Host stage preference and demographic parameters of Leptomastix dactylopii (Hymenoptera: Encyrtidae) on vine mealybug Planococcus ficus (Hemiptera: Pseudococcidae)

This study was conducted to investigate host stage preference and demographic parameters of the parasitoid species, Leptomastix dactylopii Howard (Hymenoptera: Encyrtidae), the parasitoid of vine mealybug Planococcus ficus (Signoret) (Hemiptera: Pseudococcidae), an important pest in vineyards in many countries. The results revealed that L. dactylopii was not able to develop on the 1st and 2nd nymphal instars of P. ficus and preferred female mealybugs as the host to the 3rd nymphal instar. Hind tibia length and head capsule width of both female and male parasitoids emerged from the mummies of mealybugs parasitized in the female stage were greater than the values of the parasitoids emerged from the mummies of mealybugs parasitized in the 3rd nymphal instar. Demographic parameters of the parasitoid were calculated with the use of development and reproduction data obtained from life table of L. dactylopii. The intrinsic rate of increase was determined as (r = 0.1527 d−1), finite rate of increase as (λ = 1.1650 d−1), net reproductive rate as (R0 = 46.0667 offspring), and mean generation time as (T0 = 25.0830 d). It was concluded that L. dactylopii was able to develop, especially in the female and in the 3rd nymphal instar of P. ficus and could be used for biological control of the vine mealybug.


Background
Vine mealybug Planococcus ficus (Signoret) (Hemiptera: Pseudococcidae) is one of the most important pests of Vitis spp. (Vitaceae), primarily in Mediterranean countries, as well as in South Africa, Pakistan, Argentina, California, and Mexico (Daane et al. 2012). It was reported that the vine mealybug feeds on nearly 40 plant species belonging to 24 families and 30 genera in 46 different countries (García Morales et al. 2016). P. ficus generates damage through sucking plant sap from almost all parts of the host, including vine roots (Ben-Dov 1994). Due to the development of saprophytic fungi on the honey-like substances it secretes, plant photosynthesis capacity, fruit quantity, and overall plant quality decrease (Godfrey et al. 2002). It also causes significant yield losses in vineyards, even in small populations, through serving as a vector for viral diseases (Tsai et al. 2010).
Today, broad-spectrum insecticides are generally used to control the vine mealybug. However, repeated insecticide applications throughout the production season have various negative effects on the environment, nontarget organisms, and especially natural enemies in the environment (Prabhaker et al. 2012). Because of these Page 2 of 10 Muştu et al. Egyptian Journal of Biological Pest Control (2022) 32:75 negative aspects of chemical control, biological control is commonly practiced against P. ficus. Although P. ficus has several natural enemies from different orders and families (Cocco et al. 2021). The parasitoids; Anagyrus pseudococci (Girault) and Coccidoxenoides perminutus Girault (Hymenoptera: Encyrtidae) are among the most important natural enemies used for the control of vine mealybug in various countries (Sime and Daane 2014). Leptomastix dactylopii is a solitary koinobiont endoparasitoid. It prefers the 3rd nymph and young female stages of the host (Muştu and Kılınçer 2015). Although the homeland of L. dactylopii was previously thought to be Brazil (Compere 1939), recent studies reported that it might be originated from Africa (Kol-Maimon et al. 2015). L. dactylopii has low tolerance to low temperatures.
Although L. dactylopii, a polyphagous species, parasitizes more than 20 mealybug species, its main host is the citrus mealybug (Anga and Noyes 1999). It prefers the 3rd nymph and female stages of the citrus mealybug (de Jong and van Alpen 1989). L. dactylopii was obtained during surveys conducted in vineyards of South Africa (Walton and Pringle 2004), Tunisia (Mahfoudhi and Dhouibi 2009), and Iran (Fallahzadeh et al. 2011). It was indicated in recent laboratory studies that L. dactylopii was a potential candidate to be used as a biological control agent for the vine mealybug (Marras et al. 2016).
This study was conducted to determine the host period preference of L. dactylopii on P. ficus nymph and adult stages and to calculate its life table parameters on the vine mealybug.

Insect cultures
A Planococcus ficus colony was collected from Morus alba L. (Moraceae) in Adana city, Turkey transferred to the laboratory, and placed on sprouted potatoes. Potatoes to be used as food for the vine mealybug were kept at 4 °C for 2 weeks and then sprouted in dark ovens at 15-18 °C. Mass production of mealybugs was carried out on potatoes sprouted at 25 ± 1 °C and 60 ± 10% RH. Mealybug eggs collected from ovisac females of P. ficus, with the aid of a soft-tipped brush, were smeared onto sprouted potatoes. Egg-infested potatoes were placed in 3-lt plastic jars covered with muslin mesh, with ventilation openings. Development of mealybugs was regularly checked. Culture continuity was ensured by smearing new eggs onto newly sprouted potatoes.
Leptomastix dactylopii was supplied from the Biyolojik Tarım (Biological Agriculture (Biyotar), Erzin, Hatay, Turkey) Company. The potatoes infested with mealybugs at the 3rd and unmated female stages were placed into plastic jars covered with muslin mesh and with ventilation holes. A stock culture was obtained by placing a certain number of female and male parasitoids (30 ♀ and 30 ♂) into the jars. With the resultant new parasitoids, new cultures were created under suitable conditions and the continuity of the culture was ensured. L. dactylopii was adapted by rearing it for 3 generations on P. ficus before the experiments.

Host stage preference of Leptomastix dactylopii on Planococcus ficus
In the present experiments, 1st, 2nd, and 3rd nymphal instars, and adult female stage of P. ficus were offered to the parasitoid females to parasitize in 2 different ways; with 20 separately from each stage (no-choice) and 5 from each stage together (choice).
To obtain mealybugs to be used in the experiments at the appropriate period, sprouted potatoes in plastic containers with 4 × 10 × 11 cm dimensions, covered with muslin mesh and with ventilation openings, were smeared with mealybug eggs daily, in 2 containers per day, throughout the experiments. The development and molting of mealybugs in the containers were followed through daily observations and the mealybugs that reached the appropriate stage were used in the experiments. Newly molted mealybugs were collected from the potatoes with the aid of a soft-tipped brush one day before the experiment, transferred onto vine leaves in previously prepared Petri dishes, and the mealybugs were allowed to settle on the leaves. About 1/3 of the Petri dishes (5.5 × 1.4 cm), with a ventilation opening and covered with muslin mesh, in which nymphs and adult mealybugs were transferred, were filled with 1% water agar and a disc-shaped vine leaf (Narince cultivar) was placed on the agar with the bottom surface facing up.
Each newly hatched parasitoid female to be used in the experiments was left for feeding and mating in the Petri dishes containing a male individual and 50% honey for 24 h after they became adult. Then, a female parasitoid was released into a Petri dish, previously prepared as stated above. Following a 24-h trial period, parasitoid females were removed from the Petri dishes and the Petri dishes with mealybugs were placed into the climate cabinet at 25 ± 1 °C, 60 ± 10% RH, and 16:8 (light: dark) conditions. Petri dishes were renewed every 3 days and mealybugs were transferred to new dishes with fresh leaves. After 10 days, mummified individuals were placed into Eppendorf tubes and waited for emergence. Emerged individuals were then placed into Eppendorf tubes containing 70% alcohol and the width of head capsule and the length of tibia of the left hind leg were measured. Morphometric measurements were made under a Leica stereo-binocular microscope using the Leica IM50 Image software. At the end of the experiments, the proportion Page 3 of 10 Muştu et al. Egyptian Journal of Biological Pest Control (2022) 32:75 of mummified host, proportion of non-emerged parasitoids, proportion of female parasitoids, and development time, head capsule width, and tibia lengths in each mealybug stage of the parasitoid were determined. Experiments were conducted in 20 replications.

Development, reproduction, and life table of Leptomastix dactylopii on Planococcus ficus
To obtain female mealybugs to be used in the experiments, daily mealybug cultures were generated with the method described above. Pre-ovipositional female mealybugs were collected from the potatoes with the aid of a soft-tipped brush one day before the experiments and transferred to the vine leaves in the Petri dishes, previously prepared as described above, to have 20 individuals in each Petri dish and the mealybugs were allowed to settle on leaves. Leaves were supplemented with 50% diluted honey to feed the parasitoids in Petri dishes.
Before the experiments, 30 parasitoid mummies were collected from the parasitoid stock culture and placed together into 1-lt plastic jars with muslin mesh-covered ventilation holes. The mummies were checked daily and emerged parasitoid adults were released into Petri dishes containing mealybugs, which were prepared beforehand, to have 1 ♀ and 1 ♂ parasitoid in each Petri dish. Parasitoids were removed from the test dish after 24 h and transferred to a new Petri dish prepared in the same way. From the day of emergence to death, parasitoids were transferred to new Petri dishes prepared as described above.
The Petri dishes used in the experiments were placed into climate cabinets at 25 ± 1 °C, 60 ± 10% RH, and 16:8 (light: dark) conditions and mummification was observed through daily checks. Fully mummified individuals were placed into separate Eppendorf tubes in the same climate cabinet and waited for adult parasitoids to emerge. At the end of the experiments, development time of parasitoid on vine mealybug, adult life span, number of oviposition days, number of juveniles, and female-male ratios were determined.

Statistical analysis
Since the data obtained from the experiments for host stage preference of L. dactylopii on P. ficus did not meet the parametric test conditions, a Kruskal-Wallis test was used to compare percent mummification ratios in no-choice tests and a Dunn-Bonferroni test was used to compare different groups. Independent-Samples Mann-Whitney U test was used for the analysis of proportion of non-emerged parasitoids. In choice tests, a Friedman test was used in the analysis of proportion of mummified host and a Dunn-Bonferroni test was used to determine different groups. Proportion of non-emerged parasitoids was analyzed with the use of the Related-Samples Wilcoxon Signed Rank test. SPSS 22.0.0.0 software was used for statistical analysis of host stage preference trials.
With the use of the development and reproduction data of L. dactylopii on vine mealybug, life table parameters revealing significant information about the parasitoid were obtained. These parameters were calculated based on age-stage specific two-sex life table analyses (Chi and Liu 1985;Chi 1988). The TWOSEX MSChart software was used to analyze life table raw data (Chi 2021). Age-stage-specific survival rate (s xj ) (x age and j stage), age-stage-specific fecundity (f xj ), age-specific longevity (l x ), female age-specific fecundity (m x ) and age-specific maternity (l x m x ) values were calculated.
In the age-stage-specific two-sex life table, l x and m x parameters were calculated with the use of the following equations: Net reproductive rate (R 0 ) was calculated with the use of the following equation. Net reproductive rate was obtained by multiplying age-specific longevity (l x ) by agespecific fecundity (m x ) rates.
Intrinsic rate of increase (r) (day −1 ) was calculated with the use of the following equation (Goodman 1982): The other life table parameters were calculated as follows: Finite rate of increase, (λ) (d −1 ), (λ = e r ). Mean generation time, (T) (d), T = ln(R 0 /r), (time span required to increase population size up to net reproductive rate).
Age and stage-specific life expectancy, (e xj ), was calculated with the use of the following equation (Chi and Su 2006). Age and stage-specific reproductive value (v xj ) was calculated with the use of the following equation (Tuan et al. 2014).
For calculated life table parameters, 100,000 artificial bootstraps (Bootstrapping is a statistical procedure that resamples a single dataset to create many simulated samples) (Efron and Tibshirani 1993) were produced with the use of TWOSEX MSChart (Chi 2021) software and means and standard errors of life table parameters were obtained.

Host stage preference of Leptomastix dactylopii on Planococcus ficus
In no-choice trials, L. dactylopii could not develop in the 1st and 2nd nymph stages of the vine mealybug (H = 74.738, df = 3, p ˂ 0.001). Although it was observed that there were parasitized individuals in the first 2 nymphal instars, the parasitized nymphs generally died and mummification was not encountered in any of them. Therefore, the 1st and 2nd nymphal instars of vine mealybug were considered unsuitable host stages for L. dactylopii. When the mealybug stages, in which the parasitoid can develop were examined, although the number of mummies formed in female P. ficus stage parasitized by L. dactylopii was higher than the number of mummies in the 3rd nymphal instar, difference between the 2 groups was non-significant (Z = − 18.050, p = 0.052) ( Table 1). When the number of parasitoid adults that could not emerge from the mummies were compared, there was non-significant difference between the parasitoid mummies formed in the female and the 3rd nymphal instar (U = 207.500, p = 0.841) ( Table 1). When the sex ratios of the parasitoid adults emerging from the mummies in both mealybug periods were examined, the number v xj = e r(x+1) s xj ∞ i=x e −r(i+1) k y=j s ′ iy f iy emerging from the adult mealybug mummies was higher than the number of female parasitoids emerging from the mealybug mummies parasitized in the 3rd nymphal instar (Table 1). When the development times of L. dactylopii in the female and 3rd nymph stages of vine mealybug were compared, the development of both female (U = 526.500, p = 0.003) and male (U = 1827.500, p < 0.001) parasitoids on adult mealybugs was longer than their development on the 3rd nymphal instar mealybugs (Table 2). When the morphometric characteristics of the parasitoids were examined, it was observed that the difference between the hind tibia lengths of both female parasitoids (U = 530.000, p = 0.003) and male (U = 2323.000, p = 0.003) parasitoids emerged from mealybug mummies parasitized in female and the 3rd nymphal instar was significant. Similarly, when the head capsule widths of the parasitoids that emerged from the mummies in both mealybug periods were compared, there were significant differences in head capsule widths of both female parasitoids (U = 668.500, p ˂ 0.001) and male (U = 2435.000, p ˂ 0.001) parasitoids.
Similar with the findings of the no-choice trials, in choice trials, L. dactylopii parasitized the mealybugs  Page 5 of 10 Muştu et al. Egyptian Journal of Biological Pest Control (2022) 32:75 in the female stage more than the 3rd nymphal instar (F r = 53.055, df = 3, p ˂ 0.001). In terms of adult parasitoid emergence, there were non-significant differences between the female and the 3rd nymphal instar (W = 8.500, p = 0.366), the rate of female parasitoids emerged from female mealybug mummies was higher than that of the mealybugs parasitized in the 3rd nymphal instar (Table 3).

Development, reproduction, and life table of Leptomastix dactylopii on Planococcus ficus
The data on the developmental stages of L. dactylopii are provided in Table 4. The pre-adult developmental period of L. dactylopii females was 18.80 days, while preadult developmental period of L. dactylopii males was 18.00 days. The pre-adult mortality rate of the parasitoid was 17%. L. dactylopii did not need an adult pre-ovipositional period (APOP) and could parasitize its host at the first day of emergence. The total pre-ovipositional period (TPOP) (sum of pre-adult developmental time and adult pre-ovipositional period) of L. dactylopii was 18.80 days ( Table 5). Number of oviposition days of the parasitoid was 17.07 days and fecundity was 92.13 offspring. Longevity of female and male adults was 22.07 and 19.80 days, respectively. The total life span of parasitoid females and males were 40.87 and 37.80 days, respectively.
Age and stage-specific survival rate (s xj ) indicated survival probability of a newly oviposited eggs until age x and stage j (Fig. 1). The survival probability of a newly oviposited L. dactylopii egg to an adult stage was 50% for females and 33% for males. Age-specific longevity (l x ), female age-specific fecundity (f x8 ), whole population fecundity (m x ), and age-specific maternity (l x m x ) values are presented in Fig. 2. The l x curve is a simplified version of the curve in Fig. 1 and was calculated as the sum of all surviving individuals in different periods.
The data on development and reproduction of L. dactylopii were used to calculate life table parameters (Table 6). The intrinsic rate of increase (r) of the parasitoid on P. ficus was calculated as 0.1527 d −1 and net reproductive rate (R 0 ) was calculated as 46.0667 offspring.
Life expectancy (e xj ) indicates expected life span of an individual at age x and stage j, reproductive value (v xj ) indicates the contribution of an individual at age x and period j to the future population. While the maximum life expectancy of a newly oviposited L. dactylopii egg was determined as 36.03 days (Fig. 3), the female    Muştu et al. Egyptian Journal of Biological Pest Control (2022) 32:75 parasitoid reached the highest reproductive value at day 19 (v19 = 41.62 offspring) (Fig. 4). Zinna (1959) reported that the parasitoid L. dactylopii preferred to parasitize the host P. citri generally at the 3rd instar nymphs and females and rarely parasitized the 2nd instar nymphs. de Jong and van Alphen (1989) divided P. citri into 4 size classes and reported that L. dactylopii parasitized size class 3 (consists mainly of 3rd nymph instar with some 2nd and 4th) and size class 4 (consists of pre-ovipositing females and ovipositing females) mealybugs of P. citri. They also indicated that female parasitoids were developed from oviposited female hosts and Age (d) mostly male parasitoids were developed from the 3rd nymphal instar and pre-ovipositing female mealybugs (de Jong and van Alphen 1989). Similarly, in parasitizing preference trials conducted with and without options, it was determined that L. dactylopii parasitized the female and 3rd nymphal instar of P. ficus and mealybug females were more preferred than the 3rd nymphal instar by the parasitoid. In addition, similar to the studies with P. citri, the number of female parasitoids emerging from P. ficus females was higher than those parasitized in the 3rd nymphal instar for both trials. However, although it was observed that L. dactylopii tried to parasitize vine mealybug individuals in the 1st and 2nd nymphal instar, mummification was not encountered in these stages. It was observed that the 1st and 2nd instar nymphs attempted to be parasitized by L. dactylopii immediately tried to move away from the place where they feed as a defensive response and these nymphs ultimately died out.

Discussion
In the present study, the rate of female parasitoids emerging from adult mealybugs in choice trials was higher than in no-choice trials. Since there are a limited number of female mealybugs in choice trials than in no-choice trials, the parasitoid might oviposit a greater number and laid more fertilized diploid eggs on female mealybugs in choice trials. Marras et al. (2016) reared L. dactylopii separately on P. ficus and P. citri and exposed the parasitoids to hosts from which they were reared and the other host mealybug species as a cross. The researchers reported that hind tibia lengths of the female parasitoids obtained for all groups were between 640 and 660 µm. In the present study, hind tibia lengths of male and female individuals parasitized during the adult mealybug stage were determined as 671 µm and 616 µm, respectively.
Life table parameters revealed that development of female parasitoids lasted shorter time than the males. In previous studies conducted at 25 °C with near relative humidity and lighting conditions, quite different durations were reported for the development of L. dactylopii on P. citri. Battaglia et al. (1996) reported a developmental duration of female and male individuals of L. dactylopii as 18.39 and 16.11 days, respectively. Telli and Yiğit (2004) reported pre-adult development period of L. dactylopii female as 22.85 days. Yang and Sadof (1997) reported development durations of L. dactylopii on three different plant cultivars of Coleus blumei (Bentham) (Lamiaceae) as 21.9, 20.2, and 20.7 days, and mortality rates as 6.48, 12.59 and 4.44%, respectively. In the present