Studies on the Development of Habrobracon Hebetor

Studies on the Development of Habrobracon hebetor on Diapausing and Non-diapausing Larvae of Plodia interpunctella

Abstract

Laboratory study was conducted to evaluate the development of Habrobracon hebetor(Say) (Hymenoptera: Braconidae) on diapausing and non-diapausing larvae of Plodia interpunctella (Hübner) (Lepidoptera: Pyralidae) at 28 °C, 16: 8 (L: D) photoperiod and 70% RH. The first experiment involved introducing three pairs of the wasps to different densities (1 to 30) of P. interpunctella larvae.Nearly100% mortality was obtained at all larval densities of diapausing larvae but in non-diapausing larvae the mortality consistently decreased with increase in host density. Similarly, more progeny of H. hebetor was produced on diapausing larvae compared to non-diapausing larvae (F_{5, 24} = 8.87, P < 0.0001). The numbers of eggs laid by a mated female wasp provisioned daily with fresh diapausing and non-diapausing larvae up to fifth day were 15.00 ± 2.33 and 13.33 ± 1.82, respectively. The maximum number of eggs yielded by H. hebetor provisioned daily with diapausing were on the 3^rd day whereas with non-diapausing on 5^th day. The results from this study show that diapausing larvae of P. interpunctella will be ideal for mass rearing of H. hebetor for the management of postharvest moth populations.

Keywords

Diapausing; Habrobracon hebetor; Host density; Mortality; Plodia interpunctella

Highlights

• P. interpunctella is among the most destructive pest of stored products.
• Infestation of food by P. interpunctella is caused by the larvae.
• Habrobracon hebetor proved efficient potential biological agent in the suppression of P. interpunctella.

1. Introduction

Hymenopteran parasitoids attack majority of lepidopteran and coleopteran stored product pests. Most of the parasitoids prefer 4^th and 5^th instar of lepidopteran pests (Alam et al., 2016; Amante et al., 2017) including Ephestia kuehneilla (Ali and Shishehbor, 2013; Darwish et al., 2003), Galleria mellonella (Alam et al., 2016), Plodia interpunctella (Akinkurolere et al., 2009), Corcyra cephalonica (Shao et al., 2008), Anagasta kuehneilla (Eliopoulos and Stathas, 2008; Mahdavi et al., 2013) Cadra cautella (Fukushima et al., 1998) and some species of Coleoptera like Acanthoscelides obtectus (Schmale et al., 2001), Sitophilus zeamais (Murata et al., 2016), Sitophilus granaries (Steidle and Scholler, 2001), Callosobruchus maculatus(Boateng and Kusi, 2008; Mbata et al. 2005a);  Helicoverpa armigera (Dastjerdi et al., 2008), Tuta absoluta (Doğanlar and Yiğit, 2011) and Cydia pomonella (Astanov, 1980). Parasitoids do not only paralyze and lay their eggs on host larvae but host larvae provide nourishment for developmental stages of parasitoids.

Dinarmus basalis, Anisopteromalus calandrae, Heterospilus posopidi (Ketoh et al., 2002; Schmale et al., 2001; Steidle and Scholler, 2001), Trichogramma turkestanica (Hansen and Jensen, 2002), Trichogramma pretiosum (Brower and Press, 1990), Cephalonomia hyalinipennis (Howard and Pérez‐Lachaud, 2002) and Habrobracon hebetor (Borzoui et al., 2016; Eliopoulos and Stathas, 2008; Garba et al., 2016; Ghimire and Phillips, 2014; Mbata and Shaopiro-IIan, 2010; Suma et al., 2014; Trematerra et al., 2016) are being considered as a potential biological control agents in stored product structures. Parasitoids are among the most promising and alternative to chemical pesticides for the suppression of the most destructive pantry pests (Liu et al., 2015; Mbata and Shaopiro-IIan, 2005; Mohandas et al., 2007).

Habrobracon hebetor (Say) (Hymenoptera: Braconidae) is a cosmopolitan, gregarious and koinobiont ectoparasitoid of a wide range of stored product pests especially lepidopteran species (Akinkurolere et al., 2009; Glupov and Kryukova, 2016; Liu et al., 2015; Milonas, 2005). H. hebetor is considered as the most promising and effective biocontrol agent of P. interpunctella (Shojaei et al., 2006). Infestation of stored products by P. interpunctella (Hübner) (Indianmeal moth) (Lepidoptera: Pyralidae) and indeed other postharvest moths is a prime concern and these moths among the cosmopolitan stored product pests infesting several types of processed and raw food materials including peanuts, legumes, dried fruits, seeds, spices, dried vegetables and packaged food materials  (Mbata 1985b; 1986). The larvae of Indianmeal moth enter diapause in response to low temperature and shorter photoperiod (Bell, 1997; Bell et al., 1979; Mbata 1990; Mohandas et al., 2007). Diapausing larvae develop very slowly (Na and Ryoo, 2000) and can survive for longer periods than non-diapausing larvae (William, 1964).

The management of Indianmeal moth inretail houses, bakeries, mills, food industries and other stored structures has mostly depended on the application of conventional chemical pesticides (Alam et al., 2015; Tuncbilek et al., 2011). Several chemical pesticide labels have either been eliminated or under review because they leave harmful residues in food or in the environment, or insects have developed resistance to them (Phillips and Throne 2010).  The use of methyl bromide, one of the most effective IPM tools in the management of stored product pests has been banned or restricted in several countries under the United Nations Environmental Protection (UNEP) Montreal protocol because of its ozone depleting property (Ayvaz et al., 2008; Mbata et al., 2005a; Thomas, 1996). Phosphine is an alternative fumigant to methyl bromide but presents the risk of poisoning to workers and can cause the fire (Mbata et al., 2005b; Valizadegan et al., 2012). Moreover, it has been reported that diapausing larvae of P. interpunctella were resistant to the shorter exposure of phosphine (Bell 1997). Additionally, diapausing larvae are more tolerant to heat (Sardesai, 1972), low pressure (Mbata et al., 2012) and methyl bromide (Johnson et al., 2003) than non-diapausing larvae of Indianmeal moth. Furthermore, pesticides may not be effective against residual populations of Indianmeal moth that hide in the corners and crevices of the storage and processing facilities. Thus, the moths can easily build up populations when warehouses are restocked.

Recently, biological control strategies such as the use of predators, parasitoids and pathogens are in focus as integrated pest management (IPM) tools for the management of stored product pests over conventional chemical pesticides (Eliopoulos et al., 2002; Scholler et al., 1997). These biological control strategies are not harmful to non-target species and minimize the use of chemical pesticides and their impact on environment (Lacey et al., 2001).

H. hebetor is among the parasitoids that have been investigated for their potentials as IPM tools in the management of postharvest storage moths (Ahmed et al., 1985; Brower and Press, 1990; Cline, 1989; Cline et al., 1984, Cline and Press, 1990; LeCato et al., 1977; Press et al., 1974, 1982; Ullyet, 1945). Cline et al., (1984) and Cline and Press, (1990) observed a significant reduction in the level of almond moth infestation in small experimental packages of corn meal and raisins with introduction of H. hebetor. In laboratory tests, H. hebetor effected 97% reduction of almond moth residual population in food debris (Press et al., 1982).  H. hebetor has the potential to be integrated with other biological control agents, such as entomopathogenic nematodes, for the management of populations of stored product moths (Mbata and Shapiro-Ilan 2005, 2010).

H. hebetor has some good characteristics, which include short developmental period of about 11 days at optimal conditions, high fecundity of females as a female could lay up to 100 eggs, the ability of adults to survive for long period of starvation, and ability to remain active in certain winter conditions (Alam et al., 2015; Benson, 1973; Chen et al., 2011). Additionally, H. hebetor has been found to penetrate commodity bags and exert significant host mortality (Adarkwah et al., 2014; Grieshop et al., 2006). These excellent traits nothwithstanding, it is difficult to rear large numbers of H. hebetor for mass, inundative or augmentative release in IPM programs. The major challenge in mass rearing of H. hebetor derives from the fact that the parasitoid has a narrow window to envernomize and lay eggs on its hosts, which are late instars of pyralids that pupates within three days at optimum conditions (Akinkurolere et al., 2009). For natural enemies to become widely adopted in IPM programs, methods of rearing them in large populations have to be developed. So far, this has become limiting in the adoption of H. hebetor as an IPM tool (Coudron et al., 2007; Chen et al., 2011).

At present, the only method known for the reproduction of H. hebetor is to rear them on larvae of stored product moths (Alam et al., 2015; Borzoui et al., 2016; Ghimire and Phillips, 2010). This study investigated the effect of the physiological state of the larval P. interpunctella host, diapausing or non-diapausing, on progeny production by H. hebetor.

2. Materials and methods

2.1. Rearing of Insects: Plodia interpunctella and Habrobracon hebetor

Culture of P. interpunctella was maintained in a 500 mL glass jars on mixed artificial diet of corn meal, chick laying eggs, chick starter mash, glycerol and yeast at a volumetric ratio of 4: 2: 2: 1: 1 at 28 ± 1.5 °C temperature, 70 ± 5% relative humidity and 16 h photoperiod (Mbata and Shapiro-Ilan, 2010). Ten days old (3^rd instar) Plodia interpunctella larvae were allowed to enter diapause by transferring them from 28 ± 1.5 °C to 14 ± 1.5 °C (Mbata, 1987). Cultures of diapausing and non-diapausing larvae were subsequently maintained at 14 ± 5 °C and 28 ± 1.5 °C temperatures, respectively.  For this experiment, 20-days non-diapausing and 40-days diapausing larvae were used since both larval types were at the last instar stage within these time frames.

The culture of H. hebetor used in this study was collected in 2001 from a laboratory colonies at United States Department of Agriculture, Agricultural Research Service(USDA ARS) Center for Grain and Animal Health Research, Manhattan, KS 66502, and since had been maintained at the Department of Biology, Fort Valley State University. The parasitoid was reared on last instar of P. interpunctella in 1000 mL of rearing jars at 28 ± 1.5 °C, 16 h: 8h (L : D) and 70 ± 5% rh (Mbata and Shapiro-Ilan, 2010).

2.2. Effect of Host Density on Host Mortality and Parasitoid Progeny Production

 

In order to determine the effect of host density of diapausing and non-diapausing larvae on host mortality and parasitoid progeny production, six different densities (1, 3, 5, 10, 20, and 30) of P. interpunctella were exposed to 2-d old three pairs (males and females)  of  H. hebetor. The larvae and the parasitoids were placed in 1000 mL rearing jars, which served as experimental arenas. H. hebetor were allowed in the jars to mate, paralyze and lay eggs on larvae. The jars containing both P. interpunctella larvae and wasps were placed in a cooled incubator maintained at 28 ± 1.5 °C, 70 ± 5% rh and 16: 8 (L: D) until the emergence of parasitoid progeny and the completion of development of P. interpunctella that escaped parasitism. Following the completion of development of H. hebetor progeny that lasted about 18 d, the experimental jars were checked for non-paralyzed host larvae that completed development and progeny of the wasp. Jars were kept at freezing temperature for 48 h to kill all insects. The numbers of H. hebetor progeny, adult moths were recorded.   There were five replicates of each treatment and three trials were carried with three simultaneous generations of P. interpunctella and H. hebetor.

2.3. Effect of provisioning H. hebetor fresh diapausing and non-diapausing larvae daily on host larva mortality and on H. hebetor progeny production

The effect of provisioning H. hebetor with fresh diapausing and non-diapausing larvae of P. interpunctella daily on progeny production was investigated in 1000 mL rearing jars at 28 ± 1.5 °C, 16L: 8D and 70 ± 5% rh. Two sets of 5 jars were prepared with each jar containing a pair (one male and one female) of 2-D old H. hebetor that were provisioned with5 fresh diapausing or non-diapausing host larvae for 5 successive days by transferring the parasitoids to new jars containing fresh larvae, the old jars containing larvae were placed inside a growth chamber maintained at the conditions stated aboveuntil the emergence of F₁ parasitoids. Following the completion of development of the wasps and P. interpunctella larvae that escaped parasitism, the jars were placed at freezing temperature for 48 h and the F₁ wasps and adult moths were counted. Observations recorded were number of moths representing larvae that escaped parasitism and the progeny of parasitoid. The treated were replicated 6 times.

2.4. Statistical Analysis

Statistical Analytical Software (SAS) ver. 9.4 updated in 2014 was used to analyze the data. Data analysis involved one way Analysis of Variance (ANOVA) procedure followed by Proc GLM and Tukey’s Kramer HSD test at 5% significance level (P ≤ 0.05) for the effects of diapausing and non-diapausing larvae on H hebetor progeny production and host larvae mortality. Prior to analysis, all data in percentages were arcsine of square root transformed (Steel and Torrie, 1980).

3. Results

3.1. Effect of Host Density on Host Mortality and Parasitoid Progeny Production

Experimental data on the effect of host density on host mortality and parasitoid progeny production are summarized in Tables 1, and 2 .The mean numbers of paralyzed diapausing larvae of P. interpunctella were significantly higher than those of non-diapausing larvae (F_{5, 24}=43.08, P<0.0001). The highest percentage mortality for diapausing larvae of P. interpunctella was obtained from two to ten larval density (100%) followed by twenty (91%) and thirty (86.66%) but among all densities, the lowest percentage of mortality was achieved by one density of larva (80%). In the case of non-diapausing larvae, the highest percentage of paralyzed larvae was ten (100%) but the mortality decreased as the larval density increased and was lowest at larval density of of thirty (41.33%; Table.1). When the mortality of diapausing and non-diapausing larvae exposed to H. hebetor was compared no significant difference was observed at low larval densities up to five beyond mortality became significantly higher in diapausing larvae (Table 1).

The parasitoid progeny production by H. hebetor on diapausing larvae was significantly higher than the number from the progeny produced on non-diapausing larvae (Table 2; F_{5, 24} =8.87, P<0.0001). The number of progeny production by a female wasp at 1, 3, 5, 10, 20, and 30 host density of diapausing and non-diapausing larvae of P. interpunctella was 3, 7, 13, 22, 38, 53 and 1, 4, 10, 11, 14, 22 respectively (Fig. 1).

Table 1. Influence of host larval density on themortality of P. interpunctella larvae.

Larval density	% (Means ± S.E)
	Diapausing larvae	Non-diapausing larvae
1	80 ± 20 B	100 ± 0 A
3	100 ± 0 A	100 ± 0 A
5	100 ± 0 A	80 ± 20 B
10	100 ± 0 A	60 ± 17.8 C
20	91 ± 78.1A	50 ± 11.6 C
30	86.66 ± 11 B	41 ± 8.2 CD

*Means followed by different letters are significantly different (P ≤ 0.05) using Tukey’s Kramer HSD test.

Table 2. Effect of host larval density on the number of F₁ progeny of H. hebetor reared on diapausing and non-diapausing larvae of P. interpunctella.

Population of larvae	F1 progeny of H. hebetor on larvae
	Diapausing larvae	Non-diapausing larvae
1	9.20±2.2 A,c	3.80 ± 1.74 A,c
3	22.20±9.5 A,c	11.80 ± 3.46 A,c
5	38.60±10.2 A,c	29.60 ± 6.94 A,c
10	67.00±24.04 A,ab	33.60 ± 4.24 A,b
20	113.60±30.63 A,ab	42.40 ± 3.35 B,b
30	159.20±23.58 A,a	64.80 ± 6.85 B,a

* A, B, C means followed by different letters in each row are significantly different (Tukey’s Kramer HSD test, P ≤ 0.05).

* a, b, c means with different letters within same column are significantly different (Tukey’s Kramer HSD test, P ≤ 0.05).

Fig. 1. Effect of density of diapausing and non-diapausing larvae of P. interpunctella on progeny production by a mated female of H. hebetor.

3.2. Effect of Provisioning H. hebetor to Fresh Diapausing and Non-diapausing Larvae Daily on Egg Production of H. hebetor

The effect of provisioning fresh diapausing and non-diapausing larvae daily was assessed on the number of eggs laid by wasps. The number of eggs laid by H. hebetor was significantly higher on diapausing larvae (F_{4, 25}= 4.08; P= 0.0111) than non-diapausing larvae (F_{4, 25}= 0.54, P= 0.0707). The mean numbers of eggs deposited by female wasp provisioned daily with fresh non-diapausing larvae from first to fifth day increased from 8.00 ± 1.44 to 13.33 ± 1.82 but were not significantly different (Table 3). A significantly higher mean number of eggs, was laid on the 3^rd day by female wasp provisioned daily with diapausing larvae (23.33 ± 2.33) compared to those laid by wasp provisioned with non-diapausing larvae (13.33 ± 1.82, Table 3).

Table 3. Parasitoidprogeny/day (Mean ± S.E) of H. hebetor that were provisioned with fresh diapausing and non-diapausing host larvae daily for 5 days

Day	Parasitoid progeny production (Means ± S.E)
	Diapausing	Non-diapausing
1st day	13.16±1.81 B	8.00±1.44 A
2nd day	18.33±1.68 AB	7.66±0.35 A
3rd day	23.33±1.40 A	7.50±0.91 A
4th day	17.50±2.17 AB	11.66±2.64 A
5th Day	15.00±2.33 B	13.33±1.82 A

*Means followed by same letters are not significantly different at P ≤ 0.05 using Tukey’s Kramer HSD test.

4. Discussion

Parasitoid progeny production depends on various factors in which density and nutritional value of host are the two most important determinants (Aung et al., 2011; Hofstetter and Raffa 1998). In the current study, the number of progeny of H. hebetor increased as increased the number of host. This has also been reported in other related species of parasitoids such as Uscana lariophaga Steffan (Hymenoptera: Trichogrammatidae) (Alebeek and Huis, 1997), Spalangia cameroni, S. endius, Muscidifurax raptor, and Nasonia vitripennis (Hymenoptera: Pteromalidae) (Lenger, 1967), and Campoletis chlorideae Uchida (Hymenoptera: Ichneumonidae) (Kumar et al., 2000).  Effect of host density on the progeny production of H. hebetor was also studied by Ghimire and Phillips (2010) and it was noticed that the number of parasitoid progeny increased with host density up to 50 larvae per female wasp. Jamil et al., (2015) recorded, the maximum number of H. hebetor adult emergence on the highest larval density (16) of Galleria mellonella. Similarly, Yu et al., (2003) observed that H. hebetor laid maximum number of eggs when mated female was offered up to 16 host larvae but fecundity became reduced beyond larval density of 16 per parasitoid. In this study, the numbers of progeny produced by per wasp on diapausing larvae with increasing density (from 1 to 30) were more compared to non-diapausing larvae. The highest density, 30 diapausing larvae of P. interpunctella produced higher number of H. hebetor progeny (53 adults of parasitoid) whereas same density of non-diapausing larvae produced 22 progeny of parasitoids. The reason of higher number of progeny production on diapausing larvae might be higher nutritional value of diapausing larvae that help in the development of wasp (Ad et al., 1985; Arrese and Soulages, 2010; Hahn and Denlinger, 2007). Another factor might be greater body mass and larger size of diapausing larvae that provide more surface area to parasitoid for oviposition (Hahn and Denlinger, 2007; Mbata et al., 2012). Next factor might be the stored body fat of the diapausing larvae. Generally, diapausing larvae have more stored fat than non-diapausing larvae and fat serves as a precursor in the biosynthesis of volatile semiochemicals that attract and stimulate parasitoid for oviposition (Arrese and Soulages, 2010; Hahn and Denlinger, 2007; Mossadeqh, 1980). Therefore more amount of stored fat can increased the synthesis of semiochemicals that will enhance parasitization and oviposition. These factors might be involved in the higher number of progeny production on diapausing larvae.

Generally, as host density increased, the number of host paralyzed by parasitoids decreased up to a certain density of host with constant number of female parasitoid (Yu et al., 2003; Mbata et al., 2005a; Alebeek et al., 1996). This study also showed the numbers of paralyzed larvae were decreased as density of diapausing and non-diapausing larvae increased from 1 to 30.  Approximately 80% mortality is achieved in all densities of diapausing larvae of P. interpunctella whereas in non-diapausing larvae with only two larval densities, one and three. This result clearly showed that H. hebetor can easily paralyze diapausing larvae compared to non-diapausing larvae. Several factors might have caused the higher mortality in diapausing larvae of P. interpunctella. Firstly, diapausing larvae are less active therefore they cannot easily escape from parasitoid attack compared to non-diapausing larvae. Secondly, diapausing larvae take more time for pupation that enhance parasitism whereas non-diapausing larvae have narrow windows for pupation and pupate very fast thereby escaping parasitism (Bell, 1982).

On the other hand in second experiment, the number of progeny production was increased when daily fresh host larvae were supplied to parasitoid. The obtained data from the development experiment is similar to the previous study of Eliopoulos and Stathas (2008). Eliopoulos and Stathas (2008) observed the different parameters of life of H. hebetor on five different larval densities (1, 5, 10, 15 and 30) of Anagasta kuehneilla and P. interpunctella and reported that progeny production per wasp was increased when per day 15 host larvae were provided to parasitoid. In this study, the parasitoid showed more efficacies for producing progeny very early on 3^rd day by providing daily fresh diapausing larvae of P. interpunctella whereas in non-diapausing larvae on 5^th day. This might be due to the wasp deriving better nourishment from diapausing larvae which in turn has tended faster maturation of eggs in oocytes. It is concluded that progeny of parasitoid can be yielded markedly on diapausing larvae with one pair of parasitoid within few days and also these findings suggested that diapausing larvae of P. interpunctella could be a suitable alternative for the mass rearing of H. hebetor with higher yield.

Acknowledgements

Thanks to United States Department of Agriculture, Agricultural Research Service(USDA, ARS) Manhattan, Kansas for providing parasitoid. This research was supported by National Institute of Food and Agriculture (NIFA) and United States Department of Agriculture (USDA), Evans Allen program (GEOX-7701) grant.

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