Cucumis sativus

AliK, Hafiz Muhammad Sajid, KamranK, Muhammad & AlatawiK, Fahad Jaber, 2025, Evaluation of different larval instars and predator-prey release ratio of indigenous generalist predator, common green lacewing, Chrysoperla carnea, for the management of two-spotted spider mite, Tetranychus urticae, on cucumber, Cucumis sativus crop in greenhouses, Acarologia 65 (3), pp. 841-853 : 843-844

Preparation of Cucumis sativus seedlings

Cucumis sativus seeds of Beth Alpha variety ( Italy: Reg # S0003043004500018), were sowed in pots with dimension 16 x 15 x 16cm (Ward, UK) filled with European standard peatmoss, and potting soil (Factory of Saudi Soil Development Company, Saudi Arabia), with a 2:1 ratio, respectively. The pots were placed in a controlled growth chamber (Memmert HPP750eco) at

25 ± 2 °C, 35 ± 5% RH, and 14:10 h L: D photoperiod and irrigated regularly to get seedlings. The seedlings were transferred to the new big sized pots 22 x 22 x 17cm (Ward, UK) when they reached the first true leaf stage. Then, pots moved to the greenhouse and placed them in cages

(Bio Quip, USA) to avoid unwanted insect pest infestation.

Experimental procedures

Ten females and two males of T. urticae were placed onto each 6-8 leaf C. sativus plant with a camel hairbrush. After 15 days, the mite population was estimated by randomly selecting three leaves from the upper, middle, and lower parts of each plant. All treatments were organized in the greenhouse following a randomized complete block design (RCBD) with 45

cm plant-to-plant and 60 cm with block-to-block distance. This ensured that the plant leaves did not touch the adjacent plants, and both predator and pest individuals were entirely to the respective experimental units. In Experiment 1, different developmental stages (egg and larval instars) of the predator were released on the plants corresponding to the treatments. Whereas, in Experiment 2, varying numbers of the 2 ndinstar larvae were released based on their predator-prey release ratios. The eggs and larval instars used in the experiment were <12 hours old. The eggs were acquired from the stock culture, pasted on egg cards and were directly used in the experiment. For acquiring different larval instars of the predator, a total of 200 eggs of C. carnea for each larval instar (total 600 eggs for three instars) were placed separately in each plastic cup (90ml) at different time intervals based on the differences in their developmental period i.e. three, six and eight days for st, 1 2 nd, and 3 rdinstar, respectively ( Shakoor et al. 2024).

It was done so that all three larval instars could be available simultaneously for the experiment,

i.e. rearing of 1 st, 2 nd, and 3 rdlarval instars started at 12, nine and seven days of mite inoculation.

A suitable amount of E. cautella eggs were provided as food to the neonate larvae C of. carnea .

The distinction among the larval instars (1 st, 2 nd, and 3 rd) was made by the exuviae of each instar. After 15 days of T. urticae inoculation, C. carnea was brought to the greenhouse in plastic cups and was released on their respective treatments with the camel hairbrush. The plants were placed on the inverted plate submerged inside the large plate to prevent the predator from escaping. All motile stages of T. urticae were recorded after four, eight, and 12 days of predator release by randomly selecting three leaves from the upper, middle, and lower parts of each plant. All observations were performed using 24x hand lens. The required population of the predator was calculated according to the following equation ( Wafaa and Eid 2008):

Total no. of T. urticae in treatment x Predator ratio Release No of Predator= Prey ratio

Experiment 1. Impact of releasing different developmental stages (egg and three larval instars) of Chrysoperla carnea for management of Tetranychus urticae

This experiment was conducted from February to April 2023 in the greenhouse of the

Plant Production Department, CFAS, KSU. The average temperature and relative humidity inside the greenhouse during the experiment were recorded as 21 ± 0.6 °C and 59 ± 1.5%, respectively. Temperature and relative humidity data in all experiments were collected with HOBO environmental monitors (Onset Computer Corporation, Bourne, MA, USA).

After the estimation of the T. urticae population, 15 days after inoculation, different stages of a predator, C. carnea , were released with a prey release ratio of 1: 20 in all treatments with the camel hairbrush. The treatments were: T 1 with the release of only egg cards, T 2 with only 1 st instar larvae, T 3 with only 2 ndinstar larvae, T 4 with only 3 rdinstar larvae, and T 5 without predator release (Control). Each treatment was replicated five times. The egg cards, approximately 6 cm long and 4 cm wide, were hung with the C. sativus leaves in treatment

T 1. The 1 st, 2 nd, and 3 rdinstar larvae were released on T 2, T 3, and T 4, respectively. The eggs hatching was verified during the experiment after observing the white stalked eggshells on the egg’s cards and the presence of st 1instar larvae on the plants in T 1. Furthermore, the presence of C. carnea larvae on the plants were observed throughout the experiment. All the instars consistently remained on the plants within their respective treatments.

Experiment 2. Impact of releasing different predator-prey release ratios of Chrysoperla carnea , against Tetranychus urticae

This experiment was conducted from September to November 2023 in the greenhouse of the Education Farm of KSU. The temperature and relative humidity inside the greenhouse during the experiment was recorded as an average of 24 ± 0.5 °C. and 57 ± 1.7% respectively. Four treatments represented three predators-prey release ratios and control treatment. The treatments were: T 1 with a predator-prey release ratio of 1:30, T 2 with a predator-prey release ratio of

1:60, and T 3 with a prey-release ratio of 1:90, and T 4 without predator release (Control). Each treatment was replicated 15 times.

Damage rate assessment

For both experiments, the damage rate caused by T. urticae to the C. sativus plant leaves was evaluated by visual observation based on damage symptoms/stippling/yellow spots and webbing density on the leaves. The damage rate was categorized into three levels, with stippling appearing/yellow spots on ( Suekane et al. 2012): (a) less than one-third of the leaf, (b) half of the leaf, and (c) more than half of the leaf/complete discoloration of the leaf. The webbing density was categorized into four levels: (a) localized/scattered webbing over the leaf, (b) webbing over the whole leaf, (c) webbing over leaves and leaves petioles, and (d) upper half of the plants covered with webbing.

Statistical analysis

The data obtained on T. urticae population size from both experiments were analyzed using a one-way ANOVA following a Randomized Complete Block Design (RCBD). Prior to analysis, data were analyzed for normality and homogeneity of variance using Kolmogorov-Smirnov test and Levene’s test, respectively. The data for the effect of different developmental stages of predator on the T. urticae population in different stages (within as well as among the treatments after each sampling) were transformed using log transformation. The means of untransformed data are presented in Table 1. Means were compared using Fisher’s least significant difference (LSD) at P ≤ 0.05. The observed damage symptoms and webbing density produced T. by urticae were comparatively investigated on C. sativus plants and were statistically analyzed through non-parametric Kruskal-Walli’s test. The mean scores were ranked by the Wilcoxon rank-sums test. Additionally, photographs related to observations of damage symptoms were captured by the Canon 700D DSLR camera (Canon, Japan). All analyses were run using the Computer Software SAS v.9.2 (SAS Institute, USA). The T. urticae population reduction percentage as compared to the control treatment was analyzed following the Abbott formula (1925):

Control − Treatment Reduction% = () x100 Control