Reproductive Mode and Male Mating Characteristic of Phenacoccus solenopsis (Hemiptera: Pseudococcidae)

Muhammad S. Waqas, Ali A.Z. Shoaib, Xinlai Cheng, Qianqian Zhang, Asem S.S. Elabasy and Zu-hua Shi*

Key Laboratory of Molecular Biology of Crop Pathogens and Insects, Ministry of Agriculture, Institute of Insect Sciences, Zhejiang University, 866 Yuhangtang Road, Hangzhou 310058, China

ABSTRACT

The cotton mealybug, Phenacoccus solenopsis Tinsley (Hemiptera: Pseudococcidae), is an invasive, polyphagous pest species. Reproductive biology of this mealybug species is poorly known, which hinders the development of an effective management program. In this study, the reproductive mode, male’s mating capacity, influence of female’s age and density on male’s mating capacity, as well as the influence of copulation on female’s longevity were investigated under the laboratory condition. Our results demonstrated that P. solenopsis reproduce sexually. Males mated 1-2 times in one day and 3-6 times in their lifetime, and did not show mating preference for female age or density. Although a few unmated females produced ovisacs, while they neither produced eggs nor gave birth any crawlers in the ovisacs. Unmated females lived for longer durations than the mated ones. Sexual reproduction with short lived males may imply potential for control practices that target males; for example, application of sex pheromone for trapping and killing male, or spraying of chemicals during the time of male activity, may be an effective practice for the management of this pest.

Article Information

Received 03 April 2018

Revised 24 July 2018

Accepted 12 September 2018

Available online 11 January 2019

Authors’ Contribution

MSW designed the experiments and carried out most of the experimental work under the supervision of ZHS. AAZS, XC, QZ and ASSE carried out some experiments. MSW wrote the manuscript.

Key words

Pseudococcidae, Phenacoccus solenopsis, Biology, Mealybug, Mating capacity, Reproductive mode.

DOI: http://dx.doi.org/10.17582/journal.pjz/2019.51.1.325.331

* Corresponding author: zhshi@zju.edu.cn

0030-9923/2019/0001-0325 $ 9.00/0

Copyright 2019 Zoological Society of Pakistan

Introduction

The cotton mealybug, Phenacoccus solenopsis Tinsley (Hemiptera: Pseudococcidae), a native of North America (Williams and Granara de Willink, 1992), is distributed over a wide range of agro-ecological zones in at least 24 countries (Wang et al., 2009). It has been recorded on more than 200 plant species belonging to over 55 families (Fand and Suroshe, 2015) as a polyphagous pest of many plants, causing huge economic losses to cotton and vegetables in the tropical and subtropical areas of the world (Dhawan et al., 2007; Jhala et al., 2008; Nagrare et al., 2009; Abbas et al., 2010; Arif et al., 2012). Estimated loss in seed cotton yield due to P. solenopsis was 40-50% (Nagrare et al., 2009). In China, it is distributed in 28 provinces (Wang et al., 2009).

Many studies relevant to the behavior, biology and ecology of P. solenopsis have been carried out in different parts of the world (Akintola and Ande, 2008; Dhawan et al., 2009; Fand et al., 2010; Nikam et al., 2010; Vennila et al., 2010; Tanwar et al., 2011; Ali et al., 2012; Asifa et al., 2012; Prasad et al., 2012; Suroshe et al., 2013). Phenacoccus solenopsis is reported to exhibit an ovoviviparous mode of reproduction with a very short egg incubation period ranging from a few minutes to a maximum of 2 h (Nikam et al., 2010; Vennila et al., 2010; Asifa et al., 2012; Prasad et al., 2012).

Phenacoccus solenopsis exhibits dimorphism, and sexes have distinct morphological differences. Male and female nymphs can be distinguished from their 3rd instar onwards (Nikam et al., 2010). Females have three nymphal instars before becoming adults. On the other hand, the immatures that are destined to develop as males construct a loosely-woven silky filamentous cocoon after the second moult and undergo two moultings inside the cocoon with prepupal and pupal stages, before emerging as winged adults (Dhawan et al., 2007; Vennila et al., 2010; Prasad et al., 2012; Fand et al., 2014). The adult males are short-lived and non-feeding (Silva et al., 2009), while the females are relatively long-lived depending on their reproduction status and temperatures (Vennila et al., 2010; Zhu et al., 2011; Asifa et al., 2012). Like a other mealybugs (Grasswitz and James, 2008; Cid et al., 2010), both males and females of the cotton mealybug have very limited range of movement.

Some scientists reported that this mealybugs was bisexual reproduction (Hodgson et al., 2008; Aheer et al., 2009; Prasad et al., 2012; Huang et al., 2013) whereas, some other scientists reported that parthenogenesis with ovoviviparity was dominant over the oviparous mode of reproduction (Nikam et al., 2010; Kumar et al., 2010; Vennila et al., 2010). These controversial reports definitively affect the selection of management strategy of the pest. If reproductive mode is truly bisexual, any mating absence and copulation delay may lead to destruction of the population since males have very limited range of movement. The obligate requirement of males for mating and production of offspring implies that a pheromone-based monitoring and management strategy for P. solenopsis would be the most practical. Therefore, understanding the reproductive attributes of the pest and the role played by males may generate ideas for amelioration of the pest management and for using this pest as a host in the multiplication of its natural enemies.

We carried out four experiments, the first of which was designed to confirm the reproductive mode, then, to investigate the copulating capacity of male adult together with to evaluate influence of the female age and density on male copulating capacity, and finally to investigate the influence of copulation on female longevity.

 

Materials and Methods

Mealybug and host plant

Tomato plants (Solanum lycopersicum) (Solanaceae) (Variety: Hezuo 903) were grown singularly with the field soil in 13-cm plastic pots in the greenhouse at 27 ± 1° C, 60–70% RH, and a 12:12 L:D photoperiod. All plants with 5–12 fully expanded true leaves were used in maintaining the colony of mealybugs in a climate room set at 27 ± 1° C, 60–70% RH, and a 12:12 L:D photoperiod. The detached tomato leaves from these plants were used in the experiments.

The male and female nymph of P. solenopsis were collected from the colony with Chinese calligraphy brush pen when they moulted from 2nd instar nymphs into 3rd instar, because at that time male nymphs have silky filamentous cocoons and the female nymph did not have. The isolated male and female nymphs were reared in clear plastic boxes (13×8×5 cm) with detached tomato leaves provided by petiole wrapped in water soaked cotton swab to prevent leaves from desiccation. There were less than 30 female nymphs on one leaf in one box. The box had holes covered with fine stainless steel mesh-covered holes on its top and walls for ventilation. The leaf was replaced as it looked not fresh with a new fresh one. Cotton swab was wetted with tap water as needed.

Eclosed male adults were collected every day, and reared with collecting date in a clear plastic box. Newly eclosed female adults (virgin females) were collected every day on the basis of exuviae after the complete moulting of 3rd instar into adult and reared on a detached tomato leaf in a clear plastic box labeled the collecting date. The collecting date was designated as the beginning day of their longevity.

Female’s reproduction mode

Thirty isolated virgin adult females were maintained in a box on a detached tomato leaf at 30 ± 1° C, 60–70% RH, and a 12:12 L:D photoperiod and observed daily for ovisacs until death. Number of dead females was recorded daily. Any eggs in the ovisac produced by the female was moved into a clear plastic box using detached tomato leaf as host, and reared until crawlers developed into adult. Three replicates of 30 females each were carried out for this experiment.

Male’s copulating capacity and influence of female’s density

One male was confined with 5, 10 or 15 virgin females (3 days old) in a clear plastic box (13×8×5 cm) with a tomato leaf as described above for 1 day or for male’s whole life (2.3±.5 days). subsequently, the confined females were individually reared using detached tomato leaves as host until they produced offspring or died. The number of females which produced ovisac with eggs was recorded. This experiment was repeated twenty times (males) for each female density category, and carried out at 27 ± 1° C, 60–70% RH, and a 12:12 L: D photoperiod (the same condition were used in the following experiments).

Male’s copulating capacity and influence of female age

One male was confined with ten virgin females of three different ages (8, 16, and 25 days old) in a clear plastic box with a tomato leaf until the male’s death. Then, the confined females were reared individually until they produced offspring or died. The number of females which produced ovisac with eggs was recorded. This experiment was repeated ten times (males) for each female’s age.

Influence of copulation on female’s longevity

Thirty mated females (Produced ovisacs with eggs) and thirty unmated (ovisac absent) females from the two male’s copulating capacity experiments above were reared individually until their death. Death date was recorded to calculate female’s longevity (from eclosion to death).

Statistical analysis

One-way analysis of variance (ANOVA) was carried out by using general linear model procedure PROC GLM (SAS Institute, 2009) to assess the male’s copulating capacity, influences of female’s density and age on the male’s copulating capacity, while the longevity between mated female and unmated female was compared by using independent t-test.

 

Results

Reproductive mode

Virgin females did not produce eggs. Some deaths of these virgin females were observed on day 27, but no female survived until day 34. The average female’s longevity (from eclosion to death) was 30.5±1.8 days. However, we observed empty ovisacs in the 7 out of 90 unmated female abdomen’s posterior ends.

Male’s copulating capacity and influence of female’s density

One male could mate with one to two females in 1 day, and mated with three to six females in the male’s lifespan (Table I). No difference has been found in the numbers of mated females among the three female’s densities confined either for 1 day or during the male’s lifespan (F=0.26; df=2, 57; P= 0.77 for 1 day, F=1.84, df=2, 57, P=0.17 for male’s whole lifespan).

 

Table I.- Numbers of females (mean±SD) which produced ovisacs at different densities after confinement with one male.

Female’s density	Confined time
	1 day	Whole male’s life
5	1.70±0.47 a	3.80±0.41a
10	1.75±0.44a	3.95±0.39a
15	1.80±0.41a	4.10±0.64a

Means (calculated from 20 repetitions) within a column followed by the same letters are not significantly different at 0.05 levels (Fisher LSD test).

 

Table II.- Numbers of females (mean±SD) that produced ovisacs with eggs at different ages after confinement with one male for the male’s lifespan.

Female’s age (days)	No. of copulated female
8	4.00±0.47a
16	3.80±0.42a
25	3.90±0.56a

Means (calculated from 10 repetitions) within a column followed by the same letters are not significantly different at 0.05 levels (Fisher LSD test).

 

Male’s copulating capacity and influence of female’ age

The numbers of females which produced ovisac are shown in Table II. One male could mate with three to six females during its whole life. No effects were observed for the females age wether it was young or old. No significance difference (F=0.64; df=2, 27; P= 0.53) was found at the number of mated females among the three female age categories.

Influence of copulation on female’s longevity

Figure 1 depicted that copulation had a negative impact on the survival of P. solenopsis Tinsley. Copulation significantly reduced the female longevity (N = 100, t-test, df=98, t=35.3, P <0.001) (Fig. 1). The mated females survived 19.3±1.3 days while unmated ones survived 30.5±1.8 days.

 

Discussion

Mode of reproduction

In the present study, only mated female P. solenopsis were able to produce offspring while unmated adult females were unable to do so. However, empty ovisacs were observed in the 7 out of 90 unmated female abdomen’s posterior ends. Our results unequivocally confirmed previous reports of sexual reproduction (Hodgson et al., 2008; Aheer et al., 2009; Prasad et al., 2012; Huang et al., 2013) but were different from earlier descriptions of obligate parthenogenesis (Kumar et al., 2010; Nikam et al., 2010; Vennila et al., 2010). Although obligate and facultative parthenogenesis has been reported in scale insects, most of them reproduce sexually (Normark, 2003). The congeneric of Phenacoccus madeirensis Green also has a sexually reproductive mode (Chong et al., 2003). Three mealybug species, Pseudococcus viburni (Signoret), Pseudococcus calceolariae (Maskell) and Planococcus citri (Risso), which were previously controversial on parthenogenesis traits were demonstrated to be obligate amphimictic (Silva et al., 2010). The controversial of reproductive mode in cotton mealybug maybe resulted from unintentional incorporations of males which were staying concealed during much of the daytime (Huang et al., 2013), or perhaps it is due to environmental variations such as host plant, and biological variations like female mating status (Nur, 1971). For example, Ferrisia virgata (Cockerell) showed a parthenogenetic mode of reproduction on the cocoa plant (Theobroma cocao) (Padi, 1997), while on the cotton plants (Gossypium hirsutum) it reproduced sexually (Oliveira et al., 2014). Thus, the differentiation in the reproductive mode of P. solenopsis between China and India may be caused by some factors which have not been taken into account so far (Huang et al., 2013). Probably they are different biotypes, cryptic species, or originated from different host plants. To make sure this, enormous information was needed to interpret the difference on reproductive modes reported by scientists. Silva et al. (2010) also found that more than 50% of virgin females of P. viburni and P. calceolariae formed ovisacs but only less than 16% of them laid some (1 to 8) infertile eggs. A similar phenomenon was reported also by Waterworth et al. (2011) on P. viburni. Silva et al. (2010) suggested that oviposition and the secretion of the ovisac were independent processes.

Effect of female density and age on male’s mating capacity

Female density did not affect male’s mating capacity. This result is not consistent with the citrophilus mealybug, P. calceolariae and the citrus mealybug, P. citri (Silva et al., 2013). The inconsistency in results is possibly due to the different species and methodologies used. In the present study, each male was exposed to female density at 5, 10 or 15 individuals, which already exceeded the mating capacity since males of the cotton mealybug mate only about 4 times on average with a range of 3-6 times (Table I). Whereas in Silva et al. (2013), each male was exposed to female density at 1, 2, 4, 8, or 16 individuals, the first three densities were apparently lower than the mating capacity since in 1 h of exposure to females each male of P. calceolariae could mate 3.7 times on average with a maximum of 8 times, and each male of P. citri could mate 1.6 times on average with a maximum of 4 times.

Female age did not affect male’s mating potential. This result supported the finding on P. calceolariae, but did not support their report on P. citri, where mating performance decreased with increased female age (Silva et al., 2013). They suggested that pheromone emission was inversely proportional to the age of females, with an eventual cessation of pheromone emission once females pass a certain age, and that this decrease resulted in a decreased rate of copulation with the males. However, since the virgin female mealybugs usually continue growing during their life, the larger body size may tradeoff the reduced pheromone emission with age by means of visual attraction (Franco et al., 2009). Meanwhile, the test time may be another reason for our difference. In Silva’s study, males were paired with females only for 1 h (Silva et al., 2013), whereas in our study the male was exposed to females for the male’s entire life. During shorter mating periods, males may not meet the female as frequently as compared to longer mating periods where the male may meet the female repeatedly, leading to increased opportunity for copulation.

Effect of copulation on the female longevity

Unmated P. solenopsis females live longer than mated females in our experiment (Fig. 1). This result is consistent with earlier report by Prasad et al. (2012), where the longevity of virgin female lasted about a month and that of mated females survived only about 20 days. Unmated striped mealybug Ferrisia virgata live 19 days more than mated females (Oliveira et al., 2014). This phenomenon has also been found in Callosobruchus chinensis (Linn.) (Yanagi and Miyatake, 2003) and Photinus obscurellus Legonte (South and Lewis, 2012) (Coleoptera), Ephestia kuehniella Zeller (Xu, 2010) and Ostrinia nubilalis (Hübner) (Fadamiro and Baker, 1999) (Lepidoptera), Gryllus bimaculatus De Geer (Green and Tregenza, 2009) (Orthoptera) and other many insects.

 

Conclusions

Male adults of P. solenopsis start to find their sexual partner immediately after they emerge from their cocoons, since they have a very short adult lifespan. They mate 3-6 times in their lifetime, can mate with young and old females, without displaying preference for females of any age range. For this species, only mated females are able to produce offspring, whereas unmated females are unable to do so despite living for much longer than mated females and often producing empty ovisacs. These results suggest that developing male targeted killing techniques, for example, applying the synthetic pheromones and Citrullus colocynthis extracts (Gulzar et al., 2017; Tabata and Ichiki, 2016) or spraying insecticides at the peak times of male activity, may be an effective practice for the management of this pest.

 

Acknowledgements

We acknowledge the National Basic Research Program of China (973 Program) (No. 2009CB119005 and 2006CB102005), the National 948 Program (No. 2011-G4), the National Department Benefit Research Foundation (NYHYZX20110321) for their generous financial support to this research, Mr. Siddiqui M. Abid in Medical College, Zhejiang University for his help during writing this paper.

 

Statement of conflicts of interest

No potential conflict of interest was reported by the authors.

 

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