This work is licensed under the Creative Commons Attribution 4.0 International License.
The use of biological agents, such as entomopathogenic nematodes (EPN) of the genera Heterorhabditis and Steinernema, is one of a range of tools for biological control. These nematode species are obligate parasites, lethal, and easy to apply (San-Blas, 2013). They can be mass-produced using in vivo or in vitro (solid or liquid) culture methods (Gaugler and Han, 2002; Rahoo et al., 2019), but in vivo is the method of choice for laboratory-scale production (e.g. for generating material for field trials) according to Shapiro-Ilan et al. (2002). In vivo nematode production yields nematodes with good virulence potential (Shapiro-Ilan et al., 2000). Under field conditions, application of EPN in insect host cadavers can reduce the quantity of nematodes required for control per unit area compared with their application in water or other solvents (Shapiro-Ilan et al., 2012). The typical host used to mass-produced EPN is the greater wax moth Galleria mellonella L. (Lepidoptera: Pyralidae) (Testa and Shields, 2017).
The in vivo culturing process is costly (Divya and Sankar, 2009), and it is imperative to find alternative solutions to economically rear the insects. Few experimental diets have been entirely successful in replacing their natural diet (Cohen, 2004). In addition to an assessment of the cost, it is important to know whether the diet influences the effectiveness of EPN emerging from an infected diet-reared host to kill and multiply in the target pest species. According to Zhen et al. (2018), the quality of the insect host can affect the efficacy or persistence of EPN produced in vivo. Ramakuwela et al. (2014) indicated that insect nutrition has a link to EPN production.
Many studies have tried to optimize the mass-rearing of G. mellonella, taking into account the cost and availability of diet ingredients, as well as the ability of the insect to adapt to diets without seriously affecting its development (Coskun et al., 2006; Birah et al., 2008; Kulkarni et al., 2012). The proportion and/or selection of ingredients in the diets play an important role in the development of larvae, as well as in the fitness and quality of the nematodes obtained from them (Shapiro-Ilan et al., 2008). Shapiro-Ilan et al. (2004) also reported that in vivo production yields vary greatly among different insect hosts and nematode species. For example, to rear G. mellonella, Brighenti et al. (2005) used 250 g of corn meal, 150 g of yeast extract, 100 g of soy flour, 100 g of powder milk, 200 g of honey, 200 g of glycerol, and beeswax blocks. Van Zyl and Malan (2015) tested and compared several other diets based on cereals, beeswax, or chemical compounds. They found that diet 1 (composed of 118 g wheat flour, 206 g wheat bran, 118 g milk powder, 88 g yeast, 24 g wax powder, 175 ml honey, and 175 ml glycerol) produced the heaviest larvae (0.19 g/ larva); and diet 3 (190 ml glycerol, 190 g yeast, 570 g wheat bran) produced the lightest larvae (0.08 g/larva). In the laboratory, a glycerol-based diet is generally used for G. mellonella rearing and results in good larval production. Unfortunately, several of the ingredients that constitute the diet such as glycerol, powdered milk, and yeast are expensive and are beyond the means of low-income sweet potato producers who are the main users of EPNs as biological control agents. The costs associated with rearing insects tend to make in vivo culture the least cost-efficient approach, compared with approaches such as in vitro-solid and in vitro-liquid culture (Shapiro-Ilan et al., 2004; Shapiro-Ilan et al., 2012). These considerations have created the need to find strategies to reduce the costs of rearing insects.
Commercially available dog croquettes are used to rear G. mellonella and are often composed of different ingredients of various origins (Fuchs et al., 2018). For example, Ellis et al. (2013) described G. mellonella breeding using a diet derived by mixing the following ingredients: seven parts (by volume) dry dog croquettes, one part water, and two parts honey, followed by content adjustment with vitamin A to produce whitish larvae. Van Zyl and Malan (2015) used dog croquettes to rear the insects by mixing 345 g of dry dog diet, 85 g of rolled oats, 85 g of wheat bran, 35 g of brewer’s yeast, 190.5 ml of honey, and 165 ml of glycerol.
This study was initiated to find an alternative artificial, environmentally safe, cost effective, and efficient diet for rearing G. mellonella larvae and to evaluate the effect of the diets on EPN virulence and multiplication. Specifically, the study (i) determined which among five different diets was best for rearing G. mellonella, (ii) evaluated costs of production of the diets, and (iii) assessed under laboratory conditions the virulence of five EPN isolates multiplied on G. mellonella larvae fed on the three best diets.
Materials and methods
Mass-rearing of G. mellonella and EPN multiplication
Diets
Five diets were used in this study: beeswax (BW), standard glycerol-based artificial diet (Gly), and three varieties of adult dog croquettes (DC1-DC3). Compositions of the diets are presented in Table 1.
Composition of diets and quantity of ingredients.
Diet
Diet code
Ingredients composing the diet
Quantity of ingredients (%)
Beeswax
BW
Beeswax slightly air-dried
100
Standard diet
Gly
Corn flour
18.85
Wheat flour
18.85
Soybean flour
9.42
Powdered milk
14.28
Yeast
4.76
Beeswax
15.00
Honey
9.42
Glycerol
9.42
Dog croquettes 1
DC1
Beef
4 in the nuggets with beef
Chicken
4 in the nuggets with chicken
Carrots
4 in the nuggets with carrots
Green vegetables
4 in the nuggets with green vegetables
Derivatives of vegetables, oils and fats, minerals
Not available
Dog croquettes 2
DC2
Cereals
4 in the nuggets with cereals in the form of beige pastille
Beef dehydrated
4 in the nuggets with beef in the form of red steak
Poultry dehydrated
4 in the nuggets with chicken in the form of beige steak
Derivatives of vegetable origin, meats, oils and fats, minerals
Not available
Dog croquettes 3
DC3
Cereals
40
Meats and derivatives of animal origin
4 of dehydrated beef protein
Derivatives of vegetable origin
4 of peas
Oils and fats, minerals, propylene glycol, sugar
Not available
The information on DC1, DC2, and DC3 ingredients and their respective quantities (columns 3 and 4) was taken from the packages containing the croquettes.
The BW was obtained from an apicultural site at Komiguea village in the district of Parakou, Benin. The moisture of BW was measured in the laboratory using a HTC-1 H596 LCD digital temperature and humidity meter (Zhangzhou KASUN Electronic Technology Co. Ltd, China). When the BW was too wet, it was hand-pressed to remove honey and then air-dried for 1 to 3 d in sunlight for 5 to 7 hr per day according to its moisture content. Moisture content was reduced to below 30% to hinder any development of fungi inside the breeding boxes. The insect larvae were directly reared on this natural diet according to Nurullahoglu and Susurluk (2001) and Jorjão et al. (2018).
The Gly diet was used in the laboratory for mass-rearing G. mellonella according to Shaik et al. (2017). Glycerol was used as a humectant due to its hygroscopic characteristics and also for its role as a preservative in diets (Pagliaro and Rossi, 2008). Corn and soybean were ground to obtain a flour consistency. All ingredients except honey and glycerol were weighed individually and mixed in a clean, dry aluminum container. For 1 kg of diet, 94.2 g each of glycerol and honey were used; due to their high viscosity, they were then melted in turn and mixed with other ingredients (Table 1). All ingredients were well homogenized by hand (Shaik et al., 2017). The mixture was cooled in ambient air for 1 hr and the diet introduced in breeding boxes for insect breeding. Any surplus mixture was kept in 5-l plastic boxes at 15°C.
Croquettes DC1, DC2, and DC3 were branded ‘Croquettes Adulte (Bœuf, Poulet, Légumes)’, ‘Multicroquettes Chien Adulte (au bœuf, à la volaille, aux céréales)’, and ‘Tendres croquettes Adultes (Au bœuf, aux céréales et aux légumes)’, respectively, with corresponding distributors ‘Belle France’, ‘Bien Vu!’, and ‘U’. Prior to use, the three types of dog croquettes were blended separately in a kitchen blender, mixed with 300 ml of honey per 1 kg of croquettes, and transferred into the breeding boxes for the insects to feed on. Honey was added to the diets to avoid dehydration.
Mass-rearing of G. mellonella
Larvae of G. mellonella were initially reared at ambient temperature (26.66–30.06°C) in 25-l plastic breeding boxes using the Gly diet. To prepare the cylindrical breeding boxes, a 6-cm-diameter hole was drilled in the circular surface of the boxes and used for the transfer of adult G. mellonella. The hole was covered with adhesive paper to prevent insects escaping. The lids of the breeding boxes were perforated and their internal surface was covered with fine mesh lined with white tissue to provide aeration.
The Gly diet was placed in an aluminum container, which was introduced into the breeding boxes. Then crumbled BW was placed onto the diet. In total, 15 rectangular (12 cm long and 5 cm wide) pieces of folded white paper were suspended on the white tissue that lined the lids, on the inner wall of breeding boxes at the rate of five pieces per place, using strong glue. Five pieces of folded paper were also placed directly on the diet to serve as a nesting support for insect females (Ramarao et al., 2012). Adult insects were introduced into the breeding boxes, which were then covered with black cloth and maintained in complete darkness (Mohamed et al., 2014) to promote activity in these essentially nocturnal moths. On average, the duration of the life cycle from egg to adult varies from weeks to months (Kwadha et al., 2017). Adults were collected from the breeding boxes to carry out trials with different diets.
Cost of producing G. mellonella diets
The cost of producing each diet tested was evaluated (Table 2). For Gly, the quantities of ingredients were first determined per kilogram of diet. Then, the cost of purchasing the different ingredients separately was related to the amount of ingredients needed to prepare 1 kg of diet. The total cost of production of the diet was finally estimated by adding the purchase cost of the various ingredients. The BW was supplied per kilogram upon purchase, which means that this represented the direct cost because no other ingredient was added. The dog croquettes were purchased in bags of 4 kg at supermarkets and the cost per kilogram was used in calculating the total cost of the diet with the added cost of honey required. The relationship between costs of producing G. mellonella diets and nematode population densities was determined.
Cost of production of diets tested to rear G. mellonella larvae.
Diet code
Composition
Purchase price (USD)a
Ingredients to produce 1 kg of diet
Calculated cost of diet/kg (USD)
Cost of total diet/kg (USD)
Time (wks) to feed 20 larvae of G. mellonella/kg of diet
BW
Beeswax
5.11/kg
1 kg
5.11
5.11
3
Gly
Corn flour
0.34/kg
188.57 g
0.068
Wheat flour
17.03/bag of 25 kg
188.57 g
0.14
Soy flour
0.51/kg
94.28 g
0.051
Powdered milk
45.97/cardboard box of 10 kg
142.86 g
0.66
6.30
1½
Yeast
40.86/cardboard box of 10 kg
47.24 g
0.20
Beeswax
5.11/kg
150 g
0.77
Honey
4.26/liter
94.28 g
0.40
Glycerol
42.57/liter
94.28 g
4.02
DC1
Adult dog multi-rolls + honey
6.81/bag of 4 kg dog croquettes
700 g
1.19
4
5.11 for 1 liter of honey
300 ml
1.53
2.72
DC2
Adult dog croquettes + honey
7.66/bag of 4 kg
700 g
1.35
2.88
4
5.11 for 1 liter of honey
300 ml
1.53
DC3
Tender adult dog croquettes + honey
8.43/bag of 4 kg
700 g
1.48
2.11
-
5.11 for 1 liter of honey
300 ml
1.53
aUnited States dollar; costs based on 2018 retail prices.
EPN multiplication
Galleria mellonella larvae fed with different diets were inoculated with five different nematode isolates. The effects of the different diets on virulence (mortality to the host and reproduction) of nematodes that emerged from cadavers of G. mellonella larvae were assessed.
The EPN isolates belonging to genera Heterorhabditis and Steinernema including three indigenous isolates (Aglali, Ze4, and Bembereke) and two isolates (S. carpocapsae and S. riobrave) imported from eNema, Germany were used in this study. The indigenous nematodes (Aglali and Ze4 both H. sonorensis; and Bembereke, an unidentified species of genus Steinernema) were previously extracted from soil samples collected in the southern and central parts of Benin during diagnostic surveys (Zadji et al., 2013). The imported nematode isolates S. carpocapsae and S. riobrave, known as pathogens of G. mellonella (Shapiro-Ilan et al., 2005; Christen et al., 2007), were included in the study for data comparison. For this study, all nematode isolates were multiplied in vivo using G. mellonella larvae (Kaya and Stock, 1997; Van Zyl, 2012). After inoculation with nematodes, G. mellonella cadavers were transferred into White traps (White, 1927) at 72 hr post-inoculation. Emerging infective juveniles (IJs) were harvested 10 d later and conserved at 15°C for use in experiments (Zadji et al., 2014).
Virulence of EPN emerged from infected G. mellonella larvae fed on diets
The three diets (BW, Gly, and DC1) that produced the heaviest and greatest numbers of G. mellonella larvae identified in the previous assay in the breeding experiment were used for the virulence assay. Eppendorf tubes (2 ml) were used for the EPN virulence assay (Zadji et al., 2014). Each tube was perforated to allow air exchange and was filled to 3/4 of its capacity with previously sterilized sandy soil (85°C, 72 hr) and adjusted to 10% moisture (w/w) (Zadji et al., 2014). We use the five nematode isolates that emerged from infected G. mellonella and kept at 15°C as described earlier in the EPN multiplication methodology section. Before their use, IJs were acclimatized to room temperature (28 ± 2°C) for 1 hr and their viability (movement) was checked under a stereomicroscope (20×). Under the same device, 20 IJs in 200-µl water suspensions were transferred into each Eppendorf tube with a Microlux pipette (10-100 µl). A late-stage larva of G. mellonella taken from each type of diet was individually introduced into the Eppendorf tube, which was then closed. There were 10 replicates and each comprised one Eppendorf tube per nematode isolate and per diet tested. The tubes were arranged in a completely randomized design and stored at room temperature of 28 ± 2°C in darkness as described in the study of Dolinski et al. (2006). Three days later, dead and living larvae were removed from Eppendorf tubes and counted. Then, larvae were rinsed with tap water to remove nematodes from their surfaces and transferred into 9-cm petri dishes lined with tissue paper. The larvae of the same treatment in a dish did not touch each other. They were kept in the dishes for four exposure times: 1, 2, 3, or 4 wks (T1, T2, T3, and T4, respectively). After each exposure time, half of the population of dead insects per treatment was individually dissected in a 9-cm petri dish under a stereomicroscope (20×). All nematodes inside each insect larva were collected in a 100-ml beaker by rinsing the petri dish to ensure no nematodes remained. The volume of nematode suspensions was adjusted to 25 ml by adding distilled water (Glazer and Lewis, 2000) and homogenized. For each G. mellonella larva, three sub-samples, each of 50-μ l aliquot of nematodes, were withdrawn with a Microlux pipette (10-100 µl) and placed into three different new 9-cm petri dishes. To count the number of nematodes in petri dishes, 10 ml of distilled water was added to each dish. The second half of the population of dead insects per treatment was transferred into White traps in 9-cm-diameter sterilized petri dishes (1 insect/dish) and progeny production assessed 1 wk later. The suspensions were collected in a 100-ml beaker and the volume of nematode suspensions was adjusted to 25 ml and homogenized. Nematodes were counted from three 50-µl aliquots as described previously for dissection. The nematode population densities presented in this study are the average of the three 50-μ l counts of nematodes by dissection or by White traps. The experiment was repeated once using new batches of the same nematode isolates and of G. mellonella larvae.
Effect of diets on G. mellonella development and reproduction
The five diets described above (BW, Gly, and DC1-DC3) were used to determine the most favorable diet for the development (in terms of larvae weight) and reproduction (in terms of progeny production) of G. mellonella. For each diet, 2 kg was taken and introduced into 25-l breeding boxes. In total, three breeding boxes were used for each diet. In total, 30 adult insects (25 females and 5 males) (Baimey et al., 2017) were introduced into each breeding box.
Emergence of larvae began 5 d after transfer of adults to the breeding boxes and continued daily according to diets and lasted a further 20 d. Daily monitoring was carried out to check for emergence of young larvae and larvae spinning cocoons. From the appearance of the first cocoon in the breeding boxes, the number of larvae was counted daily until there was no larva without a cocoon. For all diets tested, several larvae reached the cocooning stage almost at the same time before the first adults started to emerge. Thus, in a batch of a minimum of 50 cocoons per diet, 30 larvae were randomly collected the same day from cocoons produced from each diet and their spinning was removed. The larvae were weighed individually and the mean weight of larvae recorded. These larvae were then reintroduced into their respective breeding boxes where they formed new cocoons. The test was repeated again with another batch of larvae.
Statistical analysis
The mean mortality of insect larvae was tested for normality and homogeneity of treatment variances using Levene’s test. Mortality rates (percentage) of insects due to EPN isolates were corrected according to Abbott (1925). To stabilize the variance of means, mortality data (%) were transformed with square-root arc sine (arc sin √x) (Gomez, 1984) prior to analysis of variance (ANOVA). Nematode population density was log10(x + 1) transformed to normalize the data prior to analyses. Data were subjected to ANOVA using R (version 3.5.1). The differences between treatment means were compared at P < 0.05 using the Student–Newman–Keuls test.
A linear bootstrapping regression was performed using 1,000 replicates to test the relationship between costs of diets and nematode population density. The bootstrap package Leisch 2019 in R 3.5.2 (R Core Team 2018) was used.
Results
Galleria mellonella larvae development on each diet
Figure 1 shows the mean weight of G. mellonella larvae per diet at the cocooning stage. There were significant differences (F = 242.5, df = 4, P < 2e−16) between the weights of the larvae obtained per diet. No larvae were observed in DC3. Larvae fed on DC2 had the lowest weight (0.18 ± 0.03 g) (mean ± SD) and larvae fed on BW, DC1, and Gly showed similar (P < 0.05) higher weights (0.21 ± 0.03, 0.21 ± 0.04, and 0.22 ± 0.04 g, respectively).
Figure 1:
Weight (mean ± standard deviation) of G. mellonella larvae obtained at the cocoon spinning stage by diet tested. No larvae emerged on diet DC3, and so there were no data. Weights followed by the same letter do not significantly differ at P < 0.05. BW, beeswax; Gly, standard glycerol-based artificial diet; DC1 to DC3, three varieties of adult dog croquettes.
Influence of diet on G. mellonella larvae rearing
Except for DC3, all diets (BW, Gly, DC1, and DC2) reached the stage of cocoon spinning with different average number of G. mellonella larvae produced according to the diet (Fig. 2). The maximum average number of cocoons (224.33 ± 62.26) was for the Gly diet at 13 d after young larvae were first observed in the breeding boxes. The lowest cocoon production was for diet DC2 during the first day of emergence and 13, 16, and 17, and then 20 to 23 d. During 13 to 15 and 17 to 19 d after observation of young larvae, the number of cocoons increased on this diet, and exceeding that for the diet BW, before decreasing. Diets BW, DC1, and Gly produced the highest number of larvae (Fig. 2).
Figure 2:
Variation curve of the number of G. mellonella larvae produced for all five diets as a function of time. BW, beeswax; Gly, standard glycerol-based artificial diet; DC1 to DC3, three varieties of adult dog croquettes.
Cost of producing diets used to rear G. mellonella larvae
Table 2 shows the cost per kilogram of the different diets used to rear G. mellonella larvae. The Gly diet was the most expensive to produce at 6.30 US dollars (USD)/kg and could feed approximately 20 larvae for 1.5 wks. The cost of diet BW (5.11 USD/kg) was less than that of Gly and could be used to feed the same number of larvae for 3 wks. Diet DC3 was the least expensive of all diets (2.11 USD/kg) but did not favor any larval development. Diets DC1 and DC2 cost 2.72 and 2.88 USD/kg, respectively, and both these diets could feed 20 larvae for about 4 wks. In general, the diets incorporating dog croquettes were approximately half of the cost of the Gly and BW diets.
Relationship between diet cost of production and weight of larvae
There was a low positive correlation (r = 0.44; non-significant, P = 0.4541) between the cost of production of the different diets and the mean weight of G. mellonella larvae at the cocoon spinning stage.
Mortality caused by EPN to G. mellonella larvae fed on different diets
Significant differences in G. mellonella larval mortality were observed for diet (P < 0.001), exposure time (P < 0.01), and the interactions diet × nematode isolate (P < 0.001) and diet × nematode isolate × exposure time (P < 0.05) (Table 3).
ANOVA for mortality at ambient temperature of G. mellonella larvae using factors of exposure time, nematode isolate, diet, and their interactions.
Source of variation
F
df
P
Isolate
0.34
4
0.851
Diet
9.91
2
0.001
Exposure time
5.42
3
0.002
Diet × isolate
5.49
8
0.001
Diet × exposure time
1.87
6
0.092
Isolate × exposure time
1.14
12
0.335
Diet × isolate × exposure time
1.62
24
0.048
Source of variation significant at P < 0.05.
Diets Gly, BW, and DC1 affected the mortality caused by EPN isolates to G. mellonella larvae (Fig. 3A-D). At time periods T1 and T3 (Fig. 3A,C), there was no significant difference in mortality of larvae fed on the different diets and infected with different nematode isolates. However, at T2 (Fig. 3B) and T4 (Fig. 3D), larval mortality varied significantly (P < 0.05) when infected with nematode isolates. At T2, 100 ± 0.00% of larval mortality was observed with Bembereke (Steinernema sp.) and Ze4 (H. sonorensis) isolates fed on diet Gly; this mortality level was also observed on isolate Ze4 (H. sonorensis) fed on diet DC1. The lowest mortality rate (73.00 ± 5.77%) was for larvae fed on DC1 and inoculated with nematode isolate S. riobrave. The same nematode isolate caused the highest mortality rate (96.66 ± 5.77%) on diet BW at T4; and isolates Bembereke (Steinernema sp.) and S. riobrave caused the lowest mortality rates (70.00 ± 10.00% and 70.00 ± 0.00%, respectively) on diet BW.
Figure 3:
Mortality rate (mean ± standard deviation) of G. mellonella larvae based on diet, nematode isolate, and exposure time (T1-T4): (A) T1 (1 wk); (B) T2 (2 wks); (C) T3 (3 wks); and (D) T4 (4 wks). Mortality rates followed by the same letter do not significantly differ at P < 0.05. BW, beeswax; Gly, standard glycerol-based artificial diet; DC1, adult dog croquettes.
Population density of nematodes inside dissected G. mellonella larvae fed on each diet
Significant differences were observed for diet, nematode isolate, exposure time, and the interactions diet × nematode isolate, diet × exposure time, nematode isolate × exposure time, and diet × nematode isolate × exposure time (P < 0.001) (Table 4).
ANOVA results for population density of nematodes in G. mellonella according to different factors (diets, nematode isolate, and exposure time) by dissection.
Source of variation
F
df
P
Diet
23.68
2
0.001
Nematode isolate
31.47
4
0.001
Exposure time
24.11
3
0.001
Diet × Nematode isolate
13.90
8
0.001
Diet × Exposure time
24.54
6
0.001
Nematode isolate × Exposure time
21.18
12
0.001
Diet × Nematode isolate × Exposure time
12.48
24
0.001
Source of variation significant at P < 0.05.
Regardless of diet and exposure time, IJs were observed inside the dissected infected G. mellonella larvae (Fig. 4A-C). The highest population densities of nematodes counted in 50 µl of suspension per infected larva were obtained at T2 with nematode isolate Aglali (H. sonorensis) on BW (309.11 ± 302.35 IJs/50 μ l of suspension, 154,555 IJs/larva) (Fig. 4A) and Gly (447.83 ± 305.75 IJs/50 μ l, 223,915 IJs/larva) (Fig. 4C) and isolate Ze4 (H. sonorensis) (452.96 ± 267.15 IJs/50 μ l, 226,480 IJs/larva) on DC1 (Fig. 4B). At T4 on BW, the population densities of isolate S. riobrave increased and were the highest (P < 0.05), but those of isolates Aglali (H. sonorensis) and Ze4 (H. sonorensis) decreased. On DC1, however, S. riobrave gave the lowest population density at T1, T2, and T4, whereas at T3, the lowest population density of nematodes was for isolate Bembereke (Steinernema sp.). With diet Gly, isolates S. carpocapsae and Bembereke (Steinernema sp.) gave the lowest population densities of nematodes at T1 and T2 and during T2 to T4, respectively.
Figure 4:
Nematode population density resulting from dissection of infected G. mellonella larvae fed on BW (beeswax), DC1 (adult dog croquettes), and Gly (standard glycerol-based artificial diet) over time (T1-T4). The nematode population densities presented are the means obtained in 50 μ l of suspension/larvae: (A) BW; (B) DC1; and (C) Gly.
Population density of nematodes emerging into White traps from infected larvae fed on different diets
Except for the interaction diet × exposure time (F = 1.08, df = 6, P = 0.3691), ANOVA showed that all factors and their interactions had significant effects: P < 0.05 for diet and P < 0.001 for nematode isolate, exposure time, diet × nematode isolate, nematode isolate × exposure time, and diet × nematode isolate × exposure time (Table 5).
ANOVA results for population density of nematodes in G. mellonella according to different factors (diets, nematode isolate, and exposure time) by White traps.
Source of variation
F
df
P
Diet
4.33
2
0.013
Nematode isolate
64.61
4
0.001
Exposure time
145.79
3
0.001
Diet × Nematode isolate
6.82
8
0.001
Diet × Exposure time
1.08
6
0.369
Nematode isolate × Exposure time
11.23
12
0.001
Diet × Nematode isolate × Exposure time
4.78
24
0.001
Source of variation significant at P < 0.05.
Population density of all five tested nematode isolates decreased with exposure time during T1 to T3, with the exception of isolates Bembereke (Steinernema sp.) and S. riobrave at T2 for diet BW (Fig. 5A-C). With up to 3 wks of exposure, all nematode isolates emerged from G. mellonella larvae fed on different diets.
Figure 5:
Nematode population density based on nematode isolate and exposure time (T1-T4), over 7 d after the first appearance of nematodes in White traps for the BW (beeswax), DC1 (adult dog croquettes), and Gly (standard glycerol-based artificial diet) diets. The nematode population densities presented are the means obtained in 50 μ l of suspension/larvae: (A) BW; (B) DC1; and (C) Gly.
With respect to larvae fed on diet BW, at T1 the nematode isolate Ze4 (H. sonorensis) had the highest nematode density (553.63 ± 311.97 IJs/50 μ l, 276,815 IJs/larva) (Fig. 5A) and that of isolate Bembereke (Steinernema sp.) was the lowest (105.36 ± 84.22 IJs/50 μ l, 52,680 IJs/larva). The population density of Aglali (H. sonorensis), Ze4 (H. sonorensis), and S. carpocapsae nematode isolates gradually decreased over time. All nematode isolates, with the exception of S. riobrave, had barely emerged at T4. The isolate Aglali (H. sonorensis) no longer emerged (0.00 ± 0.00 IJs/50 μ l, 0 IJs/larva) at T4 from G. mellonella larvae. However, isolate S. riobrave continued to emerge and resulted in the highest nematode population density (242.95 ± 254.73 IJs/50 μ l, 121,475 IJs/larva); its density increased during T1 to T2, but then decreased during T2 to T3 and again increased during T3 to T4.
The larvae fed on diet DC1, which had been infected and conserved until T4, did not favor the emergence of Bembereke (Steinernema sp.) and S. carpocapsae nematode isolates. On diet DC1, the highest population density of nematodes was for isolate Ze4 (H. sonorensis) (481.02 ± 2.97 IJs/50 μ l, 240,510 IJs/larva) at T1 and the lowest density was for imported isolate S. carpocapsae (187.55 ± 0.00 IJs/50 μ l, 93,775 IJs/larva) (Fig. 5B). The population density of Bembereke (Steinernema sp.), S. carpocapsae, and S. riobrave nematode isolates decreased progressively to T4 but S. riobrave increased from T3 to T4. However, this population density was generally stable during T1 to T2 for Ze4 and Aglali (H. sonorensis) isolates before progressively declining.
Nematode isolate Aglali (H. sonorensis) had the highest population density (615.18 ± 309.63 IJs/50 μ l, 307,590 IJs/larva) at T1 on diet Gly among all diets. The lowest population density of nematodes at T1 was for isolate S. carpocapsae (87.83 ± 74.76 IJs/50 μ l, 43,915 IJs/larva). At T4, isolate Bembereke (Steinernema sp.) had the highest population density (62.17 ± 33.60 IJs/50 μ l, 31,085 IJs/larva) (Fig. 5C).
Relationship between weight of larvae and population density of nematodes emerging
There was a significant positive correlation (r = 0.79, P = 0.0015) between the weight of G. mellonella larvae and the population density of nematodes from White traps.
Relationship between costs of producing diets of G. mellonella and nematode population densities
There was a positive (slope = 0.0002 ± 0.0005 > 0) but non-significant linear relationship between costs of producing diets of G. mellonella and nematode population density (P = 0.636) (Fig. 6).
Figure 6:
Relationship between costs of producing diets of G. mellonella and nematode population densities.
Discussion
Our investigations demonstrated the influence of diet (one natural and four artificial diets) on development and survival of G. mellonella larvae. Earlier studies had carried out mass-rearing of G. mellonella mostly on artificial diets to study various biological parameters such as duration of developmental life stages, fertility, and survival before choosing the tested materials as suitable diets (Chandel et al., 2003; Coskun et al., 2006; Birah et al., 2008; Kulkarni et al., 2012; Ellis et al., 2013). The best diet developed by Birah et al. (2008) for G. mellonella comprised wheat flour (130 g), wheat bran (130 g), milk powder (130 g), maize flour (97.5 g), yeast powder (97.5 g), beeswax (26 g), honey (195 ml), and glycerol (195 ml). Constituents similar to the above but in different proportions were also tested by Huang et al. (2010). In our study, Gly contained ingredients similar to those used by Birah et al. (2008) and Huang et al. (2010) and proved to be the most effective diet because it resulted in the highest mean number of G. mellonella larvae. Yeast has been frequently reported as a very important component in G. mellonella diets (Gross et al., 1996; Singh et al., 2014; Van Zyl and Malan, 2015). Gulati and Kaushik (2004) observed that factors such as relative humidity and diet influence G. mellonella development and metamorphosis. According to Cohen (2004), imperative to the rearing of high-quality hosts and EPNs is the selection of an artificial host diet that supports development of the entire life cycle of the host and outperforms other diets in terms of host yield production, weight accumulation, and developmental rate of hosts. Despite its effectiveness, the high cost of rearing G. mellonella larvae on the Gly diet remains a very important issue to resolve. This study showed that the diets differed in terms of larvae produced, with more expensive diets producing a higher weight of G. mellonella larvae. Moreover, diet Gly required more physical effort for preparation to rear G. mellonella larvae in the laboratory than any other diet evaluated.
The BW diet also resulted in good production of G. mellonella and can therefore be used as a substitute for Gly to rear the insects. It can feed the same number of larvae as Gly and for a longer period. Beeswax is easily obtained from beekeeping sites; however, in this study it did not allow a rapid production of G. mellonella larvae and therefore cannot be recommended when rapid production (i.e. within 3 wks) of insect larvae is needed for mass production of EPN. To overcome this problem, it is necessary to switch to another diet. Another problem with using beeswax as a diet is its limited availability only in the dry season on beekeeping sites. Coskun et al. (2006) found that the insects on a beeswax diet pupated earlier because of nutritional deficiency. According to NiemierKo and Wlodawer (1950), G. mellonella larvae can obtain a large amount of energy from beeswax, but it may not provide other nutrients in sufficient quantity, such as protein, for successful development. The diet DC3 did not favor emergence of G. mellonella larvae at all. Despite the presence of cereals in that diet, as also used in diets Gly and DC2, no G. mellonella larvae were observed. There are a number of dog croquette products on the market but they cannot all be used to rear G. mellonella. These findings agree with previous observations (Chandel et al., 2003; Birah et al., 2008; Kulkarni et al., 2012), who reported that detailed knowledge of an insect’s biology is necessary to evaluate artificial diet modifications. In some cases, brands of dog food have insecticides added to the formulation as a protective treatment against fleas (Wallinga and Greer, 2000), thus making them useless for insect rearing. The diet DC3 may contain such products and this could explain the lack of development of G. mellonella larvae.
For a small-scale laboratory production intended, for example, for conservation of isolates of EPN, diet DC1 could be considered because it produced a greater number of larvae and more quickly than diet DC2. However, there are some difficulties associated with the use of dog croquettes for G. mellonella larvae rearing because of the scarcity of certain brands of croquettes on the market for extended periods. Dog croquettes DC1 and DC2 favored the development of G. mellonella larvae and this could be explained by their inclusion of beef and chicken (or other poultry) which were absent from DC3.Thus, the type of diet and its composition influenced development of G. mellonella.
Diet DC1 is economically profitable for mass-rearing G. mellonella but larval development was less rapid compared with Gly. The choice of diet to adopt in this context seems to be a function of available diets and the materials to prepare them. We recommend Gly for G. mellonella larvae mass-rearing in the laboratory when EPN are to be applied in infected insect larvae for biological control trials in the field. However, because of the high cost of this diet, BW and DC1 can be used instead of Gly if a program of larvae application in the field has previously been well established in advance. To maintain EPN isolates in the laboratory, DC1 can be used to rear G. mellonella because this diet has a low cost and the weight of larvae does not significantly differ from those fed on BW and Gly. When considering the independence of sweet potato producers in the context of biological control, beeswax will more useful than the other diets because some producers have beekeeping sites or friends raising bees from which they can easily obtain beeswax (the natural diet of the insect). For those growers for whom acquisition of beeswax is difficult, diet DC1 is preferable and DC2 is an alternative.
Concerning the mortality of larvae, it is possible that the diet fed to G. mellonella affects the efficiency in killing the host by the EPN multiplied within them. According to Finke (2002), G. mellonella larvae contain a high percentage of fat, and certain lipid components that have been shown to be conducive in increasing the developmental rate and yield of Heterorhabditis nematodes. Shin et al. (2001) analyzed the lipid content of G. mellonella exposed to different concentrations of cadmium chloride independently contaminating an artificial diet at different concentrations. Their results showed that lipid levels decreased in the cadmium-contaminated groups compared with the control group. Thus, the lipid composition and perhaps other compounds in the body of G. mellonella larvae vary with the diet consumed. According to Andaló et al. (2011), lipids represent the main source of energy for EPN; in the IJ stage, the level of such reserves can be influenced by storage, and this may affect their infectivity, while IJ is the only infective stage of nematodes. The diet used to feed G. mellonella affected population density of nematodes and this also varied with time. Therefore, the diet on which the host feeds also influences the efficacy of the parasite. Sajjan (2016) evaluated the yield of H. indica from G. mellonella larvae reared on five diets and found that the population density of nematodes for these diets followed different patterns. This could explain the highest densities of nematodes obtained from G. mellonella larvae at T1 into White traps with isolate Ze4 on BW and DC1, and Aglali on Gly, which both belong to the genus Heterorhabditis. The G. mellonella larvae fed on the BW diet continued to produce nematodes of isolate S. riobrave beyond T4.
The findings of this study suggest that diet is one of the most important factors influencing development of G. mellonella. Also, it should be noted that the choice of diet, particularly with regard to dog croquettes, for rearing the insects as well as diet composition are important factors in planning breeding of G. mellonella. We conclude that the reproductive potential of EPN and their pathogenicity vary over time depending on the diet fed to G. mellonella larvae.
