Assessing the life cycle of pests, Diaphania pulverulentalis (Hampson) and Maconellicoccus hirsutus Green, reared on transgenic mulberry

Abstract

The study aims at investigating the growth and development of two common pests in mulberry namely the leaf roller (Diaphania pulverulentalis) and mealy bug (Macconellicoccus hirsutus), reared on transgenic mulberry plants in comparison with the wild type plants V1, a ruling variety. Both the pests completed normal life cycle on all the four different transgenic plants (Ip::HVA1, Cp::HVA1, Cp::BCH1, Cp::osmotin and Ip::osmotin) expressing three different transgenes (HVA1, BCH1 and osmotin) in the presence of the marker gene NPTII. There was no significant difference in the incubation period of the eggs, growth of first to fifth instar larvae and total larval period of the leaf roller reared on transgenic and non-transgenic mulberry. The pre-pupal, pupal, adult stages and adult fecundity also did not differ. The variations in the duration of the different nymphal instars, and their total duration was not significant in the case of mealy bug, when reared on the transgenics. The adult longevity and total life span of female mealy bugs, and the pupal period and total life span of the male bugs were on par with those reared on the wild type plants. The study indicates that the life cycle of both the pests, which are common in a mulberry ecosystem, were not affected by feeding on any of the transgenic mulberry plants at any stage of their growth and development.

Introduction

Mulberry (Morus indica L.), is a non-food crop, with high commercial value in the sericulture industry, as the sole diet for the silkworm (Bombyx mori L.). Quality mulberry foliage production has a key role in the sustenance of the industry, while, abiotic stresses, especially limiting water resources pose serious threat for sustained and quality foliage production. Concerted efforts are being made to breed new mulberry varieties incorporating stress resilient traits using biotechnological interventions, which assume more relevance during the fast-changing climate scenario. Transgenic approach has already been shown to be promising to incorporate desirable stress adaptive traits into mulberry (Lal et al. 2008; Das et al. 2011; Checker et al. 2012; Saeed et al. 2015; Sajeevan et al. 2017), although the technology could not be exploited at the field level due to bio-safety regulations. For example, controlled laboratory studies using the transgenic mulberry plants expressing barley Hordeum vulgare abundant protein (HVA1), tobacco osmotin and β-carotene hydroxylase (BCH1) genes (Lal et al. 2008; Das et al. 2011; Checker et al. 2012; Saeed et al. 2015) showed improved tolerance to abiotic stresses. Osmotin gene expressed under constitutive as well as stress inducible promoter in mulberry conferred tolerance to multiple stresses, such as drought, salt and fungal pathogens, without undesirable effect on silkworm rearing and feeding (Das et al. 2011). Transgenic mulberry overexpressing HVA1 gene conferred tolerance to drought and salinity, without compromising silkworm growth, cocoon quality and yield (Lal et al. 2008). Overall, the overexpression of different transgenes along with the marker gene neomycin phosphotransferase II (NPTII), imparting resistance to kanamycin, has shown no significant impact on the silkworm growth and development (Lal et al. 2008; Das et al. 2011).

However, due to the bio-safety regulations and ecological concerns, we examined the impact of the transgene expression on the leaf roller (Diaphania pulverulentalis) and mealy bug (Maconellicoccus hirsutus), the known major pests of mulberry grown in tropical conditions (Geethabhai et al. 1997; Manjunath et al. 2006). The major aim of the study was to examine the effect of transgenes on the biology of these pests, if any. We monitored the impact of transgene on the life cycle of the insects under controlled laboratory conditions by feeding healthy leaves from transgenics plants. We report that the mulberry transgenic plants expressing barley HVA1, tobacco osmotin, BCH1 and NPTII genes do not have any effect on the biological cycle of these pests, when compared with the wild type plants V1.

Materials and methods

Plant materials

The study used transgenic mulberry (Morus indica cv. K2) plants expressing barely HVA1 (Lal et al. 2008; Checker et al. 2012), tobacco osmotin (Das et al. 2011) and β-carotene hydroxylase1 (BCH1) (Saeed et al. 2015) genes, generated at the Department of Plant Molecular Biology, University of Delhi South Campus, via Agrobacterium-mediated transformation. These lines were designated as Ip::HVA1 and Cp::HVA1 (HVA1 under inducible and constitutive promoter, respectively), Cp::BCH1 (BCH1 under constitutive promoter), and Ip::osmotin and Cp::osmotin (osmotin under inducible and constitutive promoter, respectively). These plants were transferred to the Department of Crop Physiology, University of Agricultural Sciences Bangalore, GKVK Campus, Bengaluru, following bio-safety guidelines. The transgenic lines were raised in transgenic containment facility in pots filled with garden soil. Healthy shoots were collected and used for the insect bioassays. Shoots from a popular variety V1 was used for comparative analysis.

Insect bioassays

We monitored the growth and development of commonly noticed pests of mulberry, i.e. leaf roller (Diaphania pulverulentalis) and mealy bugs (Maconellicoccus hirsutus) on feeding the transgenic mulberry plants. The biology of leaf roller was studied by adopting the methodology followed by Misra (1919). The pest was reared in glass test tubes, on terminal tips of mulberry shoots bearing 2–3 leaves. Ten test tubes were maintained for each transgenic mulberry. Eggs collected by rearing a pair of female and male moths (collected from mulberry garden) inside a cage were transferred to the shoot tips in the test tubes. The shoot tips were changed daily, until the formation of pre-pupa. Observations on egg duration, larval duration, total larval period, pre-pupal and pupal periods, adult longevity and fecundity were recorded.

Similarly, the biology of male and females of the mealybug was studied by adopting the methodology followed by Pradeep-Kumar (2013). Individual eggs collected from egg sacs of mealy bugs on mulberry plants at the Department of Sericulture, University of Agricultural Sciences Bangalore, GKVK Campus, Bengaluru, were placed on tender mulberry leaves in petri plates by using a fine camel hairbrush and a needle. Ten individuals were raised on each transgenic and non-transgenic mulberry and fresh mulberry leaves were provided every day. The hatching of these eggs was monitored, and the duration of incubation period, I, II and III nymphal instars, pupal period of male, adult longevity and total life span were observed. The data collected were analyzed by paired t test.

Results

Biology of leaf roller

The life cycle of leaf roller (D. pulverulentalis) was examined on feeding with different types of transgenic plants (Ip::HVA1, Cp::HVA1, Cp::BCH1, Cp::osmotin and Ip::osmotin) in comparison with widely accepted mulberry genotype V1. The pest could successfully complete its life cycle normally on all the transgenic mulberry lines as shown in Figs. 1 and 2. There was no significant difference in the incubation period of the eggs, growth of first to fifth instar larvae and total larval period between transgenic and wild type plants (Fig. 1). The larval duration from first to fifth instar was almost the same when the larva was reared on transgenic mulberry lines and V1 variety. Similarly, there was no significant difference in pre-pupal, pupal and adult stages between transgenic and wild type mulberry plants (Fig. 2). The fecundity of the adults varied from 98.86 to 130.65 in case of the transgenics while it ranged from 118.15 to 137.07 days, in case of the wild type plants.

Fig. 1.

Duration of egg and larval instars of the mulberry leaf roller reared on different transgenic and wild type mulberry plants. The biology of leaf roller was studied by rearing the worms in glass test tubes, on terminal tips of mulberry shoots bearing 2–3 leaves. For this, eggs were collected from a pair of female and male moths and transferred to the shoot tips and the shoot tips were changed daily, until the formation of pre-pupa. Observations on egg duration, larval duration and total larval period, were recorded analyzed by paired t test. Error bar represents standard deviation from ten replications (Ip, inducible promoter; Cp, constitutive promoter)

Fig. 2.

Pre-pupal, pupal and adult longevity of the mulberry leaf roller reared on different transgenic and wild type mulberry plants. The worms generated from a pair of female and male moths were reared on terminal tips of mulberry shoots bearing 2–3 leaves in glass test tubes. The shoot tips were changed daily, until the formation of pre-pupa. Observations on pre-pupal and pupal periods, and adult longevity was recorded and analyzed by paired t test. Error bar represents standard deviation from ten replications (Ip, inducible promoter; Cp, constitutive promoter)

Biology of mealy bug

The life cycle of mealy bug M. hirsutus on transgenic mulberry lines and V1 variety was studied for male and female bugs together up to III nymphal instar as the male and female could not be distinguished (Table 1). However, after III nymphal instar, the male bugs underwent pupal stage, while females moulted to become adults and started egg laying. The incubation period of mealy bug eggs ranged from 4.36 days in V1 variety to 5.59 days in Cp::BCH1 and Cp::osmotin lines. The I nymphal instar duration ranged from 4.27 days in Cp::HVA1 to 5.46 days in Cp::BCH1 and Ip::osmotin lines. The II nymphal instar duration ranged from 7.18 days in Cp::HVA1 lines to 9.31 days in Cp::BCH1 plants. The III nymphal instar duration ranged from 7.19 days in V1 variety to 9.38 days in Cp::BCH1 and Ip::osmotin lines. The total nymphal period ranged from 18.78 days in Cp::HVA1 to 24.15 days in Cp::BCH1 lines. The female adult longevity ranged from 20.05 days in V1 variety to 23.20 days in Ip::HVA1 lines. The total life span of female mealy bug ranged from 43.50 days in Cp::HVA1 lines to 52.04 days in Ip::HVA1. The male mealy bugs had a pupal period ranging from 4.02 days in Ip::osmotin and V1 variety to 4.07 days in Cp::osmotin lines. The adult longevity ranged from 2.01 days in Cp::HVA1 to 2.40 days in Ip::HVA1 lines. Thus, their total life span ranged from 29.27 days in Cp::HVA1 to 36.05 days in Cp::BCH1 lines.

Table 1.

Life cycle (days) of mealy bug as influenced by transgenes in mulberry

Life stages		Ip::HVA1	Cp::HVA1	Cp::BCH1	Cp::osmotin	Ip::osmotin	V1
Incubation period		5.44 ± 0.45	4.45 ± 0.38	5.59 ± 0.47	5.59 ± 0.47	4.64 ± 0.39	4.36 ± 0.33
I nymphal instar		5.31 ± 0.76	4.27 ± 0.58	5.46 ± 0.78	4.53 ± 0.65	5.46 ± 0.78	4.53 ± 0.65
II nymphal instar		8.96 ± 0.56	7.18 ± 0.39	9.31 ± 0.52	9.21 ± 0.58	7.64 ± 0.48	7.64 ± 0.48
III nymphal instar		9.13 ± 0.37	7.33 ± 0.44	9.38 ± 0.38	7.77 ± 0.32	9.38 ± 0.38	7.19 ± 0.28
Total nymphal period		23.40 ± 1.69	18.78 ± 1.41	24.15 ± 1.68	21.51 ± 1.55	22.48 ± 1.64	19.36 ± 1.41
Pupal stage (male)		4.06 ± 0.15	4.03 ± 0.37	4.03 ± 0.15	4.07 ± 0.35	4.02 ± 0.45	4.02 ± 0.29
Adult longevity	Female	23.20 ± 0.65	20.27 ± 0.71	22.17 ± 0.62	20.53 ± 0.92	20.21 ± 0.65	20.05 ± 0.73
	Male	2.40 ± 0.11	2.01 ± 0.11	2.28 ± 0.11	2.29 ± 0.10	2.04 ± 0.09	2.08 ± 0.12
Total life span	Female	52.04 ± 2.79	43.50 ± 2.50	51.91 ± 2.77	47.63 ± 3.26	47.33 ± 2.68	43.77 ± 2.47
	Male	35.30 ± 2.40	29.27 ± 2.27	36.05 ± 2.41	33.46 ± 2.47	33.18 ± 2.57	29.82 ± 2.15

Individual eggs collected from the eggs sacs of mealy bugs were placed on tender mulberry leaves in petri plates using a fine camel hairbrush and a needle. Ten individuals were raised in each and fresh leaves were provided daily. Hatching of the eggs was monitored and observations on the duration of incubation period, I, II and III nymphal instars, pupal period of male, adult longevity and total life span were observed. Experiment was performed with ten replications and analysed by paired t test (Ip, inducible promoter; Cp, constitutive promoter)

Discussion

Genetically modified or transgenic crop plants have been commercialized worldwide, after following prescribed safety procedures. New traits are being inserted in to different plants types, both annuals and perennials for targeted manipulations of specific traits, using transgenic approaches. In this study, we used different transgenic mulberry plants expressing barley HVA1, tobacco osmotin and β-carotene hydroxylase (BCH1) genes, respectively, to assess the effect of the transgene expression on two major pests of mulberry. We studied the full life cycle of leaf roller (leaf webber) and male and female mealy bug, with the observations on egg duration, larval duration, total larval period, pre-pupal and pupal periods, adult longevity and fecundity.

Under field conditions, the total larval duration of leaf roller lasts for 13–19 days (Gowda et al. 2005; Siddegowda et al. 1995), depending on the season. However, in the present study, only marginal reduction in the larval duration for both transgenic as well as wild type plants was noticed. In case of leaf roller, the total pupal period and adult longevity have been recorded to be 9–10 days and 7–14 days, respectively, in different seasons (Geethabhai et al. 1997; Gowda et al. 2005). In the present study, the total pupal period and adult longevity were found to be within the pupal durations as reported above. The fecundity was also within the expected range as reported by Rajadurai et al (1999).

The mealy bug was able to complete its life span on transgenic mulberry lines as it did on non-transgenic plants. As per previous reports, the total life span of males ranges from 29–32.8 days while that of females ranges from 42.5–45.9 days, during different seasons (Katke and Balikai 2009). In another study, Sahito et al. (2012) recorded 32.2 days and 47.7 days of life cycle for male and female mealy bugs, respectively. The results from our study were within the range as reported earlier indicating that transgene expression did not change the life cycle of mealy bug.

Conclusion

Overall, our study indicated that leaf roller and mealy bug were able to complete their life cycle on transgenic mulberry lines, as could have been on non-transgenic mulberry. The transgenic mulberry lines expressing transgenes under inducible or constitutive promoter did not affect the growth and development of pests. These data suggest that transgenic plants are suitable for further evaluation such as confined/open field trials which are essential for the commercial release of transgenic plants, as per the bio-safety guidelines.

Author contributions

MSR, and MG equally contributed for the work. NKC, JKS and KNN planned the experiments; MSR and MG executed the work and partially analysed the data; PK provided the transgenic lines; DKH partially analyzed the data; MG, DKH, KNN framed the manuscript; NKC, DKH and KNN edited and finalized the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest in the publication.