Survival, development and reproduction of Phenacoccus solenopsis (Hemiptera: Pseudococcidae) on kaolin-treated cotton

The mealybug, Phenacoccus solenopsis Tinsley (Hemiptera: Pseudococcidae) is a cotton pest widespread in several cotton growing regions of Brazil, particularly in the semi-arid region of southwestern Bahia. The impact of kaolin on survival, development and reproduction of P. solenopsis was evaluated in the laboratory. The experiment was developed in a completely randomized design with two treatments: immature or newly emerged adults of P. solenopsis sprayed with kaolin and fed with cotton leaf discs treated with kaolin suspension (with kaolin) (T1) and immature or newly emerged adults of P. solenopsis sprayed with distilled water and fed with cotton leaf discs treated with distilled water (without kaolin) (T2). The kaolin suspension shortens the life cycle, increases the reproductive potential and population growth of the cotton mealybug, P. solenopsis and, therefore, it should be used with caution on cotton plants in regions with a history of occurrence of this pest.


Introduction
Cotton plants host a high boll, leaf, root, square and stem pest complex (Allen et al., 2018). The mealybug, Phenacoccus solenopsis Tinsley (Hemiptera: Pseudococcidae) is a major cotton pest in several countries (Nagrare et al., 2016). This insect sucks cotton sap, injects toxins and secretes "honeydew" stimulating the development of black sooty moulds which adversely affect photosynthesis .
New insecticide formulations have been developed with effective physicochemical properties against P. solenopsis including natural products with potential to substitute conventional chemical insecticides (Silva and Ramalho, 2013;Silva and Silva, 2015). These products need to be biodegradable and selective, characteristics largely found in plant extracts or in minerals.
The main basic action of kaolin particle film against pest insects is due to its interference during the host plant location and acceptance process by the insect. Indeed, the white particle films of kaolin on plants alters the visual and tactile recognization and smell inducing repellence from treated foliage and a consequent feeding and oviposition by insects (Glenn and Puterka, 2005). The kaolin may affect the moisture of the cotton leaf surface and, consequently, the feeding and oviposition behavior of P. solenopsis on its leaves. However, kaolin has specific action against each pest (Silva and Ramalho, 2013) and, therefore, understanding its impact on sucking insects can minimize failures when used against these organisms.
The objective of the present study was to assess kaolin effects on the survival, development and reproduction of P. solenopsis on cotton plants.

Site and insect
Experiments were conducted at Embrapa Algodão Entomology Laboratory, Campina Grande, Paraíba State, Brazil in climatic chambers with a temperature of 25 ± 1 °C, relative humidity of 60 ± 10% and photophase of 12 hours.
The P. solenopsis specimens were collected from an experimental cotton crop of the Embrapa Algodão.

Biology and life table
One hundred adult P. solenopsis were transferred from the stock-rearing unit to leaves of the cotton cultivar BRS 286 for oviposition and obtainment of first instar nymphs of this pest. The nymphs were collected forty-eight hours after transferring the females of this mealybug to the cotton leaves.
The experiment was developed in a completely randomized design with two treatments: immature or newly emerged adults of P. solenopsis sprayed with kaolin and fed with cotton leaf discs treated with kaolin suspension (with kaolin) (T1) and immature or newly emerged adults of P. solenopsis sprayed with distilled water and fed with cotton leaf discs treated with distilled water (without kaolin) (T2). Fifty replications were used. A manual pre-compressed sprayer (Guarany Indústria e Comércio Ltda, Itu, SP, Brazil) with a 1.2 L volume capacity and empty cone nozzle was used to spray the newly emerged immature and adult P. solenopsis. The kaolin particle film was applied at a concentration of 60g of kaolin/L of water based on previous studies with sap-sucking cotton pests (Showler and Armstrong, 2007).
Fifty-five P. solenopsis newly emerged first instar nymphs were individually maintained per rearing unit of PVC tubes (2.5 cm x 2.0 cm) sealed at the base. A moistened filter paper was inserted inside the tubes and a cotton leaf disc (2.4 cm diameter) collected from the cotton leaf ventral side. The rearing units were covered with a transparent plastic film and maintained in climatic chambers until the conclusion of observations.
Biological observations were performed daily at 8:00 A.M. and 04:00 P.M. with stereoscopic microscope. The development, duration and survival of each instar and of the nymph stage, besides the pre-oviposition, oviposition and post-oviposition periods, longevity, fecundity and number of eggs/day and of eggs/female/day of P. solenopsis were evaluated. The adults of this insect were sexed based on sexual dimorphism and separated into couples with the newly emerged males and females individualized in pairs in new arenas. The fertility and sex ratio of the progeny on the cotton leaf discs, with and without kaolin, were determined. The number of P. solenopsis eggs in these arenas was quantified daily at 04:00 P.M.

Data analysis and statistics
Development data of P. solenopsis males and females were submitted to homoscedasticity tests and to the analysis of variance (ANOVA) and the means compared by LSD.
The P. solenopsis reproductive data were organized for life table calculations (Silveira et al., 1976). The reproductive rate net values (Ro= Σmx.lx -mx: total eggs/female number; lx: alive individuals/total), gross reproductive, also expressed in units of number of females per female (GRR= n x 0 mx = ∑ generation time (T= mx.lx.x/mx.lx Σ), intrinsic rate (r m = log Ro/T.0.4343), and finite (λ= antilog r m ) rate of increase were calculated. The confidence intervals of the means were estimated with these parameters determined using the SAS computer program (SAS Institute, 2008) applying the Jackknife resampling technique (Maia et al., 2000).

Results
The survival of immature P. solenopsis had no interactions with the treatment duration (Table 1) but differed between its stages. The survival of P. solenopsis second and third instars and of the pupa stage was highest and that of the first instar lowest in both treatments ( Table 2).
The duration of the P. solenopsis immature stage showed no interaction with the treatments (Table 2), but it differed between treatments and immature stages. The duration of second and third instar and of the egg to adult period of females was longer with distilled water (without kaolin) and shorter with kaolin.
The duration of pre-oviposition and oviposition periods of P. solenopsis were, respectively, shorter and longer with kaolin, but that of the post-oviposition was similar between treatments (Table 3). The number of eggs per female and their fecundity were higher with kaolin, but that of eggs per day was similar between treatments.
The duration of one generation (DG) and the period for the P. solenopsis to double its population in numbers (DT) did not differ between treatments ( Table 4). The positive r m and λ values show that P. solenopsis populations tend to grow, exponentially, in both treatments. On the other hand, the gross (GRR) and net (Ro) reproductive rates and the intrinsic (r m ) and finite (λ) rate of increase differed with higher values with the kaolin suspension, indicating that the particle film increased this mealybug biotic potential. --*Treatments: spraying the immature and newly emerged adult P. solenopsis and immersing cotton leaf discs with kaolin suspension in 60 gL -1 distilled water or with distilled water (control); **Survival: data transformed into square root of x + 0.5. DF = degrees of freedom; F = Fisher test; P = probability.

Discussion
Kaolin did not affect P. solenopsis immature stage survival. However, the second and third instar and P. solenopsis egg to adult period for females on cotton leaf discs with kaolin was shorter than without this particle film. The higher nymph vulnerability of this instar to the kaolin suspensions and water is due to the thinner wax layer of their bodies compared to older individuals. Additionally, the removal of these nymphs from the female ovisac may decrease their survival. Wax on the coccids forms a physical or chemical barrier against parasitoids, predators and the external environment (Foldi, 1983;Zhang et al., 2012). The ovisac represents a physical barrier against predatory insects and its hydrophobic properties enable the mealybug females to keep their interior morphological structure dry .
The shorter duration of the second and third instar and of the egg to adult period of P. solenopsis females with kaolin can be explained by the absorption of moisture from kaolin on the cotton leaf disk surface due to its hygroscopic properties, associated with an ambient temperature of 25 °C. Water shortage, like any other stress, has the potential to change the plant suitability for insect development, colonization, oviposition, and offspring survival (Blaney and Simmonds, 1988;Ramaswamy, 1988;Torres and Ruberson, 2006). These modifications in plant quality may favor or not insect pest damage (White, 1969;Mattson Junior and Haack, 1987;Bostanian and Racette, 2008;Markó et al., 2008;Showler and Armstrong, 2007), especially by sapsucking species (White, 1969;Miles et al., 1982;Oliveira et al., 2014). Likewise, the combination between high temperatures with low moisture content may shorten the insect development. This has been demonstrated for the Cephalcia arvensis (Panzer, 1805) (Hymenoptera: Pamphiliidae) larval development (Battisti, 1993). However, the longer duration of the egg to adult period of males compared to females seems to be common for this mealybug genus (Vennila et al., 2010;Prasad et al., 2012;Tok et al., 2016).
The longer oviposition period and the higher fecundity and number of eggs per P. solenopsis female with kaolin indicate a positive influence of this mineral particle film on the reproduction of this mealybug. This is probably due to the moisture reduction in the cotton leaf disk surfaces from kaolin water absorption. Variability in environmental moisture can also affect insect egg morphology, embryo physiology and oviposition. Low moisture content may stimulate egg cooling by evaporation, thereby protecting them from short thermal shocks and maintaining their viability (Potter, 2010). Host quality, food, light intensity and relative humidity (Vennila et al., 2010;Asifa et al., 2012;Fand et al., 2014) in addition to the kaolin particle film may affect insect fecundity. The longer longevity of females compared to males is due to the fact that males of this mealybug family present sexual dimorphism and shorter life span because they do not ingest food due to their nonfunctional and fragile mouthparts (Miller, 1991;Williams, 1991). Female longevity was 38.50 ± 2.23 days compared to the 3.50 ± 0.33 days for males, similar to the 35.6 days and 3.2 days for virgin female and mated male P. solenopsis with cotton leaves, respectively (Prasad et al., 2012).
The intrinsic rate of population increase (r m ) links Ro and DG and demonstrates the biotic potential of the species (Price, 1997). The shorter time required for P. solenopsis to double its population (TD) as well as its higher crude (TBR) and net (Ro) reproductive rate with and without kaolin, demonstrates the favorable conditions from both treatments for this mealybug to increase its population.
The population growth of P. solenopsis sprayed with kaolin suspension, fed on cotton leaf discs treated with kaolin suspension, was higher. Kaolin apparently reduces the cotton foliar surface moisture and consequently, the duration of the life cycle while increasing mealybug reproduction. Kaolin also increased the population density of Aphis gossypii (Glover, 1877) (Homoptera: Aphididae) on cotton (Showler and Armstrong, 2007) (Markó et al., 2008;Bostanian and Racette, 2008). This demonstrates the need to evaluate the kaolin effects with different environmental conditions and cotton cultivars. Kaolin particles should be used with caution

Conclusion
The kaolin suspension shortens the life cycle, increases the reproductive potential and population growth of the cotton mealybug, P. solenopsis and, therefore, it should be used with caution on cotton plants in regions with a history of occurrence of this pest.