Maier et al. also reported similar retention of total anthocyanins in gels stored for 24 weeks at 6 ◦C and 24 ◦C. The lower amount of anthocyanins recovered in the gummies stored at 4.4 ◦C compared to the same product stored at 21 ◦C may be explained by reduced extraction efficiency due to the hardening of the gel at low temperature, as opposed to degradation late during storage. Changes in the major individual anthocyanins in the gummy product stored at 4.4 ◦C and 21 ◦C over eight weeks of storage are shown in Figure S5. At 4.4 ◦C, all the individual anthocyanins decreased with storage time with retentions <50%, except for the two co-eluting anthocyanins galactoside + cyanidin-3- galactoside and malvidin-3-glucoside . The percent retentions of the rest of the anthocyanins at this storage temperature ranged from 29.3% to 49.2%. When stored at 21 ◦C, two anthocyanins did not significantly decrease with time, namely the unknown delphinidin derivative and the two co-eluting compounds galactoside + cyanidin-3- galactoside. For the rest of the anthocyanins, percent retentions ranged from 40% to 71% glucoside. In all the products, the individual anthocyanin loss did not appear to be impacted by the anthocyanidin structure or the type of sugar moiety attached .The distribution of the products according to their individual anthocyanin profile as affected by storage time can be visualized on a PCA scores plot . The first principal component explained 83.9% of the variation with all the individual anthocyanins being positively loaded on PC1. Therefore, PC1 represents the amount of individual anthocyanins.
The juice and ice pop samples had high scores on PC1 . The oatmeal bar samples also had positive scores on PC1 for the earlier storage times,blueberry plant pot whereas the oatmeal bar samples stored at 21 ◦C for eight weeks were the only oatmeal sample to havea negative score. Except for the control samples , all the graham cracker cookie samples had negative scores on PC1, regardless of the storage temperature. Finally, all gummy samples had negative scores on PC1, with scores becoming smaller with storage time. The PCA figure confirmed higher values of anthocyanins in the juice and ice pop samples, as well as, to a lesser extent, the oatmeal bars. The graham cracker cookie and gummy samples did not demonstrate high values for anthocyanins, with a clear loss of anthocyanins with storage time for the gummy samples.Percent polymeric color values typically show an inverse correlation with total anthocyanins during storage of blueberry products , and inverse correlations with each individual anthocyanins in all the products and storage temperature . Higher percent polymeric color values indicate that a higher percentage of anthocyanins are resistant to bleaching in the presence of potassium metabisulfite. Since the sulfonic acid adduct attaches at C4 on the middle heterocyclic ring, it is thought that anthocyanin–procyanidin polymers are formed via a direct condensation reaction, resulting in a C4–C8 anthocyanin–procyanidin linkage as the major polymers formed in blueberries during storage. Hence, it is possible that declines in anthocyanins during storage of the blueberry products are not true losses due to degradation, but the conversion of monomeric anthocyanins to anthocyanin–procyanidin polymers. Anthocyanins can be degraded via a hydration reaction, where the flavylium ion is converted to a hemiketal structure, which is rapidly converted to cis-chalcone, which slowly arranges to a trans-chalcone structure.
The trans-chalcone structure is highly unstable and rapidly degrades to hydroxybenzoic acid derivatives. However, we do not consider that this reaction was responsible for anthocyanin losses in the blueberry products over storage since we did not observe an increase in phenolic acid derivatives in our HPLC chromatograms at 280 nm . The stability of chlorogenic acid in the four blueberry products stored at 4.4 ◦C and 21 ◦C is shown in Figure 5. Chlorogenic acid was stable in all products over storage regardless of storage temperature,except for the juice and oatmeal bar stored at 4.4 ◦C, where levels significantly decreased . At 4.4 ◦C, the chlorogenic acid content decreased from 4.3 to 3.6 mg/g WBB powder in the juice and from 3.0 to 2.6 mg/g WBB powder in the oatmeal bar. At 21 ◦C, chlorogenic acid in the juice showed a slight increasing trend; however, this change was not statistically significant . Chlorogenic acid was also stable in the ice pop over eight weeks of storage at −20 ◦C , with an average value of 6.5 mg/g WBB powder over storage . Initial levels of chlorogenic acid were higher in all products stored at 21 ◦C compared with 4.4 ◦C storage, which may be due to the variation in processing the two sets of samples for the storage study, or possible the degradation of chlorogenic acid in the WBB powder used to prepare the products. The WBB powder used to prepare samples for the refrigerated storage study was stored at 15.5 ◦C for three months prior to preparing the samples. Blueberries contain polyphenol oxidase, which can readily oxidize chlorogenic acid. Chlorogenic acid was previously found to be stable in blueberry juice, puree, and canned berries stored for six months at 25 ◦C, but blueberry jams lost 27% of chlorogenic acid over six months of storage at 25 ◦C. Leaf footed bugs in the genus Leptoglossus Guérin-Méneville are large phytophagous insects native to the Western Hemisphere. At least 61 species are known, and several species are pests in forests or agricultural systems.
Many Leptoglossus spp. are multivoltine, which allows them to exploit multiple hosts per year. Direct damage to crops is caused when Leptoglossus spp. feed by probing their stylets into fruits and seeds, and secondary damage can occur through the transmission of pathogens at the feeding site. Field studies assessing the feeding damage of insects can provide information about the phenology of the pest and pinpoint when during the growing season insect feeding occurs, as well as determine when the crop is susceptible to damage or losses. Two species of Leptoglossus, Leptoglossus clypealis Heidemann and Leptoglossus zonatusare occasional pests feeding on almond and pistachio crops in the Central Valley of California. L. clypealis was considered to have a more limited distribution in the western United States, but is now reported to occur through the Midwest into Illinois,plastic gardening pots with some additional records from the east coast. While L. clypealis is noted in California for infesting almonds and pistachios, it has been recorded from at least twenty host plants throughout its range. L. zonatus is found in much of the Western Hemisphere ranging from Brazil into the southern United States on a wider range of host plants including citrus, pomegranates, almonds, and corn, among others. In California, Leptoglossus spp. are reported to overwinter in adult aggregations. As temperatures warm up in the spring, the adults disperse from aggregations and can be observed in almond orchards. Feeding by L. zonatus and L. clypealis on almonds results in clear sap exuding from developing fruit, known as gummosis. Early season feeding by these two species in March and April can result in almond drop, while feeding later in the growing season can directly damage almond kernels and result in losses. Both L. zonatus and L. clypealis are reported to be more abundant in the last few years, perhaps due to increased plantings of almonds in California. Approximately 1.36 million acres of almonds were cultivated in California in 2017, with an estimated value of $5.6 billion. Determining the level of damage from feeding L. clypealis and L. zonatus during the growing season in a field experiment will help determine the relative damage from each of these insects, and demonstrate when the almond crop is most vulnerable to Leptoglossus feeding, which in turn will help to determine the timing of prevention and control measures.
The objectives of this work were to determine the level of almond drop from feeding by adult L. clypealis and by L. zonatus, compare how almond drop varies during the growing season, consider almond size and its relationship to feeding damage, and quantify the final damage to almonds at harvest time from feeding by L. clypealis and L. zonatus. The effect of adult L. clypealis and L. zonatus feeding was evaluated on four almond varieties during the growing season from the end of March untilmid-August. The four experimental treatments included controls, mechanical damage to the developing almonds, feeding by adult L. clypealis and feeding by adult L. zonatus. All four treatments included an almond branch with approximately 20 almonds, covered by a sleeve cage consisting of a 5-gallon organdy mesh paint strainer closed with a large binder clip. Control branches with almonds served to determine the natural level of almond abscission during the growing season. The second treatment consisted of branches with almonds which were mechanically punctured to mimic feeding damage caused by the insect stylet probing into developing nuts. Each developing almond was punctured 4–5 times with a #1 insect pin . Puncturing almonds served an additional purpose, which was to provide an estimate of the time of shell hardening; shells typically became resistant to puncture by the end of April. The third treatment consisted of 5 adult L. clypealiswhich were allowed to feed for 4–6 d and were then removed. The fourth treatment was similar to the third but used 5 adult L. zonatus . For these treatments, insects were taken from the lab colony, and were first isolated with only water for 24 h before placing them into an experimental sleeve-cage. Each week in each almond variety, four branches were setup as controls, four were setup with punctured almonds, one branch each was setup with L. clypealis, and one with L. zonatus. For approximately eight weeks, the four treatments were replicated in the same manner on new trees in each of the four almond varieties. For Monterey and Carmel varieties in 2014, fields could not be entered on two weeks due to flood irrigation, and this resulted in six weeks of observations rather than 8. In 2015, the same experiment was repeated on the same four varieties, with the exception that feeding damage was assessed for L. zonatus but not for L. clypealis, due to an insufficient numbers of adult L. clypealis to complete the experimental replicates. Each week, data were recorded on the number of almonds fallen from branches within all cages that were setup in previous weeks. These data were used to determine the mean percent almond drop in each of the four treatments for each of the four varieties. An analysis of variance test was considered to compare means, but data were not normally distributed, even after log transformation. Thus, nonparametric Kruskal–Wallis tests were used as they do not assume a distribution for the data. Post-hoc pairwise comparisons were by Steel–Dwaas tests and were considered significant if p < 0.05. In 2015, similar comparisons were made for the mean almond drop for three treatments within each almond variety. To examine when almonds were most susceptible to almond drop from feeding by each Leptoglossus species, the mean percent almond drop was compared among the experimental weeks within each bug feeding treatment. A Chi-square goodness of fit test was used to first examine whether the percent almond drop from a Leptoglossus species was equal among the weeks of the study for each almond variety. If almond maturity had no impact on insect feeding damage, the percent drop would be equal among weeks of the study. When a significant difference among weeks was observed, subsequent tests were by Fisher’s Exact tests to compare pairs of weeks . Just before harvest, the almonds remaining in field cages were removed to conduct a final damage assessment. For each control and each branch with mechanically damaged almonds, a sub-sample of four almonds was used to assess several damage parameters. For branches caged with L. clypealis or L. zonatus, all remaining almonds were removed and used for the final damage assessment. Four parameters of feeding damage were determined, hull strikes, almond kernel necrosis, strikes on the kernel, and shriveled kernels. A strike on the hull was characterized by a black or brown spot. A strike on the kernel was the third type of damage. The fourth and final damage type was whether or not the almond kernel was shriveled. Damage was recorded for each category as presence or absence.