# 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

Hafiz Muhammad Sajid Ali, Muhammad Kamran, Fahad Jaber Alatawi

## ► To cite this version:

Hafiz Muhammad Sajid Ali, Muhammad Kamran, Fahad Jaber Alatawi. 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, 2025, 65 (3), pp.841-853. 10.24349/lf2w-eh0c. hal-05311306

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Submitted on 13 Oct 2025

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# 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

Hafiz Muhammad Sajid Ali IDª, Muhammad Kamran IDª, Fahad Jaber Alatawi IDª

## Original research

### ABSTRACT

The present research aimed to study the efficiency of the indigenous generalist predator, *Chrysoperla carnea*, for the management of the two-spotted spider mite, *Tetranychus urticae*, infesting cucumber, *Cucumis sativus* plants in greenhouses. The most effective predator stage was determined by placing egg cards and releasing different larval instars (1st, 2nd, and 3rd) with a release ratio of 1:20 for *T. urticae* management. The population reduction percentage of *T. urticae* was 5%, 41%, and 47% after four, eight, and 12 days of 1st larval instar release as compared to egg cards. Among the three larval instars, the 2nd and 3rd instars significantly reduced the *T. urticae* population after eight days, with 75% and 79% population reduction percentages, respectively. Furthermore, less visual damage and webbing density per plant of *T. urticae* were observed where 2nd instar larvae were released. The 2nd instar larva was further evaluated with three predator-prey release ratios to manage *T. urticae* population on *C. sativus* plants in the greenhouse. The *T. urticae* population reduction percentages were 66%, 61%, and 24% at the release ratio of 1:30, 1:60, and 1:90, respectively, after 12 days of predator release. Moreover, less visual damage and localized webbing on leaf per plant was observed for the release ratio of 1:30. The outcomes of the present study showed that the 2nd instar larvae of the indigenous predator, *C. carnea*, could potentially manage the *T. urticae* infestation on greenhouse crops.

**Keywords** biological control; egg cards; larval release; predator instars; predator-prey release ratios

### Introduction

The common green lacewing, *Chrysoperla carnea* (Stephens) (Neuroptera: Chrysopidae), is an efficient generalist predator used to manage different soft-bodied insect pests such as aphids, whiteflies, thrips, mealybugs, etc. (Gautam and Tesfaye 2002). *Chrysoperla carnea* is seeking more attention than other potential biocontrol agents due to its high voracity, adequate searching ability, broad food spectrum, wide distribution, high adaptability to the arid climates,

Received 09 December 2024

Accepted 06 August 2025

Published 11 August 2025

Corresponding author

Fahad Jaber Alatawi ID:
falatawi@ksu.edu.sa

Academic editor

Tsolakis, Haralabos

https://doi.org/10.24349/lf2w-eh0c

ISSN 0044-586X (print)

ISSN 2107-7207 (electronic)

<figure><img src="image_2.png" /></figure>

Ali H. M. S. et al.

Licensed under

Creative Commons CC-BY 4.0

<footnote>ªDepartment of Plant Protection, College of Food and Agriculture Sciences, King Saud University, Riyadh 11451, Saudi Arabia.</footnote>
prevailing in the Kingdom of Saudi Arabia (SA), and well-established mass-rearing techniques (Sattar *et al.* 2011; Tauber and Tauber 1993; Tauber *et al.* 2000).

*Chrysoperla carnea* has been found in different regions of SA (Hölzel 1980; Alghamdi and Sayed 2017; Letardi *et al.* 2020), in association with various spider mite pests on different cultivated and wild vegetations (unpublished data of Acarology and Biological Control Research and Development Laboratory). *Tetranychus urticae* Koch (Acari: Tetranychidae) is the major pest of greenhouse-grown crops in the country (Alatawi 2011; Alatawi and Kamran 2018), and its management is necessary. For developing effective biological control, understanding the effect of different developmental stages of insect predators on pest management is very crucial (New 2002). Each life stage of an insect predator has distinct features, such as incubation period, handling time, consumption rate, searching ability, etc. that influence its predation efficiency (New 2002). The larvae of *C. carnea* are the only stage that predate on insect pests and are known as aphid lions (Tauber *et al.* 2000). *Chrysoperla carnea* eggs are released using egg-cards, while larvae are released either by singly or automated motorized machine mixed with bran or rice husk (Tauber *et al.* 2000).

Some recent studies have documented the efficiency of *C. carnea* as a biocontrol agent of soft-bodied insect pests with a different release ratio of both eggs and larvae in the field (Iqbal *et al.* 2008; Zia *et al.* 2008; Khan *et al.* 2012; Sarwar 2013, 2014), and greenhouses (Alghamdi and Sayed 2017; Salama *et al.* 2016). Few studies have accounted for the management of spider mites by releasing *C. carnea* larval stage (Mena *et al.* 2020; Imam and Nawar 2020; Wafaa and Eid 2008; Saleh *et al.* 2017). However, *T. urticae* management on economic greenhouse crops by releasing *C. carnea* is scarcely studied.

Keeping in view the importance of the locally adapted predator to the prevailing conditions of SA, the aim of this research was: 1) to elucidate the optimal stage (eggs or larvae) of *C. carnea* to release and, 2) to evaluate the efficiency of *C. carnea* predator stage with different predator-prey release ratios for the management of *T. urticae* on *Cucumis sativus* L. (Cucurbitaceae) crop in the greenhouse.

# Materials and Methods

## Predator and prey collections and identification

The population of *Chrysoperla carnea* used in experiments was taken from the mass-reared colony of the predator at the Acarology and Biological Control Research and Development Laboratory (ABRL), Department of Plant Protection, King Saud University (KSU). *Chrysoperla carnea* was collected initially from *Washingtonia filifera* (Rafarin) H.Wendl. (Aceraceae) using an aerial net in February 2020 from the vicinity of KSU campus and has been reared in ABRL for three years. The identification was confirmed by taxonomists/ experts at the KSU, Museum of Arthropods (KSUMA), Department of Plant Protection, College of Food and Agriculture Science (CFAS), KSU, and the voucher specimens were preserved in 75% ethanol. The *C. carnea* larvae were reared by using plastic cups (90ml). A single fresh egg was placed in each plastic cup. As the first larval instar emerged, eggs of *Ephestia cautella* (Walker) (Lepidoptera: Pyralidae) were provided daily. The distinction between the 1st, 2nd, and 3rd instar larvae were made by the exuvia of larvae.

*Tetranychus urticae* population used in current experiments was collected from eggplants grown in the greenhouse in the vicinity of KSU during April 2022. A few collected specimens were brought in laboratory and mounted on (1-1.2mm thick) glass slides (25.4×76.2mm) under SZX10 stereomicroscope (Olympus, Tokyo, Japan), for species identification with the help of available taxonomic literature (Jeppson *et al.* 1975). After identification, voucher glass slide-mounted specimens were deposited in the Acarology laboratory at KSU. For mass rearing, few individuals were released on the 3-4 leaf stages *C. sativus* plants inside the growth chamber (Memmert HPP750eco) at 25 ± 2 °C, 35 ± 5% RH, and 14:10 h L: D photoperiod.
## 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 restricted 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 2nd instar 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 1st, 2nd, and 3rd instar, 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 1st, 2nd, and 3rd larval 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 of *C. carnea*. The distinction among the larval instars (1st, 2nd, and 3rd) 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):

$$ \text{Release No of Predator} = \frac{\text{Total no. of } T. urticae \text{ in treatment} \times \text{Predator ratio}}{\text{Prey ratio}} $$

## 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: T₁ with the release of only egg cards, T₂ with only 1st instar larvae, T₃ with only 2nd instar larvae, T₄ with only 3rd instar larvae, and T₅ 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 T₁. The 1st, 2nd, and 3rd instar larvae were released on T₂, T₃, and T₄, respectively. The eggs hatching was verified during the experiment after observing the white stalked eggshells on the egg's cards and the presence of 1st instar larvae on the plants in T₁. 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: T₁ with a predator-prey release ratio of 1:30, T₂ with a predator-prey release ratio of 1:60, and T₃ with a prey-release ratio of 1:90, and T₄ 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 by *T. urticae* were comparatively investigated on *C. sativus* plants and were statistically analyzed through non-parametric Kruskal-Wallis 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):

$$ \text{Reduction \%} = \left( \frac{\text{Control} - \text{Treatment}}{\text{Control}} \right) \times 100 $$
Results

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

At predator-prey release ratio of 1:20, larval releases of *C. carnea* in its different instars were comparatively effective than using egg cards to manage the *T. urticae* population on *C. sativus* plants in the greenhouse. The larval instar releases significantly suppressed the

<figure><img src="image_3.png" /><figcaption>**Figure 1** Damage symptoms/stippling/yellow spots based on the visual observation of the leaf and webbing density caused by two spotted spider mites, *Tetranychus urticae* Koch by releasing different developmental stages, egg, and larvae, of common green lacewing, *Chrysoperla carnea* (Stephens) on infesting cucumber, *Cucumis sativus* plant leaves in the greenhouse. Damage symptoms/stippling/yellow spots on the *C. sativus* plant leaves were a – less than one-third of the leaf at the time of predator release. In final observation, b – damage remained persistent i.e. less than one-third of the leaf where 2^nd^ instar larvae released, c – half of the leaf where egg, 1^st^ and 3^rd^ instar larvae released, d – more than half of leaf in control plants, e – webbing over whole leaf where egg, 1^st^ instar and 3^rd^ instar larvae, f – upper half of plant covered with webbing in control were observed.</figcaption></figure>
<table><caption>**Table 1** Population size (mean±standard error) and reduction percentage (%) of two spotted spider mites, *Tetranychus urticae* Koch per treatment, infesting *Cucumis sativus* plants after releasing different developmental stages (eggs, 1st, 2nd, and 3rd larval instars) of common green lacewing, *Chrysoperla carnea* (Stephens) in the greenhouse.</caption><thead><tr><th rowspan="2">Treatments (Ts)</th><th colspan="4">TIME (Days)</th></tr><tr><th>At the time of predator release</th><th>After four days</th><th>After eight days</th><th>After 12 days</th></tr></thead><tbody><tr><td>Egg (T<sub>1</sub>)</td><td>141.60 ± 12.8 b</td><td>220.4 ± 29 a, A</td><td>244.2 ± 35 a, A</td><td>259.8 ± 32 a, AB</td></tr><tr><td>1<sup>st</sup> instar larvae (T<sub>2</sub>)</td><td>141.60 ± 12.8 a</td><td>194.4 ± 40 a, A</td><td>185.4 ± 50 a, B</td><td>167.4 ± 24.4 a, BC</td></tr><tr><td>2<sup>nd</sup> instar larvae (T<sub>3</sub>)</td><td>141.60 ± 12.8 b</td><td>104.8 ± 25 a, B</td><td>76.8 ± 13 b, C</td><td>111.6 ± 11a, C</td></tr><tr><td>3<sup>rd</sup> instar larvae (T<sub>4</sub>)</td><td>141.60 ± 12.8 a</td><td>50.4 ± 6 b, C</td><td>64.2 ± 17 b, C</td><td>132.00 ± 38.4 a, C</td></tr><tr><td>Control (T<sub>5</sub>)</td><td>141.60 ± 12.8 c</td><td>204.4 ± 22 b, A</td><td>311.6 ± 52 a, A</td><td>313.8 ± 49 a, A</td></tr></tbody></table>

Means followed by different capital letters within the column are significantly different (RCBD followed by LSD test: $P \le 0.05$). Means followed by different small letters within the row are significantly different (RCBD followed by LSD test: $P \le 0.05$)

T. urticae population and reduced the damage rate than using egg cards ($F = 7.431$, $DF = 4$, $P \le 0.05$) (Tables 1 and 2; Figure 1). The blocking had a significant effect on T. urticae population reduction and its damage rate after four ($P = 0.0125$) and eight ($P = 0.0034$) days of predator release. The highest T. urticae population reduction percentage of 79% was found within eight days after the release of 3rd instar larvae. Afterward, the average population of T. urticae increased up to (132.3 ± 32.7) motiles per plant in 12 days (Table 1). Similarly, a 64% population reduction percentage of T. urticae was observed, with an average mean population of (76.8 ± 13) motiles per plant after 12 days of 2nd instar release. Furthermore, The T. urticae population reduction gradually increased with the release of 1st instar larvae, reaching 47% after 12 days (Table 1). In the control plants, without predator release, the highest T. urticae population was (313.8 ± 49 motiles per plant), with severe leaf damage by the T. urticae population characterized by stippling, discoloration, and webbing (Tables 1 and 2). The population reduction percentage in all treatments was calculated relative to the control.

## Damage assessment

The stippling that appeared on the *C. sativus* leaves by the *T. urticae* population was less than one-third of the leaf (rank 1) in all treatments, including control at the time of predator release (Table 2). After 12 days of predator release, stippling increased to half of the leaf (ranks 2) in the treatments where egg cards, 1st and 3rd instar larvae were released. While it remained less than one-third of the leaf (rank 1) where 2nd instar larvae were released (Table 2). Either

<table><caption>**Table 2** Assessment of damage caused by two spotted spider mites, *Tetranychus urticae* Koch based on the visual observation of the infested cucumber, *Cucumis sativus*, plant leaves and webbing density in different treatments of *Chrysoperla carnea* in the greenhouse.</caption><thead><tr><th rowspan="2">Treatments</th><th colspan="2">Visual observation</th><th colspan="2">Webbing density</th></tr><tr><th>At the time of predator release</th><th>Final observation</th><th>At the time of predator release</th><th>Final observation</th></tr></thead><tbody><tr><td>Eggs (T<sub>1</sub>)</td><td>1</td><td>2</td><td>1</td><td>2</td></tr><tr><td>1<sup>st</sup> instar larvae (T<sub>2</sub>)</td><td>1</td><td>2</td><td>1</td><td>2</td></tr><tr><td>2<sup>nd</sup> instar larvae (T<sub>3</sub>)</td><td>1</td><td>1</td><td>1</td><td>1</td></tr><tr><td>3<sup>rd</sup> instar larvae (T<sub>4</sub>)</td><td>1</td><td>2</td><td>1</td><td>2</td></tr><tr><td>Control (T<sub>5</sub>)</td><td>1</td><td>3</td><td>1</td><td>4</td></tr></tbody></table>

**Ranks for Visual observation:** Stippling appeared on; 1 = less than one-third of the leaf, 2 = half of the leaf, and 3 = more than half of the leaf/Complete discoloration of the leaf. **Ranks for Webbing density:** 1 = localized/scattered webbing, 2 = webbing over the whole leaf, 3 = webbing over the complete leaf and side branch/es, and 4 = upper half of the plants covered with webbing.
complete leaf discoloration or stippling of more than half of the leaf (rank 3) was observed in the control treatment (Figure 1). The webbing constructed by the *T. urticae* population on the *C. sativus* leaves was localized/scattered on all plants, including control (rank 1), at the time of predator release. During final observations, the webbing density increased on all plants (rank 2), except where the 2nd instar larvae were released. Localized/scattered webbing (rank 1) was observed. The upper half of the plants were covered with webbing (rank 4), in the control treatment, produced by the *T. urticae* population (Figure 1).

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

The release of *C. carnea* 2nd instar larvae with three different predator-prey release ratios of 1:30, 1:60, and 1:90 significantly (F = 4.47, DF = 3, *P* <0.0001) reduced the mean population growth rate of the *T. urticae* (Table 3) and its damage to the *C. sativus* plants leaves as compared to the control after 12 days of larval release. (Table 4 and Figure 2). The blocking showed a statistically significant (F = 4.343, *P* = 0.04) effect on *T. urticae* population reduction percentage and its damage after 12 days of 2nd instar release. The 2nd instar larvae significantly (*P* <0.0001) reduced the population growth rate in all three release ratios. However, the highest population

<figure><img src="image_4.png" /><figcaption>**Figure 2** Damage symptoms caused by two spotted spider mites, *Tetranychus urticae* Koch based on the visual appearance of the leaf and webbing density were observed using 2nd instar larvae of the common green lacewing, *Chrysoperla carnea* (Stephens) against three different predator-prey ratios (1:30, 1:60, and 1:90) on the cucumber, *Cucumis sativus* in the greenhouse. Chlorotic symptoms/Stippling/ yellow spots appeared on the leaves in final observations when 2nd instar larvae were used with a predator-prey ratio 1:30 a , 1:60 b, 1:90 c, and control d.</figcaption></figure>
<table><caption>**Table 3** Population size (means±standard error) and reduction percentage (%) of two spotted spider mites, *Tetranychus urticae* Koch infesting cucumber, *Cucumis sativus* plants after releasing 2nd instar larvae of common green lacewing, *Chrysoperla carnea* (Stephens) with three different predator-prey release ratios in the greenhouse.</caption><thead><tr><th rowspan="2">Predator-prey ratios</th><th rowspan="2">Before predator release</th><th colspan="3">TIME (Days)</th></tr><tr><th>After four days</th><th>After eight days</th><th>After 12 days</th></tr></thead><tbody><tr><td>T<sub>1</sub> (1:30)</td><td>117.07±4.18 a</td><td>101.60±10.31 a, B<br>54%</td><td>109.27±15.23 a, C<br>57%</td><td>116.00±19.19 a, C<br>66%</td></tr><tr><td>T<sub>2</sub> (1:60)</td><td>117.07±4.18 a</td><td>104.53±09.17 a, B<br>53%</td><td>117.13±14.02 a, C<br>54%</td><td>132.27±09.68 a, C<br>61%</td></tr><tr><td>T<sub>3</sub> (1:90)</td><td>117.07±4.18 c</td><td>194.40±16.74 b, A<br>13%</td><td>205.93±18.50 b, B<br>19%</td><td>255.73±13.02 a, B<br>24%</td></tr><tr><td>T<sub>4</sub> (Control)</td><td>117.07±4.18 c</td><td>223.00±13.51 b, A</td><td>253.33±11.40 b, A</td><td>336.53±18.39 a, A</td></tr></tbody></table>

Means followed by different capital letters within the column are significantly different (RCBD followed by LSD test: P≤0.05). Means followed by different small letters within the row are significantly different (RCBD followed by LSD test: P≤0.05).

reduction (66%) was significantly higher with the release ratio of 1:30 with an average mean population of (116 ± 19.19) motiles per plant followed by a ratio of 1:60 (61%) with (132.27 ± 09.68) as compared to a ratio of 1:90 (24%) with (255.73 ± 13.02), respectively (Table 3). In the control treatment, the highest average mean population (336.53 ± 18.39) motiles per plant was observed (Table 3).

## Damage assessment

The stippling appeared on the *C. sativus* leaves caused by the *T. urticae* population were less than one-third of the leaf in all treatments at the time of predator release. After 12 days, stippling increased to one-third of the leaf in the release ratio of 1:30, while it was half the leaf in the 1:60 and 1:90. Either more than half or complete discoloration was observed in the control treatment (Table 4 and Figure 2). The webbing constructed by *T. urticae* on the *C. sativus* leaves was localized/scattered in all treatments at the time of predator release. After 12 days, localized/scattered webbing was observed in both release ratios of 1:30 and 1:60 as compared to webbing over the whole leaf in 1:90. Furthermore, webbing over the complete leaf and side branches was observed in control (Figure 2).

<table><caption>**Table 4** Assessment of damage caused by two spotted spider mites, *Tetranychus urticae* Koch based on the visual observation of the infested cucumber, *Cucumis sativus*, plant leaves and webbing density in different treatments by using the 2nd instar larvae of common green lacewing, *Chrysoperla carnea* with three different predator-prey release ratios in the greenhouse.</caption><thead><tr><th rowspan="2">Treatments</th><th colspan="2">Visual appearance</th><th colspan="2">Webbing density</th></tr><tr><th>At the time of predator release</th><th>Final observation</th><th>At the time of predator release</th><th>Final observation</th></tr></thead><tbody><tr><td>T<sub>1</sub> (1:30)</td><td>1</td><td>1</td><td>1</td><td>1</td></tr><tr><td>T<sub>2</sub> (1:60)</td><td>1</td><td>2</td><td>1</td><td>1</td></tr><tr><td>T<sub>3</sub> (1:90)</td><td>1</td><td>2</td><td>1</td><td>2</td></tr><tr><td>T<sub>4</sub> (Control)</td><td>1</td><td>3</td><td>1</td><td>3</td></tr></tbody></table>

**Ranks for Visual appearance:** Stippling appeared on; **1** = less than one-third of the leaf, **2** = half of the leaf, and **3** = more than half of the leaf/Complete discoloration of the leaf. **Ranks for Webbing density:** **1** = localized/scattered webbing, **2** = webbing over the whole leaf, **3** = webbing over the complete leaf and side branch/es, and **4** = upper half of the plants covered with webbing.
Discussion

The impact of using egg cards and releasing different larval instars of the predator, *C. carnea*, was investigated to determine the most optimal larval instar and its predator-prey release ratio by conducting two different greenhouse experiments on *C. sativus* crop to manage the *T. urticae* population and its damage to the crop. *Chrysoperla carnea* larval releases were comparatively more significant than egg cards in managing the *T. urticae* population (Table 1). Similarly, less damages to the *C. sativus* plant leaves were assessed in the larval release treatment as compared to the egg card release (Table 2 and Figure 1).

*Tetranychus urticae* population increased after 12 days of the egg card release (Table 1). The environmental conditions significantly impact on the hatching and incubation period of *C. carnea* eggs (Tauber *et al.* 2000; Sarwar 2014; Takalloozadeh 2015) (Table 1). Laboratory studies revealed that the incubation period of *C. carnea* eggs increased as the temperature decreased: 3.2 days at 32 °C, 6 days at 20 °C and 12 days at 15 °C with 50 ± 10% relative humidity (Ranjbar and Nemati 2020), while the average incubation period is 2.26 days at 25 ± 2 °C and 60 ± 5% relative humidity (Takalloozadeh 2015; Sattar *et al.* 2011). The average daily temperature and relative humidity in the present study were 21 ± 0.6 °C and 59 ± 5%, respectively.

Among the three larval instars, the 2^nd^ instar larvae voraciously consumed the *T. urticae* population and contributed to maximum reduction, similarly to the 3^rd^ instar larvae. According to Hassanpour *et al.* (2009), the highest *T. urticae* population reduction percentage is due to the larger size and low handling time of these stages. Similar results were reported by Sattar (2022) for the 3^rd^ instar of *C. carnea* release in the field, against the cotton-sucking pests was found after one week. Overall, after 12 days, the 2^nd^ and 3^rd^ larval instars showed a significant *T. urticae* population reduction percentage (Table 1). It has been reported that developmental period of 3^rd^ instar larvae is increased till 17 days when fed with motiles of *T. urticae* before it died (Kasap *et al.* 2003), but the nutritional quality of spider mites could have played a potential role in this prolonged duration (Schmidt *et al.* 2012). The 2^nd^ instar larvae remained on the plants for a longer duration compared to the 3^rd^ instar larvae and contributed more effectively to the population reduction percentage of *T. urticae*. The 2^nd^ instar larvae of *C. carnea* showed the highest *T. urticae* population reduction after nine days in the greenhouse on *Phaseolus vulgaris* L. (Fabaceae) plants (Saleh *et al.* 2017). Furthermore, the releasing of *C. carnea* 2^nd^ instar larvae exhibited 96.70% and 100% population reduction percentage of *Aphis gossypii* Glover (Hemiptera: Aphididae) after four and seven days, respectively, at a release ratio of 1:30 (Abdel-Salam *et al.* 2005). Similar results were also reported against the same aphid species on *Solanum melongena* L. (Solanaceae) (Gurbanov 1984). Similarly, the 2^nd^ instar larvae of *Chrysoperla plorabunda* (Fitch) (Neuroptera: Chrysopidae) reduced 84% of the population of Russian green peach aphids, *Diuraphis noxia* (Mordvilko) (Hemiptera: Aphididae), after eight days of the predator release on *Capsicum annum*, *Solanum lycopersicum*, and *S. melongena* (all three from Solanaceae) (Messina and Sorenson 2001).

The 1^st^ instar larvae of *C. carnea* depicted the lowest population reduction percentage of *T. urticae*. This could be due to different biological and ecological aspects. It has a low predation rate due to long handling time, and less nutritional requirement owing to a small body size (Hassanpour *et al.* 2009). Additionally, the glandular trichomes on *C. sativus* leaves may have reduced the predation rate and searching ability of the 1^st^ instar larvae (Elsey 1974). It has been reported that the 1^st^ instar *C. carnea* larvae have a lower predation rate and searching speed on tobacco leaves than on cotton (Elsey 1974). The lowest population reduction percentage on tobacco and cotton leaf by the 1^st^ instar larvae of *C. carnea* was consistent with the effect of trichomes on the predation rate and searching speed (Fordyce and Agrawal 2001).

The plants infested by *T. urticae*, which were subsequently managed by larval releases of *C. carnea*, showed significantly less damage than control plants (Table 2). As a generalist predator, the predation efficiency of *C. carnea* could be affected by the webbing of *T. urticae*, especially at the time of 1^st^ instar emergence as happens for other generalist predators (Shimoda *et al.* 2010; Mirza *et al.* 2023).
However, among the different motile stages of *T. urticae*, the 2^nd^ instar larvae preferred the immature stages for foraging instead of the eggs (Sengonca and Coeppicus 1985). Based on data collected in the present experiment, there are non-significant differences in the *T. urticae* population among different intervals of 1:30 and 1:60 ratios, as shown (Table 3). If eggs of *T. urticae* were being predated, there would be a serious decline in the *T. urticae* population. However, the population maintained or increased very slowly. It means the predator preferred the available motile stages while eggs hatched and added more immatures to the overall *T. urticae* population. The feeding potential of different *C. carnea* larvae, revealed that larvae of *C. carnea* preferred the protonymph stage (Sengonca and Coeppicus 1985). Similarly, the females of the phytoseiid generalist predators, *Amblyseius andersoni* (Chant), *Euseius findlandicus* (Oudemans), and *Euseius hibisci* (Chant), exhibited moderately more preference for the larvae of *T. urticae* over the eggs (Blackwood *et al.* 2001). Likewise, the immature stage of the phytoseiid predator, *Neoseiulus longispinosus* (Evans), demonstrated a preference for the nymphal stage of *Tetranychus neocaledonicus* André over its eggs (Jyothis and Ramani 2019). Similar damage on plants caused by *T. urticae* population in the present study, has been observed in both predator-prey release ratios. This could be an indication of the high searching ability of the larvae.

At 1:90, *T. urticae* showed an exponential population growth rate after 12 days with the lowest reduction percentage of 24% (Table 3). The exponential growth of *T. urticae* created more complex/dense webbing than in 1:30 and 1:60 experiments, hindering the movement of the predator larvae. Initially, the 2^nd^ instar larvae of *C. carnea* reduced the *T. urticae* population by primarily foraging on immatures (Sengonca and Coeppicus 1985). However, surviving *T. urticae* females dispersed to new leaves and established new colony patches (Van and Yurewicz 1998). These dispersed females contributed to exponential population growth in the new leaves and constructed more complex/complicated webbing which hinder the movement of the predator. The webs serve as an anti-predator function, with complex, dense webbing providing better protection from predators (Dittmann and Schausberger 2017). The predator's foraging to *T. urticae* was limited due to webbing over the whole abaxial surface of *C. sativus* leaves. In this situation, the generalist predator cannot cope with the complex, dense webbing produced by the *T. urticae* population. Consequently, the predator relocates to find the new patches inhabited by the *T. urticae* population with comparatively low webbing. However, without the predator, the population of *T. urticae* thrives and exploits the plants (Kuchlein 1966; Sabelis 1982).

Similarly, in the damage assessment of this experiment, webbing was observed over the whole leaf at 1:90 (predator/prey ratio) after the 12 days of the predator release (Table 4). The *T. urticae* damage to *C. sativus* plants is directly related to the visual yellow spots (stippling) and webbing density (Tables 3 and 4, Figure 2). The plants infested with *T. urticae* managed by higher *C. carnea* release ratios, had significantly less damage than the control. In support of the present experiment, *Phytoseiulus persimilis* Athias-Henriot released with release ratios of 1:10 and 3:10 showed less damage and stippling at high release ratios on the Lima bean (*Phaseolus lunatus* L.) crops in the greenhouse (Amoah *et al.* 2016).

The outcomes of the present study indicate that the use of *C. carnea* larval instars is more effective than releasing its egg card. However, egg cards could be used at low levels of spider mite infestations. As compared to all three instars of *C. carnea*, the 2^nd^ instar larvae have the potential for the management of *T. urticae* in greenhouses between the release ratio of 1:30 to 1:60. Simultaneously, these release ratios significantly reduced the visual and webbing damage of *T. urticae* on cucumber plants. In this way, these predators could provide pest management for an extended time. Clearly, repeated releases of 2^nd^ instar larvae to explore time intervals and cost-effective techniques for the management of *T. urticae* and other greenhouse pests would be desirable.
## Funding

This project was funded by the National Plan for Science, Technology, and Innovation (MAARIFAH), King Abdul-Aziz City for Science and Technology, Kingdom of Saudi Arabia, Award Number (3-17-07-001-0005).

## Data Availability Statement

All necessary data for the manuscript is provided.

## Acknowledgments

We are thankful to the researchers of Acarology and Biological Control Research and Development Laboratory, Department of Plant Protection, King Saud University, for their help in the experimentation.

## Conflicts of Interest

The authors declare no conflict of interest.

## ORCID

Hafiz Muhammad Sajid Ali https://orcid.org/0000-0001-7110-6433

Muhammad Kamran https://orcid.org/0000-0001-6084-203X

Fahad Jaber Alatawi https://orcid.org/0000-0002-6824-2650

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