceptibility in the Watsonville, CA population was identified, Gress and Zalom performed a resistance risk assessment by continuing to expose adults from this population to a discriminating dose of spinosad using a standard glass vial bio-assay protocol developed for D. suzukii . After implementing this technique for 5 generations, Gress and Zalom found that LC50 values increased by 86% for males and 49% for females. This selection protocol, however, is highly labor intensive and costly, making it impractical for long-term selection programs which are needed to generate and maintain resistant lines. Additionally, this method requires that researchers kill off much of their adult population each generation, increasing the risk that selection lines will be lost in the process. Here, we describe a simple larval bio-assay that overcomes these challenges and requires little effort or cost beyond what is already needed for basic laboratory stock maintenance. We then perform a series of larval dose-response bio-assays to calibrate the selection protocol and identify baseline susceptibility for three commonly used insecticides. Finally, we implement the larval bio-assay protocol to perform resistance risk assessments using the same Watsonville, CA field strain previously tested by Gress and Zalom. Changes in the susceptibility at both the larval and adult life stages were quantified, and results from this study are compared to those obtained with the original adult selection method.First, groups of n = 20 mated females were transferred into plastic 6 oz Drosophila stock bottles containing fresh Bloomington standard Drosophila cornmeal diet. Females were allowed to lay eggs for four days before being transferred into new bottles with fresh diet. Once females were removed, 400 μL of insecticide solution was pipetted onto the surface of the diet, and each bottle was shaken sideto-side to distribute the solution. This ensured that all larvae were uniformly exposed to the insecticide, both through external contact and internally while feeding on the treated diet. Control bottles were treated with either ddH2O or acetone, depending on the insecticide treatment , but otherwise were handled using the same protocol as experimental bottles. All bottles were then re-plugged with a cotton stopper and stored in a climate-controlled walk in chamber at 22–23˚C and 14–10 light-dark cycle. Because the first larvae typically crawl out of the diet to pupate after 6 days under these conditions , treatment timing is critical to ensure that all larvae are exposed. Bottles were monitored for up to 18 days following treatment for the emergence of adult D. suzukii, and all newly enclosed adults were transferred to fresh bottles and counted.To quantify baseline susceptibility of D. suzukii larvae, we used the larval bio-assay protocol to perform dose-response analyses for three insecticide AIs, each representing a different insecticide class, commonly used against this pest. This work was conducted using a susceptible laboratory strain of D. suzukii originally collected from USDA Wolfskill Germplasm Repository in Winters, CA in fall of 2017. Wolfskill is an experimental, mixed-fruit orchard, and crops at this location receive no insecticide treatment. This strain of D. suzukii was previously used as a susceptible control by Gress and Zalom when quantifying resistance in the Watsonville population. The three formulated insecticides used in this study were malathion , spinosad , and zeta-cypermethrin .