Pathogenicity of Heterorhabditis indica against developmental stages of Eudocima materna L. (Lepidoptera, Erebidae)

Virulence of the entomopathogenic nematode, Heterorhabditis indica against larvae (3rd, 4th and 5th larval instars) and pupae of citrus fruit piercing moth, Eudocima materna L. (Lepidoptera: Erebidae) was evaluated under laboratory conditions. The experiments included pathogenicity assessment of H. indica against larvae and pupae using a range of concentrations (10, 25, 50, 100 and 200 infective juveniles (IJs) larva−1 and pupa−1) as well as evaluation of their reproductive potential on different larval instars with different concentrations (50, 100, 200, 400 and 600 IJs larva−1). Application of increasing concentrations of IJs induced higher mortality on larval instars and pupae with mean mortality ranging from 26.6 to 100% (LC50- 3rd (14.43 IJs larva−1), 4th (17.08 IJs larva−1), 5th (23.63 IJs larva−1)) and 10–70% (LC50- 85.91 IJs pupa−1) after 48 h post-exposure, respectively. H. indica successfully reproduced in the 3rd, 4th and 5th larval instars of E. materna and their offspring emerged from the cadavers. The highest reproduction was recorded in 5th instar larvae (1,082,855 IJs larva−1) at 600 IJs larva−1 in E. materna. The present study suggests that H. indica strain had the potential for biological control of E. materna.


Background
Fruit piercing moths, Eudocima spp. (Lepidoptera: Erebidae), are considered as serious pests of fruit crops grown throughout the tropical and sub-tropical belt from Africa, Asia, Australia and to the Pacific Islands (Leong and Kueh 2011). The adult moths infest ripened fruits of various horticultural crops by piercing with its barbed proboscis which becomes an entry point for bacterial and fungal infections, finally causing rotting and premature dropping of infected fruits (Kamala Jayanthi et al. 2015). Damaged fruits are unmarketable and pose a threat of further pathogenic breakdown to healthy fruits in the packed lots (Fay and Halfpapp 2006). In India, 2 species, Eudocima materna and E. fullonica cause significant economic damage, in mandarins, sweet oranges and kinnow, particularly during August-December, with their peak activity during September-October (Rao and George 2018).
Adult moth lays oval-shaped and shiny pale green collared eggs on wild plants and weed hosts grown in and around citrus orchards (Shivankar and Singh 2000). Larvae are semi-loopers with stout appearance, feed on alternate weed hosts for about 4-5 weeks, progressing through 5 instars and pupate within a silken cocoon between webbed leaves and the leaves may stay on the tree or become desiccated and fall to the ground. The emergence of adult from the pupa takes place after 2-3 weeks. There are 2-3 generations in a year Page 2 of 8 Kumar et al. Egyptian Journal of Biological Pest Control (2022) 32:65 depending on the availability of weed hosts (Chhagan and McKenna 2019). Insecticidal sprays, poison baits or the traps are traditional components of integrated citrus fruit piercing moth management strategies (Kamala Jayanthi et al. 2010). Since this pest species starts infesting citrus fruits at maturity/harvesting stage, application of chemical insecticides is not encouraged at this crop stage. Very few options are available for the management of this pest species even by using of poison baits or light traps, they are not much effective (Shivankar and Singh 2000). Thus, the use of entomopathogenic nematodes (EPNs) of the genera Steinernema and Heterorhabditis offers an ecofriendly alternative to chemicals and as biological control agents against different insect pests. EPNs have been reported to be pathogenic to larval and pupal stages of various groups of insect pests. Therefore, evaluation of the efficacy of different concentrations of Heterorhabditis indica against E. materna larvae (3rd, 4th and 5th instars) and pupae under laboratory conditions were aimed. Also, investigation of the reproductive potential of H. indica in E. materna larval instars was evaluated. E. materna complete its entire life cycle on wild plants and weed hosts which are generally grown on the bunds of the orchards and nearby localities; it is very imperative to carry out the field application of H. indica at these sites. In recent years, significant progress has been made in developing EPN formulations, especially for above ground applications such as combining EPNs with polymers or surfactants or sprayable gels (Shapiro-Ilan et al. 2012). The present work is the first investigation on using EPNs against fruit piercing moth larvae and pupae and provides some preliminary evidence for potential use of H. indica for biological control of E. materna.

Insect culture
The 1st and 2nd instar larvae of E. materna were initially collected from the weed host, T. cordifolia in and around the research farm (21° 08′ 44′′ N 79° 01′ 32′′ E) of ICAR-Central Citrus Research Institute, Nagpur, Maharashtra, India, during August, 2020. The larvae were reared on T. cordifolia leaves in a plastic tray covered with a muslin cloth at 25 ± 1 °C. The developed pupae were transferred to plastic containers (1000 ml) covered with a black muslin cloth. The newly emerged adults were transferred to a nylon cage (2 m × 2 m x 3 m size) at 25 ± 1 °C with 70% RH and a 12:12 h light/dark photoperiod; adults were fed with Nagpur mandarin fruits and a synthetic diet prepared by dipping of cotton balls in a honey solution (1(honey):1(water)) with vitamin-E capsules. The newly deposited eggs by adult female moths were collected with the help of a wet painting brush and carefully placed in plastic containers under lukewarm condition. Eggs were allowed to hatch and the newly emerged larvae were reared on T. cordifolia leaves for further development to various larval instars. Larvae were separated into 3rd, 4th and 5th instars based on their size, weight and head capsule width and subsequently used for efficacy and reproduction assays. For pupal bioassay, pupae were collected from the dried T. cordifolia leaves which were cultured in the lab. E. materna were successfully reared up to 5 generations (August 2020-January 2021) on weed host, T. cordifolia and larvae from second generation onwards were used for experimental purpose (Fig. 1). The average size (length (L), width (W)) of 3rd, 4th, 5th larval instars and pupae used were 21. 24, 2.19; 33.85, 3.97; 51.84, 8.41 and 27.74, 9.61 mm, respectively, whereas the average weights were 112.13, 444.12, 1420.03 and 1330.02 mg, respectively.

EPN culture
EPN species, Heterorhabditis indica, was obtained from the Biological Control Centre, ICAR-IISR, Pravaranagar, Maharashtra, India. Infective juveniles (IJs) of H. indica were continuously propagated on last instar larvae of rice moth, Corcyra cephalonica (Kaya and Stock 1997). IJs harvested on a white trap (White 1927) were stored in sterile distilled water in tissue culture flasks at 15 °C for about one week before use. Only IJs with survival rates greater than 90% were used for relevant experiments.

Pathogenicity of H. indica against larval instars of E. materna
In order to examine the susceptibility of larval stages (3rd, 4th and 5th) of E. materna, IJs at concentrations 10, 25, 50, 100 and 200 IJs larva −1 were suspended in 150 μl of water and distributed evenly on the insect breeding dish (50 × 15 mm) lined with sterile filter paper previously moistened with 350 μl of sterile distilled water for each treatment. In control treatments, only 500 μl of distilled water was added to the dishes. Larvae of E. materna were then individually placed in the dishes and pieces of fresh T. cordifolia leaves were provided as food; then, the dishes were sealed with parafilm and incubated at 25 ± 1 °C. Ten individuals of each larval instar were used for each concentration and the experiment was conducted thrice (n = 10; r = 3). Larval mortalities were recorded after 24 and 48 h of treatment, followed by dissection of cadavers under the stereomicroscope for confirmation of nematode infection (Aryal et al. 2022).

Pupal susceptibility in a plastic container assay
To study the infectivity of H. indica to pupae of E. materna, different concentrations of IJs (10, 25, 50, 100 and 200 IJs pupa -1 ) were suspended in 2.5 ml of distilled water and distributed evenly in the 100 ml plastic containers (height: 7 cm; diameter: 5.5 cm; soil capacity: 100 cm 3 ) containing the mixture of 90 cm 3 autoclaved sand and soil (1:1). Distilled water alone was added to the Control treatments. The single pupa was placed at 1 cm depth in each container. The containers were closed and incubated at 24 ± 2 °C and 60 ± 5% RH. Each treatment contained 10 pupae and the assay was repeated once. The adult eclosion rate was examined on a daily basis for 15 days. The non-emerged pupae were collected and washed in distilled water and dissected in sterile distilled water under a stereomicroscope for confirmation of nematode infection.

Reproduction potential of H. indica in larval instars
The larval instars (3rd, 4th and 5th) of E. materna infected with IJs of H. indica at different concentrations (50, 100, 200, 400 and 600 IJs larva −1 ) were selected for reproduction assay. A total of ten numbers of each larval instar for each IJ concentration were used in this assay. Five cadavers were selected randomly, rinsed with distilled water to remove nematode adhering to the body surface. The cadavers were individually transferred onto the white trap and incubated at 25 ± 1 °C. The IJs that emerged from each cadaver were collected fifteen days after treatment in centrifuge tubes separately and the concentration of IJs was determined by serial dilution method as described by Glazer and Lewis (2000).

Statistical analysis
To assess the number of IJs required for killing 50% lethal concentration (LC 50 ) and 90% (LC 90 ) of the various larval instars and pupae of E. materna after 48 h post-inoculation at 95% confidence intervals at P ≤ 0.05, larval and pupal mortality resulted from H. indica and their concentrations was subjected to probit analysis (Finney 1971) using online tool OPSTAT. The mean reproduction rate of H. indica in different larval instars of E. materna at different concentrations was compared by Duncan's test at P ≤ 0.05 using SPSS 21 software.

Pathogenicity of H. indica at different concentrations against E. materna larval instars
Pathogenicity assessment indicated that H. indica was capable of infecting and killing different larval instars (3rd, 4th and 5th) of E. materna (Fig. 2). The larval mortality was influenced by 3rd, 4th and 5th instars as nematode concentrations increased with mean mortality ranging from 26.6-100%, when they treated with 10 to 200 IJs larva −1 of the tested H. indica strain (Fig. 3). There were significant differences in mortality among different IJ concentrations on 3rd, 4th and 5th larval instars

Pupal susceptibility to H. indica
Laboratory assay showed that pupae of E. materna appeared susceptible to H. indica. Insect mortality recorded with 1-3 days old infected pupae revealed significant differences and greater mortality (> 60%) at concentrations of 100 and 200 IJs pupa −1 (Figs. 2, 4). Fully formed pupae were less susceptible to nematodes than 3rd, 4th and 5th instar larvae. > 90% insect mortality was recorded when nematodes were applied on 3rd, 4th and 5th instar larvae, while insect mortality induced on 1-3 days old pupae showed a maximum 70% mortality at higher concentrations (200 IJs pupa −1 ). The LC 50 and LC 90 of H. indica for pupae after 48 h of treatment were 85.91 and 617.54 IJs pupa −1 , respectively (Table 1).

Nematode reproductive potential on E. materna larval instars
The results indicate that H. indica successfully reproduced in the 3rd, 4th and 5th larval instars of E. materna and their offspring were emerged from the larval cadavers. Non-significant difference was detected among different concentrations of IJs inoculated to 4th instar larvae on the reproduction of H. indica (F = 1.16; df = 4; P < 0.356). However, significant difference was observed between 50 to 200 IJs and 400-600 IJs concentrations inoculated to 3rd instar larvae on the reproduction of H. indica (F = 38.07; df = 4; P < 0.001). Significant difference was also detected among the 5 IJ concentrations inoculated to 5th instar larvae on the reproduction of H. indica (F = 12.57; df = 4; P < 0.001). The reproduction of H. indica in 3rd instar larvae was significantly lower than that of 4th and 5th instar larvae. Although the reproductive potential of H. indica in the larvae of E. materna significantly increased as the rate of IJs increased, the number of IJs per 5th instar larvae at the  higher concentration (1,082,855; 600 IJs larva −1 ) was significantly higher than at the lower concentrations (Fig. 5).

Discussion
EPNs are successfully used in different parts of the world to control different insect pests. Susceptibility of EPNs to targeted insect pest can vary greatly among strains of the same species. Hence, an in vitro study on infectivity of EPNs is a prerequisite in a biocontrol program for a particular insect pest prior to field studies (Ricci et al. 1996). Based on the available literature scan and to the best of our knowledge, obtained data provide the first insight into the pathogenicity of EPNs against citrus fruit piercing moth larvae and pupae.
The present investigation showed that H. indica strain was highly virulent to E. materna larvae (3rd, 4th and 5th instars) and caused larval mortality under laboratory conditions. EPNs virulence was dependent on the larval stage of the host, E. materna. H. indica was highly virulent in 3rd instar larvae than the 4th and 5th larval instars. However, > 80% mortality was observed after 48 h of treatment in all the larval instars at 50 to 200 IJs concentrations. Several studies have shown that the larval stages of lepidopteran pests are susceptible to infection by different EPNs (Salari et al. 2021). Recently, Aryal et al. (2022) reported significant mortality of Queensland fruit fly (Bactrocera tryoni) larvae using H. indica strains (Hi.ECCH, Hi.HRN and Hi.HIE2) under in vitro conditions. Obtained results are consistent with the earlier studies and indicated that H. indica is highly virulent to larval stages of E. materna. Pupae of lepidopteran pests are also susceptible to EPN (Mhatre et al. 2020). Also, H. indica was highly lethal to E. materna pupae (70% mortality at 200 IJs pupa −1 ). In similar studies, Yan et al. (2020) also reported that H. indica was highly virulent to lepidopteran pest, Spodoptera litura pupae. Recently, Aryal et al. (2022) reported the significant effect of H. indica strains (Hi.ECCH, Hi.HRN and Hi.HIE2) against pupae of B. tryoni. In contrast, Kaya and Hara (1981) reported that some lepidopteran insect pest pupae were less susceptible to EPNs. Similarly, Batalla-Carrera et al. (2010) reported low mortality (10%) in pupal stages of the tomato leaf miner, Tuta absoluta, whereas Kary et al (2019) reported no mortality in pupal stages of diamond back moth, Plutella xylostella with EPNs. From the present study, it can be concluded that the pupae of E. materna were susceptible to H. indica.
The reproductive potential of EPNs is an important feature for their persistence and pathogenicity against targeted insect pests (Blanco-Pérez et al. 2017). It not only causes insect pest mortality, but it also impacts the ability of EPNs to tackle the subsequent generations of the targeted pests (Patil et al. 2019). However, the reproduction rate of EPNs is influenced by different life stages of the host insects (Park et al. 2001). Obtained results indicated that the reproduction rate of H. indica was significantly dependent on the larval stage of E. materna. Specifically, the reproduction rate of H. indica was higher in 5th instar larvae than in 3rd and 4th larval instars. This result corroborates with the previous study that showed the reproduction rate of H. indica was higher in the later instars of S. litura (Acharya et al. 2020) and H. bacteriophora in Heliothis virescens (Gulzar et al. 2020). Furthermore, laboratory trials with diverse EPN strains are necessary to evaluate EPNs performance against the target pest, allowing the best performing strains to be selected for further field testing.

Conclusions
Efficacy of H. indica on larvae and pupae of E. materna was demonstrated under laboratory conditions. Given the difficulties and limitations in management of citrus fruit piercing moth in Citrus and other crops, the EPNs could be efficiently use against this pest species within the framework of biological control initiatives. Since the efficacy of H. indica on larvae and pupae E. materna under laboratory conditions was tested, its efficacy and mode of applications should also be tested further under field conditions for subsequent incorporation of EPNs in biological control programs. The information generated in this study would certainly be useful for better designing of management strategies for the management of fruit piercing moth in citrus and other crops.