Food Suitability and Population Dynamics of Lorryia formosa (Acari: Tydeidae)

Food Suitability and Population Dynamics of Lorryia formosa (Acari: Tydeidae) Abstract Lorryia formosa Cooreman (Acari: Tydeidae) is a species of mite commonly associated with citrus in many countries including the United States. A survey report in 1957 suggested phytophagous nature, while other studies claimed that L. formosa populations are associated with honeydew producing insects and sooty mold and it acts as a sanitizing agent. We investigated the effect of various diets on the survival and progeny production of L. formosa on excised leaves and the survival and potential to cause feeding damage to leaves of potted plants in a greenhouse study. A 2-yr field survey of a mandarin orchard was also conducted to elucidate the seasonal infestation, damage potential and population structure of L. formosa in a natural habitat. Results showed that all L. formosa adults and immatures died in less than 14 d on excised leaves, did not survive beyond 7 d on potted citrus plants alone, and caused no observable feeding damage to leaves or fruit. When sugar water, honeydew, or cottony cushion scale, Icerya purchasi Maskell (Hemiptera: Margarodidae), was present, adults and immatures survived the duration of the experiments and produced additional generations. The field survey showed that all stages of L. formosa were present in a mandarin orchard throughout the year and insecticide applications affected but did not eliminate mite populations. Fruit generally had a greater percentage infestation of mites (44.8 ± 4.0) than leaves (16.0 ± 4.7). These studies confirmed that L. formosa cannot sustain a population on leaf tissue alone and is nondamaging to citrus in California. Lorryia formosa, citrus, population structure, honeydew, sooty mold Lorryia formosa Cooreman (Acari: Tydeidae) is a mite species in the family Tydeidae that is found in citrus orchards worldwide (Smirnoff 1957, Aguilar-Piedra 2001, Badii et al. 2001). This species was originally described from Morocco where it had been reported in large numbers from citrus trees infested with Saissetia oleae (Olivier) (Hemiptera: Coccidae) and mites were reported to be associated with S. oleae nymphs producing honeydew (Smirnoff 1957). In the United States, (Aguilar-Piedra 2001) reported L. formosa as the most common Tydeidae mite in citrus orchards in Florida, Texas, and Louisiana. However, its occurrence in California, the second largest citrus-producing state, which contributed 41% of the total citrus production in the United States in 2015 (NASS 2015), has not been previously reported. Interceptions of L. formosa on California citrus exported to Australia and New Zealand suggests that this species is present in California citrus orchards. Australia and New Zealand have determined that L. formosa has not established in their countries and declared it to be a phytosanitary risk making L. formosa a species of export concern for California citrus growers. Though this species may be a part of the natural fauna in citrus orchards, it has not been reported as a pest of concern by citrus growers in California or in other parts of the world. The first and the only mention of the possible phytophagous nature of L. formosa was reported by Smirnoff (1957), in which the author suggested that this species, because it is present in large numbers, may have an economic impact on citrus. However, the author also reported that they did not observe any feeding damage or other pathological damage in areas where the mites aggregated. Damage symptoms observed, such as premature sclerification of green branches and development of dark rings in fruit, may have been a result of S. oleae feeding or caused by other pests such as thrips. L. formosa numbers were positively correlated with the abundance of honeydew from feeding stages of S. oleae and sooty mold (Smirnoff 1957). Other studies also reported association of L. formosa with honeydew producing insects but suggested a complex feeding behavior (Mendel and Gerson 1982, Aguilar-Piedra 2001). Field surveys conducted in Florida, Texas, Louisiana, and eight different countries reported that L. formosa does not cause any harm to citrus trees and laboratory experiments showed that L. formosa has a complex diet, feeding on fungus, honeydew released by homopteran insects, and pollen (Aguilar-Piedra 2001). However, interceptions of L. formosa in California citrus and ambiguous reports of possible damage makes it a species of concern to the California citrus industry. The current study was conducted to determine the feeding habits, potential for citrus damage, and the seasonal abundance of L. formosa in California. In the first objective we determined the suitability of different diets; leaf, honeydew, sugar water, cottony cushion scale [Icerya purchasi Maskell (Hemiptera: Margarodidae)], and sooty mold to support L. formosa population survival and progeny production on excised mandarin leaves. Our second objective was to determine survival and feeding damage (if any) on potted rough lemon plants in a greenhouse experiment. We also conducted a 2-yr field survey of a mandarin orchard to elucidate the seasonal abundance and population structure of L. formosa in Ventura County, California. Materials and Methods Suitability of Different Diets for Survivorship and Progeny Production Adult L. formosa were collected from a ‘Gold Nugget’ mandarin (Citrus reticulata Blanco) orchard grafted on Valencia orange rootstock, located in Saticoy in Ventura County, CA (Lat. 34° 16ʹ15.22ʺ N, Long. 119° 2ʹ 59.38ʺ W), on 8 July 2014. Mixed ages of adult L. formosa were utilized for the diet preference experiment within 48 h of collection. Fresh untreated, fully expanded leaves, approximately 5 × 10 cm, collected from ‘Owari’ satsuma mandarin trees (Citrus unshiu Marcovitch) at the Kearney Agricultural Research and Extension Center, Parlier, CA, were used for this experiment. The leaves were washed with tap water and placed lower side up on a 2.5-cm thick sponge in a plastic container 22.9 × 35.6 × 7.6 cm (l by b by h) (Rubbermaid, Atlanta, GA). Fifty milliliters of tap water were added to saturate the sponge to maintain relative humidity. The edges of the leaves were covered with a strip of absorbent wadding (Curity absorbent wadding, Kendall, Boston, MA) to provide water for the leaves and ensure leaves were held in place. There were six treatments: 1) no additional food—leaf only, 2) honeydew—three drops of honeydew produced by cottony cushion scale were transferred using an artist brush (5/0 Atlas brush and Co. Inc, Cleveland, OH), 3) sugar water—two drops of 30% sugar solution per leaf using a 15-mm Pasteur pipette (Fisher Scientific 15 mm), 4) honeydew + sugar water as described earlier, 5) cottony cushion scale (two first-instar nymphs placed on the leaf 1 week prior to infesting the leaves with mites), and 6) honeydew + sugar water + two cottony cushion scales + sooty mold (sooty mold was collected from leaves infested by a colony of cottony cushion scale and moved with an artist brush). Honeydew, sugar solution, or sooty mold was placed on the leaves close to the midrib where mites usually aggregated prior to adding mites and once every week in the second and third week. Cottony cushion scale was reared inside a greenhouse maintained at 23 ± 2°C, ambient relative humidity, and photoperiod of 12:12 (L:D) h on Pittosporum tobira (Thunb) ‘variegata’ potted plants. For each treatment, 15 adult L. formosa were transferred to each leaf and the containers containing infested leaves were placed in an incubator maintained at 25 ± 2°C, 70 ± 5% RH, and 14:10 (L:D) h. Each treatment had four replications in separate containers. The number of live adults, cumulative eggs deposited, and immatures that developed were counted three times a week for 26 d. Cumulative egg production data were analyzed for day 5, before eggs began to hatch in any of the treatments. The cumulative numbers of immatures on day 7, 14, and 21 were compared between treatments to determine the suitability of various diets to support population development. Because the eggs were not removed from the experimental arena and began hatching, and because females continued to lay more eggs, fecundity data for weeks 2 and 3 were not analyzed. The experimental design for determining the suitability of different diets for survivorship and progeny production by L. formosa was a completely randomized design with four replications. Statistical procedures were accomplished using Statistical Analysis System software (SAS Institute 2016). PROC MIXED was used for a two-way ANOVA to determine the effects of diet and time on survival and suitability to produce progeny and one-way ANOVA was conducted to determine the effects of diet on survival for each date and the effect of time on survival. Percentage survival data were transformed using the arcsine square root (x) transformation, whereas the number of progeny produced was transformed using the square root (x) transformation to stabilize variances before analysis. Untransformed means and SEs are reported. A Tukey Honest Significant Difference (HSD) test (P = 0.05) was used to determine if there were significant differences between treatments for survivorship of female mites and progeny production. Assessment of Feeding Damage Caused by L. formosa on Potted Plants One-year-old rough lemon plants (Citrus jambhiri Lush.) potted with a mixture of vermiculite and soil and kept at rearing conditions of 25 ± 1°C, 65% RH, and 12:12 (L:D) h were used for the experiment. There were three plants in each of the four treatments: 1) a control with no mites or cottony cushion scale, 2) plants with cottony cushion scale only, 3) plants infested with mites only, and 4) plants infested with both cottony cushion scale and mites. For treatments with cottony cushion scale, 5–10 scales (different life stages) were transferred 1 wk prior to transferring mites. Prior to the experiment, each plant was trimmed to contain only eight leaves. For treatments with mites, five leaves per tree were randomly selected and labeled and 20 L. formosa adults were transferred to each leaf using a soft artist brush. Transfer of the mites was conducted under a stereo microscope (M 3Z Kombistereo, Wild Leitz, Heerburg, Switzerland). Leaves were examined each week and eggs, immatures, and adults on each leaf were counted. The total number of mites (all life stages) observed per leaf in each week was then rated as 0, 1–10, 11–20, or >20 mites per plant. In addition to the five leaves onto which mites were transferred, the other three leaves were also checked for the presence of mites. On week 12, all leaves were excised and the total number of each life stage of the mites was counted from the eight original as well as all newly grown leaves. After counting the number of adults, nymphs, and eggs, the mites were carefully removed, and leaves were observed under the stereoscope for any visual feeding damage. A completely randomized design was used to determine the survival and potential feeding damage caused by L. formosa on potted plants. One-way ANOVA analysis using Statgraphics (StatPoint Technologies, Inc.; Statgraphics 2013) was used to analyze the differences between treatments for the total number of mites per leaf at week 1. Data for weeks 2–12 for the treatment 3 were not included in the analysis, because we did not observe any mites during those weeks. For cottony cushion scale-infested plants, the total number of mites per leaf observed was compared between treatments for each week sampled through week 12. The mean number of mites per leaf (sum of adults, eggs, and immatures) was transformed using the square root (x) transformation to stabilize variances before analysis. Untransformed means and standard errors are reported. Fisher’s least significant difference test (P = 0.05) was used to determine the difference in mean number of mites found between all treatments at week 1 and between weeks for cottony cushion scale and mite-infested plants. Seasonal L. formosa Abundance and Population Structure in a Mandarin Orchard A field study was conducted from January 2015 to December 2016 in a ‘Gold Nugget’ mandarin orchard described above (8.9 ac) located in Saticoy in Ventura County. Insecticides were applied to the orchard for routine management of various arthropod pests such as Asian citrus psyllid, Diaphornia citri Kuwayama (Hemiptera: Psyllidae). These treatments included 0.29 liters/ha thiamethoxam (Actara 15 WDG, Syngenta Crop Protection, Greensboro, NC) in 1,893 liters of water applied on 15 October 2015; 0.21 liter/ha spinetoram (Delegate WG Dow Agrosciences, Indianapolis, IN) in 946 liters of water applied on 17 March 2016; 0.73 liter/ha abamectin (Timectin 0.15 EC, Tide International USA, Irvine, CA) combined with 23.38 liter/ha mineral oil (Omni Oil 6E, Helena Chemical Company, Collierville, TN) in 1,893 liters of water applied on 12 August 2016; and 0.40 liters/ha thiamethoxam in 946 liters of water applied on 20 September 2016. A fungicide application of 5.6 kg/ha aluminum Tris (O-ethyl phosphonate) (Aliette WDG Fungicide, Bayer CropScience, Research Triangle Park, NC) in 378 liters of water was applied on 20 January 2016. To determine the percentage infestation of leaves and fruit, 30 trees were randomly selected (10 trees per row from each of the three evenly spaced rows), flagged, and monitored monthly. Five fruits and five leaves per tree were randomly selected and examined in the field using a 10× lens (Bausch & Lomb Coddington Magnifier) for the presence of L. formosa adults. Each fruit or leaf was rated as 0, 1 to 10, or >10 mites. Other life stages were not evaluated as they were too small to be accurately identified in the field using a hand lens. This procedure was repeated every month from January 2015 through December 2016. In 2015, the fruit data were not collected from harvest on 19 May until new fruit set in July. To determine the L. formosa population structure in a mandarin orchard, an additional 30 trees were randomly selected each month for assessment. Five leaves and one fruit (current year or previous season) that showed evidence of L. formosa infestation were collected from each tree. Ensuring that mites were present on the leaf or fruit (hereafter referred to as sample) for the population structure study was an essential procedure, because of the limited number of samples that were allowed from each tree. Fruit collected in May, June, and July developed during the previous season. Collected leaves and fruit were placed in a paper bag and stored in an ice chest and brought back to the laboratory at the Kearney Agricultural Research and Extension Center. Each sample (leaf or fruit) was examined under a stereo microscope and the number of L. formosa eggs, immatures (larvae + nymphs), and adults were counted. The experimental design for the percentage infestation and population structure of L. formosa was a completely randomized design. Statistical procedures were accomplished using Statistical Analysis System software (SAS Institute 2016). PROC GLM was used for ANOVA to determine if the percentage infestation by L. formosa varied according to plant parts, months, or years sampled. For the analysis, percentage infestation data for each month were pooled to generate one data point per month for each plant part and transformed using the arcsine square root (x) transformation. For the L. formosa population structure, PROC GLM was used for ANOVA to determine if the mean number of mites (total), and mean number of eggs, immatures, and adults sampled varied according to plant parts, months, and years sampled. Data for leaves were averaged for each tree and only one data point for leaf or fruit per tree was used in the analysis, for a total of 720 data points for each plant part (30 trees × 2 yr × 12 mo). The number of mites per sample was transformed using the square root (x) transformation to stabilize variances before analysis. Untransformed means and standard errors are reported to simplify interpretation. We used Tukey’s HSD test (P = 0.05) to determine differences among mean numbers of mites sampled on fruit and leaves in different months and years. Correlation between the number of L. formosa with rainfall or temperature was analyzed by the simple regression procedure of the Statgraphics (Statpoint Technologies, Inc.; Statgraphics 2013). Weather data were obtained from California Irrigation Management Information System (CIMIS), California Department of Water Resources. Results Suitability of Different Diets for Survivorship and Progeny Production by L. formosa Survivorship The response of L. formosa to both diet and time varied significantly during the 26-d experiment (F = 3.35; df = 55,192; P < 0.0001; Table 1). Diet had a significant effect on the percentage survival of L. formosa (F = 56.39; df = 5,192; P < 0.0001). Irrespective of diet, survivorship significantly declined over time for all treatments (F = 100.9; df = 11,192; P < 0.0001). Percentage survivorship of mites was similar in all treatments for day 0, day 2, and day 5, but on day 7, the leaf only treatment had significantly lower survivorship than the other treatments. Survivorship of mites in the leaf only treatment sharply decreased on day 7 (26.1%), and none of the mites were alive on day 12. Percentage survivorship of mites on day 7 and afterward was significantly lower for the leaf-only treatment compared to all other treatments through day 19. Table 1. Mean percentage survivorship (±SE) of L. formosa provided various diets on excised mandarin leaves over a period of 26 d (n = 15) Mean % survivorship of L. formosa (± SE) Treatments Day 0 Day 2 Day 5 Day 7 Day 9 Day 12 Day 14 Day 16 Day 19 Day 21 Day 23 Day 26 Leaf only 100 ± 0.0a1A2 95.6 ± 4.4abA 88.9 ± 5.9abA 26.1 ± 3.9dC 15.2 ± 4.3deC 0.0 ± 0.0 eC 0.0 ± 0.0eB 0.0 ± 0.0eC 0.0 ± 0.0eC 0.0 ± 0.0eB 0.0 ± 0.0eB 0.0 ± 0.0e C Honeydew 100 ± 0. 0aA 86.7 ± 6.1abA 85.1 ± 6.4abA 81.8 ± 6.9abAB 76.8 ± 8.9bAB 73.5 ± 11.9bAB 70.1 ± 13.5bcA 67.0 ± 12.0bcA 55.5 ± 12.2bcA 42.3 ± 10.3cdA 25.8 ± 13.4deA 14.5 ± 7.0deAB Sugar water 100 ± 0.0aA 90 ± 4.3abA 88.5 ± 3.2abA 86.9 ± 2.7abAB 75.2 ± 7.5bAB 73.5 ± 7.3bAB 68.5 ± 7.6bcA 63.8 ± 6.6bcAB 50.6 ± 6.7cA 39.0 ± 11.8cdA 34.0 ± 11.4cd A 26.2 ± 6.5d A Honeydew + sugar water 100 ± 0.0aA 95.0 ± 5.0abA 95.0 ± 5.0abA 90.7 ± 4.2abA 87.6 ± 3.5abA 87.6 ± 3.5abA 70.8 ± 8.1bcA 66.7 ± 9.7bcAB 52.0 ± 10.2cA 47.3 ± 7.3cdA 37.9 ± 5.3d A 31.5 ± 3.3d A Cottony cushion scale 100 ± 0.0aA 84.4 ± 12.4abA 80.0 ± 13.9abA 68.9 ± 9.7bcB 57.8 ± 4.4bcB 55.5 ± 2.2bcB 46.6 ± 6.7cdA 40.0 ± 6.7cdB 24.4 ± 5.9deB 24.4 ± 5.9deAB 15.5 ± 2.2deAB 4.5 ± 2.2e BC Honeydew + sugar water + cottony cushion scale + sooty mold 100 ± 0.0aA 100 ± 0.0aA 100 ± 0.0aA 82.5 ± 4.9abAB 75.2 ± 10.4bAB 70.6 ± 12.3bcAB 64.7 ± 10.8bcA 52.0 ± 7.7cAB 39.4 ± 6.4cdAB Leaf collapsed Leaf collapsed Leaf collapsed Mean % survivorship of L. formosa (± SE) Treatments Day 0 Day 2 Day 5 Day 7 Day 9 Day 12 Day 14 Day 16 Day 19 Day 21 Day 23 Day 26 Leaf only 100 ± 0.0a1A2 95.6 ± 4.4abA 88.9 ± 5.9abA 26.1 ± 3.9dC 15.2 ± 4.3deC 0.0 ± 0.0 eC 0.0 ± 0.0eB 0.0 ± 0.0eC 0.0 ± 0.0eC 0.0 ± 0.0eB 0.0 ± 0.0eB 0.0 ± 0.0e C Honeydew 100 ± 0. 0aA 86.7 ± 6.1abA 85.1 ± 6.4abA 81.8 ± 6.9abAB 76.8 ± 8.9bAB 73.5 ± 11.9bAB 70.1 ± 13.5bcA 67.0 ± 12.0bcA 55.5 ± 12.2bcA 42.3 ± 10.3cdA 25.8 ± 13.4deA 14.5 ± 7.0deAB Sugar water 100 ± 0.0aA 90 ± 4.3abA 88.5 ± 3.2abA 86.9 ± 2.7abAB 75.2 ± 7.5bAB 73.5 ± 7.3bAB 68.5 ± 7.6bcA 63.8 ± 6.6bcAB 50.6 ± 6.7cA 39.0 ± 11.8cdA 34.0 ± 11.4cd A 26.2 ± 6.5d A Honeydew + sugar water 100 ± 0.0aA 95.0 ± 5.0abA 95.0 ± 5.0abA 90.7 ± 4.2abA 87.6 ± 3.5abA 87.6 ± 3.5abA 70.8 ± 8.1bcA 66.7 ± 9.7bcAB 52.0 ± 10.2cA 47.3 ± 7.3cdA 37.9 ± 5.3d A 31.5 ± 3.3d A Cottony cushion scale 100 ± 0.0aA 84.4 ± 12.4abA 80.0 ± 13.9abA 68.9 ± 9.7bcB 57.8 ± 4.4bcB 55.5 ± 2.2bcB 46.6 ± 6.7cdA 40.0 ± 6.7cdB 24.4 ± 5.9deB 24.4 ± 5.9deAB 15.5 ± 2.2deAB 4.5 ± 2.2e BC Honeydew + sugar water + cottony cushion scale + sooty mold 100 ± 0.0aA 100 ± 0.0aA 100 ± 0.0aA 82.5 ± 4.9abAB 75.2 ± 10.4bAB 70.6 ± 12.3bcAB 64.7 ± 10.8bcA 52.0 ± 7.7cAB 39.4 ± 6.4cdAB Leaf collapsed Leaf collapsed Leaf collapsed 1Means in each row followed by different lowercase letters are significantly different (Tukey’s LSD, P = 0.05) after arcsine square root transformation. Untransformed means and SEs are reported. 2Means in each column followed by different uppercase letters are significantly different (Tukey LSD, P < 0.05) after arcsine square root transformation. Untransformed means and SEs are reported. View Large Table 1. Mean percentage survivorship (±SE) of L. formosa provided various diets on excised mandarin leaves over a period of 26 d (n = 15) Mean % survivorship of L. formosa (± SE) Treatments Day 0 Day 2 Day 5 Day 7 Day 9 Day 12 Day 14 Day 16 Day 19 Day 21 Day 23 Day 26 Leaf only 100 ± 0.0a1A2 95.6 ± 4.4abA 88.9 ± 5.9abA 26.1 ± 3.9dC 15.2 ± 4.3deC 0.0 ± 0.0 eC 0.0 ± 0.0eB 0.0 ± 0.0eC 0.0 ± 0.0eC 0.0 ± 0.0eB 0.0 ± 0.0eB 0.0 ± 0.0e C Honeydew 100 ± 0. 0aA 86.7 ± 6.1abA 85.1 ± 6.4abA 81.8 ± 6.9abAB 76.8 ± 8.9bAB 73.5 ± 11.9bAB 70.1 ± 13.5bcA 67.0 ± 12.0bcA 55.5 ± 12.2bcA 42.3 ± 10.3cdA 25.8 ± 13.4deA 14.5 ± 7.0deAB Sugar water 100 ± 0.0aA 90 ± 4.3abA 88.5 ± 3.2abA 86.9 ± 2.7abAB 75.2 ± 7.5bAB 73.5 ± 7.3bAB 68.5 ± 7.6bcA 63.8 ± 6.6bcAB 50.6 ± 6.7cA 39.0 ± 11.8cdA 34.0 ± 11.4cd A 26.2 ± 6.5d A Honeydew + sugar water 100 ± 0.0aA 95.0 ± 5.0abA 95.0 ± 5.0abA 90.7 ± 4.2abA 87.6 ± 3.5abA 87.6 ± 3.5abA 70.8 ± 8.1bcA 66.7 ± 9.7bcAB 52.0 ± 10.2cA 47.3 ± 7.3cdA 37.9 ± 5.3d A 31.5 ± 3.3d A Cottony cushion scale 100 ± 0.0aA 84.4 ± 12.4abA 80.0 ± 13.9abA 68.9 ± 9.7bcB 57.8 ± 4.4bcB 55.5 ± 2.2bcB 46.6 ± 6.7cdA 40.0 ± 6.7cdB 24.4 ± 5.9deB 24.4 ± 5.9deAB 15.5 ± 2.2deAB 4.5 ± 2.2e BC Honeydew + sugar water + cottony cushion scale + sooty mold 100 ± 0.0aA 100 ± 0.0aA 100 ± 0.0aA 82.5 ± 4.9abAB 75.2 ± 10.4bAB 70.6 ± 12.3bcAB 64.7 ± 10.8bcA 52.0 ± 7.7cAB 39.4 ± 6.4cdAB Leaf collapsed Leaf collapsed Leaf collapsed Mean % survivorship of L. formosa (± SE) Treatments Day 0 Day 2 Day 5 Day 7 Day 9 Day 12 Day 14 Day 16 Day 19 Day 21 Day 23 Day 26 Leaf only 100 ± 0.0a1A2 95.6 ± 4.4abA 88.9 ± 5.9abA 26.1 ± 3.9dC 15.2 ± 4.3deC 0.0 ± 0.0 eC 0.0 ± 0.0eB 0.0 ± 0.0eC 0.0 ± 0.0eC 0.0 ± 0.0eB 0.0 ± 0.0eB 0.0 ± 0.0e C Honeydew 100 ± 0. 0aA 86.7 ± 6.1abA 85.1 ± 6.4abA 81.8 ± 6.9abAB 76.8 ± 8.9bAB 73.5 ± 11.9bAB 70.1 ± 13.5bcA 67.0 ± 12.0bcA 55.5 ± 12.2bcA 42.3 ± 10.3cdA 25.8 ± 13.4deA 14.5 ± 7.0deAB Sugar water 100 ± 0.0aA 90 ± 4.3abA 88.5 ± 3.2abA 86.9 ± 2.7abAB 75.2 ± 7.5bAB 73.5 ± 7.3bAB 68.5 ± 7.6bcA 63.8 ± 6.6bcAB 50.6 ± 6.7cA 39.0 ± 11.8cdA 34.0 ± 11.4cd A 26.2 ± 6.5d A Honeydew + sugar water 100 ± 0.0aA 95.0 ± 5.0abA 95.0 ± 5.0abA 90.7 ± 4.2abA 87.6 ± 3.5abA 87.6 ± 3.5abA 70.8 ± 8.1bcA 66.7 ± 9.7bcAB 52.0 ± 10.2cA 47.3 ± 7.3cdA 37.9 ± 5.3d A 31.5 ± 3.3d A Cottony cushion scale 100 ± 0.0aA 84.4 ± 12.4abA 80.0 ± 13.9abA 68.9 ± 9.7bcB 57.8 ± 4.4bcB 55.5 ± 2.2bcB 46.6 ± 6.7cdA 40.0 ± 6.7cdB 24.4 ± 5.9deB 24.4 ± 5.9deAB 15.5 ± 2.2deAB 4.5 ± 2.2e BC Honeydew + sugar water + cottony cushion scale + sooty mold 100 ± 0.0aA 100 ± 0.0aA 100 ± 0.0aA 82.5 ± 4.9abAB 75.2 ± 10.4bAB 70.6 ± 12.3bcAB 64.7 ± 10.8bcA 52.0 ± 7.7cAB 39.4 ± 6.4cdAB Leaf collapsed Leaf collapsed Leaf collapsed 1Means in each row followed by different lowercase letters are significantly different (Tukey’s LSD, P = 0.05) after arcsine square root transformation. Untransformed means and SEs are reported. 2Means in each column followed by different uppercase letters are significantly different (Tukey LSD, P < 0.05) after arcsine square root transformation. Untransformed means and SEs are reported. View Large The highest percentage survival of mites was observed for the honey dew + sugar water treatment for each evaluation date for all 26 d; this was significantly different from the leaf-only treatment from day 7 to day 26 and from the cottony cushion scale treatment for days 7, 9, 12, 16, 19, and 26 but was similar to the honeydew or sugar water treatments. Through day 19, all the treatments with honeydew and/or sugar water supported adults and their progeny similarly. The cottony cushion scale treatment had significantly lower percentage survival of mites compared to the sugar + honeydew treatment on day 7, 9, 12, and 19. On day 21, leaves with the honeydew + sugar water + cottony cushion scale + sooty mold treatment collapsed, and no data were collected. Progeny production The cumulative number of eggs produced by females did not differ significantly between treatments on day 5 (F = 2.13; df = 5,16; P = 0.1144) (Table 2). Similarly, the cumulative number of immatures (larvae + nymphs) produced by females in all treatments were statistically similar on day 7 (F = 0.59; df = 5,16; P = 0.708) but differed significantly between treatments on day 14 (F = 6.91; df = 5,16; P = 0.0013) and day 21 (F = 8.68; df = 5,16; P = 0.0004). The data showed that honeydew, sugar water, cottony cushion scale, and honeydew + sugar water supported immature mites through day 21 and honeydew + sugar water supported the largest number of mites. When honeydew, sugar water, cottony cushions scale, and sooty mold were combined, the leaves collapsed on day 21 (Table 1) and the mite population declined to 0.0. Despite a similar number of eggs recorded in the first week in the leaf only treatment, larvae did not survive past day 7 when no sugar supplement (honeydew, sugar water, or cottony cushion scale) was provided (Table 2). Table 2. Mean cumulative number of egg and immature mites per leaf (±SE) produced by L. formosa provided various diets on excised leaves Mean number of mites per leaf (±SE) Eggs Immatures Treatments Day 5 Day 7 Day 14 Day 21 Leaf only 6.0 ± 1.5a1 2.0 ± 0.6a 0.0 ± 0.0c 0.0 ± 0.0c Honeydew 5.5 ± 1.8a 3.3 ± 0.9a 4.0 ± 0.6b 4.0 ± 2.5b Sugar water 3.8 ± 1.3a 2.3 ± 0.7a 3.8 ± 0.9b 4.3 ± 1.2b Honeydew + sugar water 5.0 ± 0.2a 1.8 ± 0.7a 7.2 ± 0.8a 7.0 ± 1.2ab Cottony cushion scale 8.3 ± 5.4a 1.7 ± 0.5a 2.3 ± 1.6bc 3.7 ± 1.6b Honeydew + sugar water + cottony cushion scale + sooty mold 1.7 ± 1.4a 0.75 ± 0.7a 1.5 ± 0.8bc 0.0 ± 0.0c Mean number of mites per leaf (±SE) Eggs Immatures Treatments Day 5 Day 7 Day 14 Day 21 Leaf only 6.0 ± 1.5a1 2.0 ± 0.6a 0.0 ± 0.0c 0.0 ± 0.0c Honeydew 5.5 ± 1.8a 3.3 ± 0.9a 4.0 ± 0.6b 4.0 ± 2.5b Sugar water 3.8 ± 1.3a 2.3 ± 0.7a 3.8 ± 0.9b 4.3 ± 1.2b Honeydew + sugar water 5.0 ± 0.2a 1.8 ± 0.7a 7.2 ± 0.8a 7.0 ± 1.2ab Cottony cushion scale 8.3 ± 5.4a 1.7 ± 0.5a 2.3 ± 1.6bc 3.7 ± 1.6b Honeydew + sugar water + cottony cushion scale + sooty mold 1.7 ± 1.4a 0.75 ± 0.7a 1.5 ± 0.8bc 0.0 ± 0.0c 1Means within a column followed by different letters are significantly different (Tukey HSD, P = 0.05) after square root transformation. Untransformed means and SEs are reported. View Large Table 2. Mean cumulative number of egg and immature mites per leaf (±SE) produced by L. formosa provided various diets on excised leaves Mean number of mites per leaf (±SE) Eggs Immatures Treatments Day 5 Day 7 Day 14 Day 21 Leaf only 6.0 ± 1.5a1 2.0 ± 0.6a 0.0 ± 0.0c 0.0 ± 0.0c Honeydew 5.5 ± 1.8a 3.3 ± 0.9a 4.0 ± 0.6b 4.0 ± 2.5b Sugar water 3.8 ± 1.3a 2.3 ± 0.7a 3.8 ± 0.9b 4.3 ± 1.2b Honeydew + sugar water 5.0 ± 0.2a 1.8 ± 0.7a 7.2 ± 0.8a 7.0 ± 1.2ab Cottony cushion scale 8.3 ± 5.4a 1.7 ± 0.5a 2.3 ± 1.6bc 3.7 ± 1.6b Honeydew + sugar water + cottony cushion scale + sooty mold 1.7 ± 1.4a 0.75 ± 0.7a 1.5 ± 0.8bc 0.0 ± 0.0c Mean number of mites per leaf (±SE) Eggs Immatures Treatments Day 5 Day 7 Day 14 Day 21 Leaf only 6.0 ± 1.5a1 2.0 ± 0.6a 0.0 ± 0.0c 0.0 ± 0.0c Honeydew 5.5 ± 1.8a 3.3 ± 0.9a 4.0 ± 0.6b 4.0 ± 2.5b Sugar water 3.8 ± 1.3a 2.3 ± 0.7a 3.8 ± 0.9b 4.3 ± 1.2b Honeydew + sugar water 5.0 ± 0.2a 1.8 ± 0.7a 7.2 ± 0.8a 7.0 ± 1.2ab Cottony cushion scale 8.3 ± 5.4a 1.7 ± 0.5a 2.3 ± 1.6bc 3.7 ± 1.6b Honeydew + sugar water + cottony cushion scale + sooty mold 1.7 ± 1.4a 0.75 ± 0.7a 1.5 ± 0.8bc 0.0 ± 0.0c 1Means within a column followed by different letters are significantly different (Tukey HSD, P = 0.05) after square root transformation. Untransformed means and SEs are reported. View Large Survival and feeding damage by L. formosa on potted plants Mites could be found on potted plants seven days after they were transferred to leaves; however, the number of mites present on the on the cottony cushion scale-infested plants was significantly greater than that on the cottony cushion scale-free plants (F = 13.51, df = 3,8; P = 0.0017). Only one adult L. formosa was recovered from the cottony cushion scale-free plants and no mites were observed in this treatment from weeks 2–12 (Fig. 1). In contrast, densities of L. formosa stages ranging from 1–4 per leaf were observed during weeks 2–12 (Fig. 1B). Although the mean number mites per leaf on cottony cushion scale-infested plants fluctuated, ANOVA results showed that they were statistically similar between weeks (F = 1.01; df = 11,24; P = 0.46; Fig. 1). We observed that mites aggregated near cottony cushion scale, honeydew, or sooty mold growing on honeydew on the lower side of the leaf near midrib (Fig. 2A). After 12 wk of exposure, cottony cushion scale-infested leaves with mite aggregations did not show any feeding damage when cleaned and observed under the stereoscope (40×). No feeding damage or any other damage was observed for untreated plants. Fig. 1. View largeDownload slide Mean number of L. formosa stages/leaf on potted rough lemon plants with and without cottony cushion scale (CCS) and L. formosa over a period of 12 wk. Fig. 1. View largeDownload slide Mean number of L. formosa stages/leaf on potted rough lemon plants with and without cottony cushion scale (CCS) and L. formosa over a period of 12 wk. Fig. 2. View largeDownload slide L. formosa infesting (A) a cottony cushion scale-infested leaf on potted rough lemon plant and (B) a field-collected mandarin leaf showing adults and immatures along the mid vein and close to sooty mold. Fig. 2. View largeDownload slide L. formosa infesting (A) a cottony cushion scale-infested leaf on potted rough lemon plant and (B) a field-collected mandarin leaf showing adults and immatures along the mid vein and close to sooty mold. Seasonal L. formosa abundance and population structure in a mandarin orchard Population abundance. Seasonal abundance data showed that L. formosa was present in the mandarin orchard on leaves and/or fruit during every sampling period (January 2015 through December 2016; Fig. 3). The percentage infestation of leaves and fruit by L. formosa was significantly lower on most dates in 2016 compared to 2015 (Fig. 3) (F = 8.97; df = 1, 43; P < 0.0001). The proportion of leaves infested with mites declined by 91, 75, 60, and 43% after the applications of thiamethoxam (2015), spinetoram, abamectin, and thiamethoxam (2016), respectively (Fig 3). Insecticides may have affected the mite population directly or indirectly by reducing honeydew-producing insects. Fig. 3. View largeDownload slide Percentage infestation of leaves and fruit by L. formosa sampled from January to December in years 2015 and 2016 in a mandarin orchard. Fruit samples were not collected in May and June of 2015. Arrows indicate pesticide treatments: dotted arrow—fungicide and solid arrow—insecticide. Fig. 3. View largeDownload slide Percentage infestation of leaves and fruit by L. formosa sampled from January to December in years 2015 and 2016 in a mandarin orchard. Fruit samples were not collected in May and June of 2015. Arrows indicate pesticide treatments: dotted arrow—fungicide and solid arrow—insecticide. The percentage infestation of fruit averaged over all time periods (44.8 ± 4%), was significantly higher compared to leaves (16.0 ± 4.7%) (F = 22.61; df = 1,43; P < 0.0001), suggesting that adult mites preferred to reside on fruit. In addition, the percentage of fruit with >10, 1–10, and 0 mites per sample were 9.9, 32.3, and 57.7%, whereas that for leaves were 3.7, 13, and 83.3%, respectively, indicating greater population development on fruit. Field observations showed that mites preferred the shaded side of the tree, the inside canopy leaves, the midrib of the lower side of a leaf with sooty mold (Fig. 2B), and the proximal end of fruit in and around the calyx. Population structure The mean total number of mites (sum of eggs, immatures, and adults per sample) varied in the months sampled (F = 31.31, df = 11, 1392; P < 0.0001) and plant parts sampled (F = 26.58; df = 1,1392; P < 0.0001) but were similar in both years (F = 1.2; df =1,1392; P = 0.2737). The mean number of eggs and immatures per leaf or fruit did not indicate any consistent preference of fruit compared to leaves; however, adults were found in higher densities on fruit (Fig. 4A–C). The data showed a consistent seasonal peak from June to August for immatures in both years; however, such trends were not observed for eggs or adults (Fig. 4A–C). Nymphal and egg mite densities, declined after insecticide treatments (Fig. 4A–C), though whether this was due to a direct effect of the toxicants or the decline in honeydew producing insects is not known. The numbers of eggs and nymphs per sample fluctuated between months in both years and were lowest in November and December of 2015 but highest in December of 2016. We observed a significant correlation between rainfall and the total number of L. formosa sampled per month (r = 0.49; P = 0.015), but no significant correlation between temperature and the number of L. formosa sampled (r = 0.08; P = 0.70) (Fig. 5). Fig. 4. View largeDownload slide Mean number of eggs (A), immatures (B), and adults (C) of L. formosa per leaf or fruit on samples collected during January to December in years 2015 and 2016. Arrows indicate pesticide treatments: dotted arrow—fungicide and solid arrow—insecticide. Fig. 4. View largeDownload slide Mean number of eggs (A), immatures (B), and adults (C) of L. formosa per leaf or fruit on samples collected during January to December in years 2015 and 2016. Arrows indicate pesticide treatments: dotted arrow—fungicide and solid arrow—insecticide. Fig. 5. View largeDownload slide Average temperature, total precipitation, and mean population numbers of L. formosa in fruit and leaf samples for each month in years 2015 and 2016. Fig. 5. View largeDownload slide Average temperature, total precipitation, and mean population numbers of L. formosa in fruit and leaf samples for each month in years 2015 and 2016. The mean number of eggs per leaf or fruit varied in the months (F = 10.49; df = 11, 1392; P < 0.0001) and years (F = 180.29, df = 1,1392; P < 0.0001) but were similar for fruit and leaves (F = 3.51; df = 1,1392; P = 0.06), suggesting that L. formosa adults did not prefer fruit over leaves for egg laying (Fig. 4A). Egg laying occurred year-round and the highest mean number of eggs, 28.7 ± 5.1 eggs/fruit, was recorded in December in 2016 (Fig. 4A). Immatures densities also varied between months (F = 64.75; df = 11,1392; P < 0.0001), years (F = 50.8; df = 1,1392; P < 0.0001), and plant parts sampled (F = 7.98; df = 1,1392; P = 0.0048) (Fig. 4B) and were the most abundant life stage sampled, reaching densities of more than 15 immatures per fruit. Similarly, the number of adults varied according to months (F = 8.08; df = 11,1392; P < 0.0001), years (F = 33.14; df = 1,1392; P < 0.0001), and plant parts sampled (F = 44.56; df = 1,1392; P < 0.0001) (Fig. 4C). Although the highest numbers of eggs and immatures were recorded for December 2016, the number of adults did not show the same increase. Honeydew-producing insects, namely, cottony cushion scale, soft scale, aphids, and whiteflies were present year-round in the orchard and in abundance during the months of April to August/September and sooty mold was associated with the presence of these insects. Discussion L. formosa did not survive on citrus leaves without a supplemental diet in the form of sugary substances and caused no damage to citrus. High adult mortality on excised leaves free of sugary substances and no recovery of L. formosa from the cottony cushion scale-free potted plants in the greenhouse experiment validates that L. formosa cannot maintain or continue development on citrus leaves alone and will eventually die because of a lack of suitable food to support development. Aguilar-Piedra (2001) concluded that L. formosa has a complex feeding behavior but is not a phytophagous species and causes no harm to citrus trees based on experiments using different diets including sugar water, honeydew, pollens, fungal pathogens, and predation of Aculops pelekassi (Keifer) (Acari: Eriophyidae), Phyllocoptruta oleivora (Ashmead) (Acari: Eriophyidae), and Panconychus citri (McGregor) (Acari: Tetranychidae). Similarly, Mendel and Gerson (1982) supported the detrivorous nature of L. formosa and recognized it as a sanitizing agent that helps to control sooty mold in citrus groves. Moreover, Hernandes et al. (2006) emphasized that though reported in high numbers from base of leaflets, L. formosa did not cause any damage to the rubber trees. In another study, researchers maintained healthy colonies of L. formosa on orange leaves; however, they did not report any feeding damage (Badii et al. 2001). Although in high numbers, there was no feeding or other pathological damage of infested leaves where L. formosa aggregated and populations positively correlated with the honeydew producing insect S. oleae (Smirnoff 1957). Honeydew, sugar water, cottony cushion scale, or the combination of these treatments applied to leaves in our study supported longer-term survivorship and progeny production compared to the leaf only treatment. These data support earlier reports that L. formosa populations are associated with honeydew producing insects and sooty mold (Smirnoff 1957, Mendel and Gerson 1982, Aguilar-Piedra 2001). Aguilar-Piedra also showed that L. formosa feeds on sugar water and suggested that sugar water may serve as an alternative source of food for laboratory rearing (Aguilar-Piedra 2001). We showed that L. formosa survived and reproduced on plants that were infested with cottony cushion scale, which provided additional food sources in the form of honeydew and sooty mold, but not on plants free of additional food source. These data confirm that L. formosa cannot survive for more than 1–2 wk on citrus leaves without additional sources of food. Field sampling studies showed L. formosa was present throughout the year in a mandarin orchard in Ventura county, CA. This species is gregarious in nature and high densities of motile stages were reported to cause a visible yellow bleaching of the green leaf (Smirnoff 1957). However, we did not observe any bleaching after 12 wk of observation in our greenhouse experiment or during any month of the 2-yr field study. We confirmed that the mites preferred interior canopy leaves, the shady side of the tree with higher sooty mold growth, and were found on the lower part of the midrib of leaves or hiding in sepals of the fruit (Aguilar-Piedra 2001). In the United States, L. formosa has been previously reported from citrus in Florida, Texas, and Louisiana (Aguilar-Piedra 2001). The highest density of mites (110 mites per leaf—motile stages only) in Florida was reported in April, and the population declined in the summer months. Smirnoff (1957) reported large populations of Lorryia in Morocco in May. In our study, the highest number of motile stages (immatures + adults) observed was in the month of July when 16.9 ± 1.6 mites per sample were recorded. The population consisted predominantly of eggs and immatures and adults were only recorded in low numbers (the highest number recorded was 4.4 per fruit in July 2015). The percentage infestation of fruit and leaves in 2015 was higher in all months compared to 2016, which may be because of insecticides applied in 2016. However, the average number of mites per sample was similar in both years. Based on these studies, L. formosa populations cannot survive for extended periods of time when they lack supplemental food in the form of sugary substances and the do not cause any feeding damage to citrus. Field data showed that L. formosa was present in mandarin orchard in Ventura County, CA, throughout the year, and that insecticide applications affected but did not eliminate mite populations. Despite their year-round presence in the field, the laboratory and greenhouse studies showed that L. formosa cannot survive for more than 2 wk without supplemental food suggesting that field populations are supported by honeydew producing insects. Also, the absence of leaf or fruit damage suggests that this species is not a pest and so it should not be considered a species of phytosanitary concern by importing countries. Acknowledgments We thank Monty Lo for technical support and Tom Roberts for providing a field site to collect samples. The California Citrus Research Board funded this work. References Cited Aguilar-Piedra , H. G . 2001 . Tydeidae of citrus from selected countries: distribution, seasonal occurrence, relative abundance, and feeding habits (Acari: Prostigmata) . pp 200. Ph.D dissertation. University of Florida , Gainesville, FL. Badii , M. H. , A. E. Flores , G. Ponce , J. Landeros , and H. Quieoz . 2001 . Does the Lorryia formosa Cooreman (Acari: Prostigmata: Tydeidae) population visit or reside on citrus foliage ? pp. 413 – 418 . In R. B. Halliday , D. E. Walter , H. C. Proctor , R. A. Norton and M. J. Colloff (eds.), Proceedings of the 10th International Congress of Acarology . CSIRO Publishing , Melbourne , Australia. Hernandes , F. A. , R. J. Feres , and F. Nomura . 2006 . Biological cycle of Lorryia formosa (Acari, Tydeidae) on rubber tree leaves: a case of thelytoky . Exp. Appl. Acarol . 38 : 237 – 242 . Google Scholar CrossRef Search ADS PubMed Mendel , Z. , and U. Gerson . 1982 . Is the mite Lorryia formosa Cooreman (Prostigmata: Tydeidae) a sanitizing agent in citrus groves? Acta . Ecol. Ecol. Appl . 3 : 47 – 51 . (NASS) National Agricultural Statistics Service. 2015 . Citrus fruit 2015 summary . https://www.cacitrusmutual.com/wp-content/uploads/2015/12/2015-USDA-Citrus-Fruits-Summary.pdf (Accessed on 17 April 2016 ). SAS Institute . 2016 . The SAS system for windows, version 9.4 . SAS Institute , Cary, NC . Smirnoff , W. A . 1957 . An undescribed species of Lorryia (Acarina: Tydeidae) causing injury to citrus trees in Morocco . J. Econ. Entomol . 50 : 361 – 362 . Google Scholar CrossRef Search ADS Statgraphics . 2013 . Statgraphics centurion XVI . StatPoint Technologies, Inc, The Plains, VA . © The Author(s) 2018. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Environmental Entomology Oxford University Press

Food Suitability and Population Dynamics of Lorryia formosa (Acari: Tydeidae)

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Entomological Society of America
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Abstract

Abstract Lorryia formosa Cooreman (Acari: Tydeidae) is a species of mite commonly associated with citrus in many countries including the United States. A survey report in 1957 suggested phytophagous nature, while other studies claimed that L. formosa populations are associated with honeydew producing insects and sooty mold and it acts as a sanitizing agent. We investigated the effect of various diets on the survival and progeny production of L. formosa on excised leaves and the survival and potential to cause feeding damage to leaves of potted plants in a greenhouse study. A 2-yr field survey of a mandarin orchard was also conducted to elucidate the seasonal infestation, damage potential and population structure of L. formosa in a natural habitat. Results showed that all L. formosa adults and immatures died in less than 14 d on excised leaves, did not survive beyond 7 d on potted citrus plants alone, and caused no observable feeding damage to leaves or fruit. When sugar water, honeydew, or cottony cushion scale, Icerya purchasi Maskell (Hemiptera: Margarodidae), was present, adults and immatures survived the duration of the experiments and produced additional generations. The field survey showed that all stages of L. formosa were present in a mandarin orchard throughout the year and insecticide applications affected but did not eliminate mite populations. Fruit generally had a greater percentage infestation of mites (44.8 ± 4.0) than leaves (16.0 ± 4.7). These studies confirmed that L. formosa cannot sustain a population on leaf tissue alone and is nondamaging to citrus in California. Lorryia formosa, citrus, population structure, honeydew, sooty mold Lorryia formosa Cooreman (Acari: Tydeidae) is a mite species in the family Tydeidae that is found in citrus orchards worldwide (Smirnoff 1957, Aguilar-Piedra 2001, Badii et al. 2001). This species was originally described from Morocco where it had been reported in large numbers from citrus trees infested with Saissetia oleae (Olivier) (Hemiptera: Coccidae) and mites were reported to be associated with S. oleae nymphs producing honeydew (Smirnoff 1957). In the United States, (Aguilar-Piedra 2001) reported L. formosa as the most common Tydeidae mite in citrus orchards in Florida, Texas, and Louisiana. However, its occurrence in California, the second largest citrus-producing state, which contributed 41% of the total citrus production in the United States in 2015 (NASS 2015), has not been previously reported. Interceptions of L. formosa on California citrus exported to Australia and New Zealand suggests that this species is present in California citrus orchards. Australia and New Zealand have determined that L. formosa has not established in their countries and declared it to be a phytosanitary risk making L. formosa a species of export concern for California citrus growers. Though this species may be a part of the natural fauna in citrus orchards, it has not been reported as a pest of concern by citrus growers in California or in other parts of the world. The first and the only mention of the possible phytophagous nature of L. formosa was reported by Smirnoff (1957), in which the author suggested that this species, because it is present in large numbers, may have an economic impact on citrus. However, the author also reported that they did not observe any feeding damage or other pathological damage in areas where the mites aggregated. Damage symptoms observed, such as premature sclerification of green branches and development of dark rings in fruit, may have been a result of S. oleae feeding or caused by other pests such as thrips. L. formosa numbers were positively correlated with the abundance of honeydew from feeding stages of S. oleae and sooty mold (Smirnoff 1957). Other studies also reported association of L. formosa with honeydew producing insects but suggested a complex feeding behavior (Mendel and Gerson 1982, Aguilar-Piedra 2001). Field surveys conducted in Florida, Texas, Louisiana, and eight different countries reported that L. formosa does not cause any harm to citrus trees and laboratory experiments showed that L. formosa has a complex diet, feeding on fungus, honeydew released by homopteran insects, and pollen (Aguilar-Piedra 2001). However, interceptions of L. formosa in California citrus and ambiguous reports of possible damage makes it a species of concern to the California citrus industry. The current study was conducted to determine the feeding habits, potential for citrus damage, and the seasonal abundance of L. formosa in California. In the first objective we determined the suitability of different diets; leaf, honeydew, sugar water, cottony cushion scale [Icerya purchasi Maskell (Hemiptera: Margarodidae)], and sooty mold to support L. formosa population survival and progeny production on excised mandarin leaves. Our second objective was to determine survival and feeding damage (if any) on potted rough lemon plants in a greenhouse experiment. We also conducted a 2-yr field survey of a mandarin orchard to elucidate the seasonal abundance and population structure of L. formosa in Ventura County, California. Materials and Methods Suitability of Different Diets for Survivorship and Progeny Production Adult L. formosa were collected from a ‘Gold Nugget’ mandarin (Citrus reticulata Blanco) orchard grafted on Valencia orange rootstock, located in Saticoy in Ventura County, CA (Lat. 34° 16ʹ15.22ʺ N, Long. 119° 2ʹ 59.38ʺ W), on 8 July 2014. Mixed ages of adult L. formosa were utilized for the diet preference experiment within 48 h of collection. Fresh untreated, fully expanded leaves, approximately 5 × 10 cm, collected from ‘Owari’ satsuma mandarin trees (Citrus unshiu Marcovitch) at the Kearney Agricultural Research and Extension Center, Parlier, CA, were used for this experiment. The leaves were washed with tap water and placed lower side up on a 2.5-cm thick sponge in a plastic container 22.9 × 35.6 × 7.6 cm (l by b by h) (Rubbermaid, Atlanta, GA). Fifty milliliters of tap water were added to saturate the sponge to maintain relative humidity. The edges of the leaves were covered with a strip of absorbent wadding (Curity absorbent wadding, Kendall, Boston, MA) to provide water for the leaves and ensure leaves were held in place. There were six treatments: 1) no additional food—leaf only, 2) honeydew—three drops of honeydew produced by cottony cushion scale were transferred using an artist brush (5/0 Atlas brush and Co. Inc, Cleveland, OH), 3) sugar water—two drops of 30% sugar solution per leaf using a 15-mm Pasteur pipette (Fisher Scientific 15 mm), 4) honeydew + sugar water as described earlier, 5) cottony cushion scale (two first-instar nymphs placed on the leaf 1 week prior to infesting the leaves with mites), and 6) honeydew + sugar water + two cottony cushion scales + sooty mold (sooty mold was collected from leaves infested by a colony of cottony cushion scale and moved with an artist brush). Honeydew, sugar solution, or sooty mold was placed on the leaves close to the midrib where mites usually aggregated prior to adding mites and once every week in the second and third week. Cottony cushion scale was reared inside a greenhouse maintained at 23 ± 2°C, ambient relative humidity, and photoperiod of 12:12 (L:D) h on Pittosporum tobira (Thunb) ‘variegata’ potted plants. For each treatment, 15 adult L. formosa were transferred to each leaf and the containers containing infested leaves were placed in an incubator maintained at 25 ± 2°C, 70 ± 5% RH, and 14:10 (L:D) h. Each treatment had four replications in separate containers. The number of live adults, cumulative eggs deposited, and immatures that developed were counted three times a week for 26 d. Cumulative egg production data were analyzed for day 5, before eggs began to hatch in any of the treatments. The cumulative numbers of immatures on day 7, 14, and 21 were compared between treatments to determine the suitability of various diets to support population development. Because the eggs were not removed from the experimental arena and began hatching, and because females continued to lay more eggs, fecundity data for weeks 2 and 3 were not analyzed. The experimental design for determining the suitability of different diets for survivorship and progeny production by L. formosa was a completely randomized design with four replications. Statistical procedures were accomplished using Statistical Analysis System software (SAS Institute 2016). PROC MIXED was used for a two-way ANOVA to determine the effects of diet and time on survival and suitability to produce progeny and one-way ANOVA was conducted to determine the effects of diet on survival for each date and the effect of time on survival. Percentage survival data were transformed using the arcsine square root (x) transformation, whereas the number of progeny produced was transformed using the square root (x) transformation to stabilize variances before analysis. Untransformed means and SEs are reported. A Tukey Honest Significant Difference (HSD) test (P = 0.05) was used to determine if there were significant differences between treatments for survivorship of female mites and progeny production. Assessment of Feeding Damage Caused by L. formosa on Potted Plants One-year-old rough lemon plants (Citrus jambhiri Lush.) potted with a mixture of vermiculite and soil and kept at rearing conditions of 25 ± 1°C, 65% RH, and 12:12 (L:D) h were used for the experiment. There were three plants in each of the four treatments: 1) a control with no mites or cottony cushion scale, 2) plants with cottony cushion scale only, 3) plants infested with mites only, and 4) plants infested with both cottony cushion scale and mites. For treatments with cottony cushion scale, 5–10 scales (different life stages) were transferred 1 wk prior to transferring mites. Prior to the experiment, each plant was trimmed to contain only eight leaves. For treatments with mites, five leaves per tree were randomly selected and labeled and 20 L. formosa adults were transferred to each leaf using a soft artist brush. Transfer of the mites was conducted under a stereo microscope (M 3Z Kombistereo, Wild Leitz, Heerburg, Switzerland). Leaves were examined each week and eggs, immatures, and adults on each leaf were counted. The total number of mites (all life stages) observed per leaf in each week was then rated as 0, 1–10, 11–20, or >20 mites per plant. In addition to the five leaves onto which mites were transferred, the other three leaves were also checked for the presence of mites. On week 12, all leaves were excised and the total number of each life stage of the mites was counted from the eight original as well as all newly grown leaves. After counting the number of adults, nymphs, and eggs, the mites were carefully removed, and leaves were observed under the stereoscope for any visual feeding damage. A completely randomized design was used to determine the survival and potential feeding damage caused by L. formosa on potted plants. One-way ANOVA analysis using Statgraphics (StatPoint Technologies, Inc.; Statgraphics 2013) was used to analyze the differences between treatments for the total number of mites per leaf at week 1. Data for weeks 2–12 for the treatment 3 were not included in the analysis, because we did not observe any mites during those weeks. For cottony cushion scale-infested plants, the total number of mites per leaf observed was compared between treatments for each week sampled through week 12. The mean number of mites per leaf (sum of adults, eggs, and immatures) was transformed using the square root (x) transformation to stabilize variances before analysis. Untransformed means and standard errors are reported. Fisher’s least significant difference test (P = 0.05) was used to determine the difference in mean number of mites found between all treatments at week 1 and between weeks for cottony cushion scale and mite-infested plants. Seasonal L. formosa Abundance and Population Structure in a Mandarin Orchard A field study was conducted from January 2015 to December 2016 in a ‘Gold Nugget’ mandarin orchard described above (8.9 ac) located in Saticoy in Ventura County. Insecticides were applied to the orchard for routine management of various arthropod pests such as Asian citrus psyllid, Diaphornia citri Kuwayama (Hemiptera: Psyllidae). These treatments included 0.29 liters/ha thiamethoxam (Actara 15 WDG, Syngenta Crop Protection, Greensboro, NC) in 1,893 liters of water applied on 15 October 2015; 0.21 liter/ha spinetoram (Delegate WG Dow Agrosciences, Indianapolis, IN) in 946 liters of water applied on 17 March 2016; 0.73 liter/ha abamectin (Timectin 0.15 EC, Tide International USA, Irvine, CA) combined with 23.38 liter/ha mineral oil (Omni Oil 6E, Helena Chemical Company, Collierville, TN) in 1,893 liters of water applied on 12 August 2016; and 0.40 liters/ha thiamethoxam in 946 liters of water applied on 20 September 2016. A fungicide application of 5.6 kg/ha aluminum Tris (O-ethyl phosphonate) (Aliette WDG Fungicide, Bayer CropScience, Research Triangle Park, NC) in 378 liters of water was applied on 20 January 2016. To determine the percentage infestation of leaves and fruit, 30 trees were randomly selected (10 trees per row from each of the three evenly spaced rows), flagged, and monitored monthly. Five fruits and five leaves per tree were randomly selected and examined in the field using a 10× lens (Bausch & Lomb Coddington Magnifier) for the presence of L. formosa adults. Each fruit or leaf was rated as 0, 1 to 10, or >10 mites. Other life stages were not evaluated as they were too small to be accurately identified in the field using a hand lens. This procedure was repeated every month from January 2015 through December 2016. In 2015, the fruit data were not collected from harvest on 19 May until new fruit set in July. To determine the L. formosa population structure in a mandarin orchard, an additional 30 trees were randomly selected each month for assessment. Five leaves and one fruit (current year or previous season) that showed evidence of L. formosa infestation were collected from each tree. Ensuring that mites were present on the leaf or fruit (hereafter referred to as sample) for the population structure study was an essential procedure, because of the limited number of samples that were allowed from each tree. Fruit collected in May, June, and July developed during the previous season. Collected leaves and fruit were placed in a paper bag and stored in an ice chest and brought back to the laboratory at the Kearney Agricultural Research and Extension Center. Each sample (leaf or fruit) was examined under a stereo microscope and the number of L. formosa eggs, immatures (larvae + nymphs), and adults were counted. The experimental design for the percentage infestation and population structure of L. formosa was a completely randomized design. Statistical procedures were accomplished using Statistical Analysis System software (SAS Institute 2016). PROC GLM was used for ANOVA to determine if the percentage infestation by L. formosa varied according to plant parts, months, or years sampled. For the analysis, percentage infestation data for each month were pooled to generate one data point per month for each plant part and transformed using the arcsine square root (x) transformation. For the L. formosa population structure, PROC GLM was used for ANOVA to determine if the mean number of mites (total), and mean number of eggs, immatures, and adults sampled varied according to plant parts, months, and years sampled. Data for leaves were averaged for each tree and only one data point for leaf or fruit per tree was used in the analysis, for a total of 720 data points for each plant part (30 trees × 2 yr × 12 mo). The number of mites per sample was transformed using the square root (x) transformation to stabilize variances before analysis. Untransformed means and standard errors are reported to simplify interpretation. We used Tukey’s HSD test (P = 0.05) to determine differences among mean numbers of mites sampled on fruit and leaves in different months and years. Correlation between the number of L. formosa with rainfall or temperature was analyzed by the simple regression procedure of the Statgraphics (Statpoint Technologies, Inc.; Statgraphics 2013). Weather data were obtained from California Irrigation Management Information System (CIMIS), California Department of Water Resources. Results Suitability of Different Diets for Survivorship and Progeny Production by L. formosa Survivorship The response of L. formosa to both diet and time varied significantly during the 26-d experiment (F = 3.35; df = 55,192; P < 0.0001; Table 1). Diet had a significant effect on the percentage survival of L. formosa (F = 56.39; df = 5,192; P < 0.0001). Irrespective of diet, survivorship significantly declined over time for all treatments (F = 100.9; df = 11,192; P < 0.0001). Percentage survivorship of mites was similar in all treatments for day 0, day 2, and day 5, but on day 7, the leaf only treatment had significantly lower survivorship than the other treatments. Survivorship of mites in the leaf only treatment sharply decreased on day 7 (26.1%), and none of the mites were alive on day 12. Percentage survivorship of mites on day 7 and afterward was significantly lower for the leaf-only treatment compared to all other treatments through day 19. Table 1. Mean percentage survivorship (±SE) of L. formosa provided various diets on excised mandarin leaves over a period of 26 d (n = 15) Mean % survivorship of L. formosa (± SE) Treatments Day 0 Day 2 Day 5 Day 7 Day 9 Day 12 Day 14 Day 16 Day 19 Day 21 Day 23 Day 26 Leaf only 100 ± 0.0a1A2 95.6 ± 4.4abA 88.9 ± 5.9abA 26.1 ± 3.9dC 15.2 ± 4.3deC 0.0 ± 0.0 eC 0.0 ± 0.0eB 0.0 ± 0.0eC 0.0 ± 0.0eC 0.0 ± 0.0eB 0.0 ± 0.0eB 0.0 ± 0.0e C Honeydew 100 ± 0. 0aA 86.7 ± 6.1abA 85.1 ± 6.4abA 81.8 ± 6.9abAB 76.8 ± 8.9bAB 73.5 ± 11.9bAB 70.1 ± 13.5bcA 67.0 ± 12.0bcA 55.5 ± 12.2bcA 42.3 ± 10.3cdA 25.8 ± 13.4deA 14.5 ± 7.0deAB Sugar water 100 ± 0.0aA 90 ± 4.3abA 88.5 ± 3.2abA 86.9 ± 2.7abAB 75.2 ± 7.5bAB 73.5 ± 7.3bAB 68.5 ± 7.6bcA 63.8 ± 6.6bcAB 50.6 ± 6.7cA 39.0 ± 11.8cdA 34.0 ± 11.4cd A 26.2 ± 6.5d A Honeydew + sugar water 100 ± 0.0aA 95.0 ± 5.0abA 95.0 ± 5.0abA 90.7 ± 4.2abA 87.6 ± 3.5abA 87.6 ± 3.5abA 70.8 ± 8.1bcA 66.7 ± 9.7bcAB 52.0 ± 10.2cA 47.3 ± 7.3cdA 37.9 ± 5.3d A 31.5 ± 3.3d A Cottony cushion scale 100 ± 0.0aA 84.4 ± 12.4abA 80.0 ± 13.9abA 68.9 ± 9.7bcB 57.8 ± 4.4bcB 55.5 ± 2.2bcB 46.6 ± 6.7cdA 40.0 ± 6.7cdB 24.4 ± 5.9deB 24.4 ± 5.9deAB 15.5 ± 2.2deAB 4.5 ± 2.2e BC Honeydew + sugar water + cottony cushion scale + sooty mold 100 ± 0.0aA 100 ± 0.0aA 100 ± 0.0aA 82.5 ± 4.9abAB 75.2 ± 10.4bAB 70.6 ± 12.3bcAB 64.7 ± 10.8bcA 52.0 ± 7.7cAB 39.4 ± 6.4cdAB Leaf collapsed Leaf collapsed Leaf collapsed Mean % survivorship of L. formosa (± SE) Treatments Day 0 Day 2 Day 5 Day 7 Day 9 Day 12 Day 14 Day 16 Day 19 Day 21 Day 23 Day 26 Leaf only 100 ± 0.0a1A2 95.6 ± 4.4abA 88.9 ± 5.9abA 26.1 ± 3.9dC 15.2 ± 4.3deC 0.0 ± 0.0 eC 0.0 ± 0.0eB 0.0 ± 0.0eC 0.0 ± 0.0eC 0.0 ± 0.0eB 0.0 ± 0.0eB 0.0 ± 0.0e C Honeydew 100 ± 0. 0aA 86.7 ± 6.1abA 85.1 ± 6.4abA 81.8 ± 6.9abAB 76.8 ± 8.9bAB 73.5 ± 11.9bAB 70.1 ± 13.5bcA 67.0 ± 12.0bcA 55.5 ± 12.2bcA 42.3 ± 10.3cdA 25.8 ± 13.4deA 14.5 ± 7.0deAB Sugar water 100 ± 0.0aA 90 ± 4.3abA 88.5 ± 3.2abA 86.9 ± 2.7abAB 75.2 ± 7.5bAB 73.5 ± 7.3bAB 68.5 ± 7.6bcA 63.8 ± 6.6bcAB 50.6 ± 6.7cA 39.0 ± 11.8cdA 34.0 ± 11.4cd A 26.2 ± 6.5d A Honeydew + sugar water 100 ± 0.0aA 95.0 ± 5.0abA 95.0 ± 5.0abA 90.7 ± 4.2abA 87.6 ± 3.5abA 87.6 ± 3.5abA 70.8 ± 8.1bcA 66.7 ± 9.7bcAB 52.0 ± 10.2cA 47.3 ± 7.3cdA 37.9 ± 5.3d A 31.5 ± 3.3d A Cottony cushion scale 100 ± 0.0aA 84.4 ± 12.4abA 80.0 ± 13.9abA 68.9 ± 9.7bcB 57.8 ± 4.4bcB 55.5 ± 2.2bcB 46.6 ± 6.7cdA 40.0 ± 6.7cdB 24.4 ± 5.9deB 24.4 ± 5.9deAB 15.5 ± 2.2deAB 4.5 ± 2.2e BC Honeydew + sugar water + cottony cushion scale + sooty mold 100 ± 0.0aA 100 ± 0.0aA 100 ± 0.0aA 82.5 ± 4.9abAB 75.2 ± 10.4bAB 70.6 ± 12.3bcAB 64.7 ± 10.8bcA 52.0 ± 7.7cAB 39.4 ± 6.4cdAB Leaf collapsed Leaf collapsed Leaf collapsed 1Means in each row followed by different lowercase letters are significantly different (Tukey’s LSD, P = 0.05) after arcsine square root transformation. Untransformed means and SEs are reported. 2Means in each column followed by different uppercase letters are significantly different (Tukey LSD, P < 0.05) after arcsine square root transformation. Untransformed means and SEs are reported. View Large Table 1. Mean percentage survivorship (±SE) of L. formosa provided various diets on excised mandarin leaves over a period of 26 d (n = 15) Mean % survivorship of L. formosa (± SE) Treatments Day 0 Day 2 Day 5 Day 7 Day 9 Day 12 Day 14 Day 16 Day 19 Day 21 Day 23 Day 26 Leaf only 100 ± 0.0a1A2 95.6 ± 4.4abA 88.9 ± 5.9abA 26.1 ± 3.9dC 15.2 ± 4.3deC 0.0 ± 0.0 eC 0.0 ± 0.0eB 0.0 ± 0.0eC 0.0 ± 0.0eC 0.0 ± 0.0eB 0.0 ± 0.0eB 0.0 ± 0.0e C Honeydew 100 ± 0. 0aA 86.7 ± 6.1abA 85.1 ± 6.4abA 81.8 ± 6.9abAB 76.8 ± 8.9bAB 73.5 ± 11.9bAB 70.1 ± 13.5bcA 67.0 ± 12.0bcA 55.5 ± 12.2bcA 42.3 ± 10.3cdA 25.8 ± 13.4deA 14.5 ± 7.0deAB Sugar water 100 ± 0.0aA 90 ± 4.3abA 88.5 ± 3.2abA 86.9 ± 2.7abAB 75.2 ± 7.5bAB 73.5 ± 7.3bAB 68.5 ± 7.6bcA 63.8 ± 6.6bcAB 50.6 ± 6.7cA 39.0 ± 11.8cdA 34.0 ± 11.4cd A 26.2 ± 6.5d A Honeydew + sugar water 100 ± 0.0aA 95.0 ± 5.0abA 95.0 ± 5.0abA 90.7 ± 4.2abA 87.6 ± 3.5abA 87.6 ± 3.5abA 70.8 ± 8.1bcA 66.7 ± 9.7bcAB 52.0 ± 10.2cA 47.3 ± 7.3cdA 37.9 ± 5.3d A 31.5 ± 3.3d A Cottony cushion scale 100 ± 0.0aA 84.4 ± 12.4abA 80.0 ± 13.9abA 68.9 ± 9.7bcB 57.8 ± 4.4bcB 55.5 ± 2.2bcB 46.6 ± 6.7cdA 40.0 ± 6.7cdB 24.4 ± 5.9deB 24.4 ± 5.9deAB 15.5 ± 2.2deAB 4.5 ± 2.2e BC Honeydew + sugar water + cottony cushion scale + sooty mold 100 ± 0.0aA 100 ± 0.0aA 100 ± 0.0aA 82.5 ± 4.9abAB 75.2 ± 10.4bAB 70.6 ± 12.3bcAB 64.7 ± 10.8bcA 52.0 ± 7.7cAB 39.4 ± 6.4cdAB Leaf collapsed Leaf collapsed Leaf collapsed Mean % survivorship of L. formosa (± SE) Treatments Day 0 Day 2 Day 5 Day 7 Day 9 Day 12 Day 14 Day 16 Day 19 Day 21 Day 23 Day 26 Leaf only 100 ± 0.0a1A2 95.6 ± 4.4abA 88.9 ± 5.9abA 26.1 ± 3.9dC 15.2 ± 4.3deC 0.0 ± 0.0 eC 0.0 ± 0.0eB 0.0 ± 0.0eC 0.0 ± 0.0eC 0.0 ± 0.0eB 0.0 ± 0.0eB 0.0 ± 0.0e C Honeydew 100 ± 0. 0aA 86.7 ± 6.1abA 85.1 ± 6.4abA 81.8 ± 6.9abAB 76.8 ± 8.9bAB 73.5 ± 11.9bAB 70.1 ± 13.5bcA 67.0 ± 12.0bcA 55.5 ± 12.2bcA 42.3 ± 10.3cdA 25.8 ± 13.4deA 14.5 ± 7.0deAB Sugar water 100 ± 0.0aA 90 ± 4.3abA 88.5 ± 3.2abA 86.9 ± 2.7abAB 75.2 ± 7.5bAB 73.5 ± 7.3bAB 68.5 ± 7.6bcA 63.8 ± 6.6bcAB 50.6 ± 6.7cA 39.0 ± 11.8cdA 34.0 ± 11.4cd A 26.2 ± 6.5d A Honeydew + sugar water 100 ± 0.0aA 95.0 ± 5.0abA 95.0 ± 5.0abA 90.7 ± 4.2abA 87.6 ± 3.5abA 87.6 ± 3.5abA 70.8 ± 8.1bcA 66.7 ± 9.7bcAB 52.0 ± 10.2cA 47.3 ± 7.3cdA 37.9 ± 5.3d A 31.5 ± 3.3d A Cottony cushion scale 100 ± 0.0aA 84.4 ± 12.4abA 80.0 ± 13.9abA 68.9 ± 9.7bcB 57.8 ± 4.4bcB 55.5 ± 2.2bcB 46.6 ± 6.7cdA 40.0 ± 6.7cdB 24.4 ± 5.9deB 24.4 ± 5.9deAB 15.5 ± 2.2deAB 4.5 ± 2.2e BC Honeydew + sugar water + cottony cushion scale + sooty mold 100 ± 0.0aA 100 ± 0.0aA 100 ± 0.0aA 82.5 ± 4.9abAB 75.2 ± 10.4bAB 70.6 ± 12.3bcAB 64.7 ± 10.8bcA 52.0 ± 7.7cAB 39.4 ± 6.4cdAB Leaf collapsed Leaf collapsed Leaf collapsed 1Means in each row followed by different lowercase letters are significantly different (Tukey’s LSD, P = 0.05) after arcsine square root transformation. Untransformed means and SEs are reported. 2Means in each column followed by different uppercase letters are significantly different (Tukey LSD, P < 0.05) after arcsine square root transformation. Untransformed means and SEs are reported. View Large The highest percentage survival of mites was observed for the honey dew + sugar water treatment for each evaluation date for all 26 d; this was significantly different from the leaf-only treatment from day 7 to day 26 and from the cottony cushion scale treatment for days 7, 9, 12, 16, 19, and 26 but was similar to the honeydew or sugar water treatments. Through day 19, all the treatments with honeydew and/or sugar water supported adults and their progeny similarly. The cottony cushion scale treatment had significantly lower percentage survival of mites compared to the sugar + honeydew treatment on day 7, 9, 12, and 19. On day 21, leaves with the honeydew + sugar water + cottony cushion scale + sooty mold treatment collapsed, and no data were collected. Progeny production The cumulative number of eggs produced by females did not differ significantly between treatments on day 5 (F = 2.13; df = 5,16; P = 0.1144) (Table 2). Similarly, the cumulative number of immatures (larvae + nymphs) produced by females in all treatments were statistically similar on day 7 (F = 0.59; df = 5,16; P = 0.708) but differed significantly between treatments on day 14 (F = 6.91; df = 5,16; P = 0.0013) and day 21 (F = 8.68; df = 5,16; P = 0.0004). The data showed that honeydew, sugar water, cottony cushion scale, and honeydew + sugar water supported immature mites through day 21 and honeydew + sugar water supported the largest number of mites. When honeydew, sugar water, cottony cushions scale, and sooty mold were combined, the leaves collapsed on day 21 (Table 1) and the mite population declined to 0.0. Despite a similar number of eggs recorded in the first week in the leaf only treatment, larvae did not survive past day 7 when no sugar supplement (honeydew, sugar water, or cottony cushion scale) was provided (Table 2). Table 2. Mean cumulative number of egg and immature mites per leaf (±SE) produced by L. formosa provided various diets on excised leaves Mean number of mites per leaf (±SE) Eggs Immatures Treatments Day 5 Day 7 Day 14 Day 21 Leaf only 6.0 ± 1.5a1 2.0 ± 0.6a 0.0 ± 0.0c 0.0 ± 0.0c Honeydew 5.5 ± 1.8a 3.3 ± 0.9a 4.0 ± 0.6b 4.0 ± 2.5b Sugar water 3.8 ± 1.3a 2.3 ± 0.7a 3.8 ± 0.9b 4.3 ± 1.2b Honeydew + sugar water 5.0 ± 0.2a 1.8 ± 0.7a 7.2 ± 0.8a 7.0 ± 1.2ab Cottony cushion scale 8.3 ± 5.4a 1.7 ± 0.5a 2.3 ± 1.6bc 3.7 ± 1.6b Honeydew + sugar water + cottony cushion scale + sooty mold 1.7 ± 1.4a 0.75 ± 0.7a 1.5 ± 0.8bc 0.0 ± 0.0c Mean number of mites per leaf (±SE) Eggs Immatures Treatments Day 5 Day 7 Day 14 Day 21 Leaf only 6.0 ± 1.5a1 2.0 ± 0.6a 0.0 ± 0.0c 0.0 ± 0.0c Honeydew 5.5 ± 1.8a 3.3 ± 0.9a 4.0 ± 0.6b 4.0 ± 2.5b Sugar water 3.8 ± 1.3a 2.3 ± 0.7a 3.8 ± 0.9b 4.3 ± 1.2b Honeydew + sugar water 5.0 ± 0.2a 1.8 ± 0.7a 7.2 ± 0.8a 7.0 ± 1.2ab Cottony cushion scale 8.3 ± 5.4a 1.7 ± 0.5a 2.3 ± 1.6bc 3.7 ± 1.6b Honeydew + sugar water + cottony cushion scale + sooty mold 1.7 ± 1.4a 0.75 ± 0.7a 1.5 ± 0.8bc 0.0 ± 0.0c 1Means within a column followed by different letters are significantly different (Tukey HSD, P = 0.05) after square root transformation. Untransformed means and SEs are reported. View Large Table 2. Mean cumulative number of egg and immature mites per leaf (±SE) produced by L. formosa provided various diets on excised leaves Mean number of mites per leaf (±SE) Eggs Immatures Treatments Day 5 Day 7 Day 14 Day 21 Leaf only 6.0 ± 1.5a1 2.0 ± 0.6a 0.0 ± 0.0c 0.0 ± 0.0c Honeydew 5.5 ± 1.8a 3.3 ± 0.9a 4.0 ± 0.6b 4.0 ± 2.5b Sugar water 3.8 ± 1.3a 2.3 ± 0.7a 3.8 ± 0.9b 4.3 ± 1.2b Honeydew + sugar water 5.0 ± 0.2a 1.8 ± 0.7a 7.2 ± 0.8a 7.0 ± 1.2ab Cottony cushion scale 8.3 ± 5.4a 1.7 ± 0.5a 2.3 ± 1.6bc 3.7 ± 1.6b Honeydew + sugar water + cottony cushion scale + sooty mold 1.7 ± 1.4a 0.75 ± 0.7a 1.5 ± 0.8bc 0.0 ± 0.0c Mean number of mites per leaf (±SE) Eggs Immatures Treatments Day 5 Day 7 Day 14 Day 21 Leaf only 6.0 ± 1.5a1 2.0 ± 0.6a 0.0 ± 0.0c 0.0 ± 0.0c Honeydew 5.5 ± 1.8a 3.3 ± 0.9a 4.0 ± 0.6b 4.0 ± 2.5b Sugar water 3.8 ± 1.3a 2.3 ± 0.7a 3.8 ± 0.9b 4.3 ± 1.2b Honeydew + sugar water 5.0 ± 0.2a 1.8 ± 0.7a 7.2 ± 0.8a 7.0 ± 1.2ab Cottony cushion scale 8.3 ± 5.4a 1.7 ± 0.5a 2.3 ± 1.6bc 3.7 ± 1.6b Honeydew + sugar water + cottony cushion scale + sooty mold 1.7 ± 1.4a 0.75 ± 0.7a 1.5 ± 0.8bc 0.0 ± 0.0c 1Means within a column followed by different letters are significantly different (Tukey HSD, P = 0.05) after square root transformation. Untransformed means and SEs are reported. View Large Survival and feeding damage by L. formosa on potted plants Mites could be found on potted plants seven days after they were transferred to leaves; however, the number of mites present on the on the cottony cushion scale-infested plants was significantly greater than that on the cottony cushion scale-free plants (F = 13.51, df = 3,8; P = 0.0017). Only one adult L. formosa was recovered from the cottony cushion scale-free plants and no mites were observed in this treatment from weeks 2–12 (Fig. 1). In contrast, densities of L. formosa stages ranging from 1–4 per leaf were observed during weeks 2–12 (Fig. 1B). Although the mean number mites per leaf on cottony cushion scale-infested plants fluctuated, ANOVA results showed that they were statistically similar between weeks (F = 1.01; df = 11,24; P = 0.46; Fig. 1). We observed that mites aggregated near cottony cushion scale, honeydew, or sooty mold growing on honeydew on the lower side of the leaf near midrib (Fig. 2A). After 12 wk of exposure, cottony cushion scale-infested leaves with mite aggregations did not show any feeding damage when cleaned and observed under the stereoscope (40×). No feeding damage or any other damage was observed for untreated plants. Fig. 1. View largeDownload slide Mean number of L. formosa stages/leaf on potted rough lemon plants with and without cottony cushion scale (CCS) and L. formosa over a period of 12 wk. Fig. 1. View largeDownload slide Mean number of L. formosa stages/leaf on potted rough lemon plants with and without cottony cushion scale (CCS) and L. formosa over a period of 12 wk. Fig. 2. View largeDownload slide L. formosa infesting (A) a cottony cushion scale-infested leaf on potted rough lemon plant and (B) a field-collected mandarin leaf showing adults and immatures along the mid vein and close to sooty mold. Fig. 2. View largeDownload slide L. formosa infesting (A) a cottony cushion scale-infested leaf on potted rough lemon plant and (B) a field-collected mandarin leaf showing adults and immatures along the mid vein and close to sooty mold. Seasonal L. formosa abundance and population structure in a mandarin orchard Population abundance. Seasonal abundance data showed that L. formosa was present in the mandarin orchard on leaves and/or fruit during every sampling period (January 2015 through December 2016; Fig. 3). The percentage infestation of leaves and fruit by L. formosa was significantly lower on most dates in 2016 compared to 2015 (Fig. 3) (F = 8.97; df = 1, 43; P < 0.0001). The proportion of leaves infested with mites declined by 91, 75, 60, and 43% after the applications of thiamethoxam (2015), spinetoram, abamectin, and thiamethoxam (2016), respectively (Fig 3). Insecticides may have affected the mite population directly or indirectly by reducing honeydew-producing insects. Fig. 3. View largeDownload slide Percentage infestation of leaves and fruit by L. formosa sampled from January to December in years 2015 and 2016 in a mandarin orchard. Fruit samples were not collected in May and June of 2015. Arrows indicate pesticide treatments: dotted arrow—fungicide and solid arrow—insecticide. Fig. 3. View largeDownload slide Percentage infestation of leaves and fruit by L. formosa sampled from January to December in years 2015 and 2016 in a mandarin orchard. Fruit samples were not collected in May and June of 2015. Arrows indicate pesticide treatments: dotted arrow—fungicide and solid arrow—insecticide. The percentage infestation of fruit averaged over all time periods (44.8 ± 4%), was significantly higher compared to leaves (16.0 ± 4.7%) (F = 22.61; df = 1,43; P < 0.0001), suggesting that adult mites preferred to reside on fruit. In addition, the percentage of fruit with >10, 1–10, and 0 mites per sample were 9.9, 32.3, and 57.7%, whereas that for leaves were 3.7, 13, and 83.3%, respectively, indicating greater population development on fruit. Field observations showed that mites preferred the shaded side of the tree, the inside canopy leaves, the midrib of the lower side of a leaf with sooty mold (Fig. 2B), and the proximal end of fruit in and around the calyx. Population structure The mean total number of mites (sum of eggs, immatures, and adults per sample) varied in the months sampled (F = 31.31, df = 11, 1392; P < 0.0001) and plant parts sampled (F = 26.58; df = 1,1392; P < 0.0001) but were similar in both years (F = 1.2; df =1,1392; P = 0.2737). The mean number of eggs and immatures per leaf or fruit did not indicate any consistent preference of fruit compared to leaves; however, adults were found in higher densities on fruit (Fig. 4A–C). The data showed a consistent seasonal peak from June to August for immatures in both years; however, such trends were not observed for eggs or adults (Fig. 4A–C). Nymphal and egg mite densities, declined after insecticide treatments (Fig. 4A–C), though whether this was due to a direct effect of the toxicants or the decline in honeydew producing insects is not known. The numbers of eggs and nymphs per sample fluctuated between months in both years and were lowest in November and December of 2015 but highest in December of 2016. We observed a significant correlation between rainfall and the total number of L. formosa sampled per month (r = 0.49; P = 0.015), but no significant correlation between temperature and the number of L. formosa sampled (r = 0.08; P = 0.70) (Fig. 5). Fig. 4. View largeDownload slide Mean number of eggs (A), immatures (B), and adults (C) of L. formosa per leaf or fruit on samples collected during January to December in years 2015 and 2016. Arrows indicate pesticide treatments: dotted arrow—fungicide and solid arrow—insecticide. Fig. 4. View largeDownload slide Mean number of eggs (A), immatures (B), and adults (C) of L. formosa per leaf or fruit on samples collected during January to December in years 2015 and 2016. Arrows indicate pesticide treatments: dotted arrow—fungicide and solid arrow—insecticide. Fig. 5. View largeDownload slide Average temperature, total precipitation, and mean population numbers of L. formosa in fruit and leaf samples for each month in years 2015 and 2016. Fig. 5. View largeDownload slide Average temperature, total precipitation, and mean population numbers of L. formosa in fruit and leaf samples for each month in years 2015 and 2016. The mean number of eggs per leaf or fruit varied in the months (F = 10.49; df = 11, 1392; P < 0.0001) and years (F = 180.29, df = 1,1392; P < 0.0001) but were similar for fruit and leaves (F = 3.51; df = 1,1392; P = 0.06), suggesting that L. formosa adults did not prefer fruit over leaves for egg laying (Fig. 4A). Egg laying occurred year-round and the highest mean number of eggs, 28.7 ± 5.1 eggs/fruit, was recorded in December in 2016 (Fig. 4A). Immatures densities also varied between months (F = 64.75; df = 11,1392; P < 0.0001), years (F = 50.8; df = 1,1392; P < 0.0001), and plant parts sampled (F = 7.98; df = 1,1392; P = 0.0048) (Fig. 4B) and were the most abundant life stage sampled, reaching densities of more than 15 immatures per fruit. Similarly, the number of adults varied according to months (F = 8.08; df = 11,1392; P < 0.0001), years (F = 33.14; df = 1,1392; P < 0.0001), and plant parts sampled (F = 44.56; df = 1,1392; P < 0.0001) (Fig. 4C). Although the highest numbers of eggs and immatures were recorded for December 2016, the number of adults did not show the same increase. Honeydew-producing insects, namely, cottony cushion scale, soft scale, aphids, and whiteflies were present year-round in the orchard and in abundance during the months of April to August/September and sooty mold was associated with the presence of these insects. Discussion L. formosa did not survive on citrus leaves without a supplemental diet in the form of sugary substances and caused no damage to citrus. High adult mortality on excised leaves free of sugary substances and no recovery of L. formosa from the cottony cushion scale-free potted plants in the greenhouse experiment validates that L. formosa cannot maintain or continue development on citrus leaves alone and will eventually die because of a lack of suitable food to support development. Aguilar-Piedra (2001) concluded that L. formosa has a complex feeding behavior but is not a phytophagous species and causes no harm to citrus trees based on experiments using different diets including sugar water, honeydew, pollens, fungal pathogens, and predation of Aculops pelekassi (Keifer) (Acari: Eriophyidae), Phyllocoptruta oleivora (Ashmead) (Acari: Eriophyidae), and Panconychus citri (McGregor) (Acari: Tetranychidae). Similarly, Mendel and Gerson (1982) supported the detrivorous nature of L. formosa and recognized it as a sanitizing agent that helps to control sooty mold in citrus groves. Moreover, Hernandes et al. (2006) emphasized that though reported in high numbers from base of leaflets, L. formosa did not cause any damage to the rubber trees. In another study, researchers maintained healthy colonies of L. formosa on orange leaves; however, they did not report any feeding damage (Badii et al. 2001). Although in high numbers, there was no feeding or other pathological damage of infested leaves where L. formosa aggregated and populations positively correlated with the honeydew producing insect S. oleae (Smirnoff 1957). Honeydew, sugar water, cottony cushion scale, or the combination of these treatments applied to leaves in our study supported longer-term survivorship and progeny production compared to the leaf only treatment. These data support earlier reports that L. formosa populations are associated with honeydew producing insects and sooty mold (Smirnoff 1957, Mendel and Gerson 1982, Aguilar-Piedra 2001). Aguilar-Piedra also showed that L. formosa feeds on sugar water and suggested that sugar water may serve as an alternative source of food for laboratory rearing (Aguilar-Piedra 2001). We showed that L. formosa survived and reproduced on plants that were infested with cottony cushion scale, which provided additional food sources in the form of honeydew and sooty mold, but not on plants free of additional food source. These data confirm that L. formosa cannot survive for more than 1–2 wk on citrus leaves without additional sources of food. Field sampling studies showed L. formosa was present throughout the year in a mandarin orchard in Ventura county, CA. This species is gregarious in nature and high densities of motile stages were reported to cause a visible yellow bleaching of the green leaf (Smirnoff 1957). However, we did not observe any bleaching after 12 wk of observation in our greenhouse experiment or during any month of the 2-yr field study. We confirmed that the mites preferred interior canopy leaves, the shady side of the tree with higher sooty mold growth, and were found on the lower part of the midrib of leaves or hiding in sepals of the fruit (Aguilar-Piedra 2001). In the United States, L. formosa has been previously reported from citrus in Florida, Texas, and Louisiana (Aguilar-Piedra 2001). The highest density of mites (110 mites per leaf—motile stages only) in Florida was reported in April, and the population declined in the summer months. Smirnoff (1957) reported large populations of Lorryia in Morocco in May. In our study, the highest number of motile stages (immatures + adults) observed was in the month of July when 16.9 ± 1.6 mites per sample were recorded. The population consisted predominantly of eggs and immatures and adults were only recorded in low numbers (the highest number recorded was 4.4 per fruit in July 2015). The percentage infestation of fruit and leaves in 2015 was higher in all months compared to 2016, which may be because of insecticides applied in 2016. However, the average number of mites per sample was similar in both years. Based on these studies, L. formosa populations cannot survive for extended periods of time when they lack supplemental food in the form of sugary substances and the do not cause any feeding damage to citrus. Field data showed that L. formosa was present in mandarin orchard in Ventura County, CA, throughout the year, and that insecticide applications affected but did not eliminate mite populations. Despite their year-round presence in the field, the laboratory and greenhouse studies showed that L. formosa cannot survive for more than 2 wk without supplemental food suggesting that field populations are supported by honeydew producing insects. Also, the absence of leaf or fruit damage suggests that this species is not a pest and so it should not be considered a species of phytosanitary concern by importing countries. Acknowledgments We thank Monty Lo for technical support and Tom Roberts for providing a field site to collect samples. The California Citrus Research Board funded this work. References Cited Aguilar-Piedra , H. G . 2001 . Tydeidae of citrus from selected countries: distribution, seasonal occurrence, relative abundance, and feeding habits (Acari: Prostigmata) . pp 200. Ph.D dissertation. University of Florida , Gainesville, FL. Badii , M. H. , A. E. Flores , G. Ponce , J. Landeros , and H. Quieoz . 2001 . Does the Lorryia formosa Cooreman (Acari: Prostigmata: Tydeidae) population visit or reside on citrus foliage ? pp. 413 – 418 . In R. B. Halliday , D. E. Walter , H. C. Proctor , R. A. Norton and M. J. Colloff (eds.), Proceedings of the 10th International Congress of Acarology . CSIRO Publishing , Melbourne , Australia. Hernandes , F. A. , R. J. Feres , and F. Nomura . 2006 . Biological cycle of Lorryia formosa (Acari, Tydeidae) on rubber tree leaves: a case of thelytoky . Exp. Appl. Acarol . 38 : 237 – 242 . Google Scholar CrossRef Search ADS PubMed Mendel , Z. , and U. Gerson . 1982 . Is the mite Lorryia formosa Cooreman (Prostigmata: Tydeidae) a sanitizing agent in citrus groves? Acta . Ecol. Ecol. Appl . 3 : 47 – 51 . (NASS) National Agricultural Statistics Service. 2015 . Citrus fruit 2015 summary . https://www.cacitrusmutual.com/wp-content/uploads/2015/12/2015-USDA-Citrus-Fruits-Summary.pdf (Accessed on 17 April 2016 ). SAS Institute . 2016 . The SAS system for windows, version 9.4 . SAS Institute , Cary, NC . Smirnoff , W. A . 1957 . An undescribed species of Lorryia (Acarina: Tydeidae) causing injury to citrus trees in Morocco . J. Econ. Entomol . 50 : 361 – 362 . Google Scholar CrossRef Search ADS Statgraphics . 2013 . Statgraphics centurion XVI . StatPoint Technologies, Inc, The Plains, VA . © The Author(s) 2018. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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Environmental EntomologyOxford University Press

Published: Apr 5, 2018

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