Study Area

Sampling was done from 1^st April 2021 till 30^th April 2022 in district Mardan, Khyber Pakhtunkhwa Pakistan. Mardan region is connected to the district Malakand on the North, district Buner on the North-East, district Swabi on the East, district Charsadda on the West, and district Nowshera on the South. Mardan is located at 34 12’0N 721’60E with an altitude of 283 meters above sea level ArcGIS (version 10.5) was used for mapping of collection points (Fig. 1).

Trap Net

The trap net was designed from mosquito mesh using PVC (polyvinyl chloride) pipes with standard size (length 1 foot, width 1 foot, and height 1 foot). having an opening (8-inch ring) over the trap net for the entrance of the H. illucens fly (Fig. 2).

Attractant

Kitchen waste (50 grams), cow dung (50 grams), rotten fruits (50 grams), and chicken waste (50 grams) were used as an attractant [14]. All these attractants were kept in each trap net for four days at four different localities of district Mardan i.e. Sultan Abad, Rashaka, Baghdada, and Gul Maira. Temperature and humidity were recorded by using a thermohygrometer model, HTC ̶ 2 at each site, early in the morning, in the afternoon, and late at night (Fig. 3 and 7).

Eggies Design

The Eggies (Cotton corrugated channel to collect the insect egg) were designed from corrugated/ channeled cardboard (weighing 128 grams, having length and width of 8 cm and 25 cm respectively) that were gripped together with a rubber band and kept over each attractant inside the trap net so that H. illucens may lay eggs and will be collected then. The trap net was checked regularly after 12 hours for H. illucens confirmation and for the observation of their egg clutch in cardboard channels (Fig. 4).

Fig. 2.

Trap net.

Fig. 3.

Hermetia illucens collection from dumping site at District Mardan.

Control Environment

Temperature (24-30ºC), humidity (60%-95%), and sodium light (100 watts) were maintained in the laboratory for the survival of H. illucens and to ensure higher yield in the lab.

The temperature was maintained by using an inverter while a humidifier model, 3L, HM300 was used for the maintenance of humidity in the required range.

Incubation of Harvested Egg Clutches and Larval Feeding

To prevent microbial contamination, the egg clutch and H. illucens were transferred to the rearing unit [24, 25]. The number of egg clutches sired in the grooves of the cardboard traps was counted initially and the final weight was determined using digital balance. The egg clutches harvested from different sources, for example, fruit waste (1000g), chicken waste (1000g), cow dung (1000g), and kitchen waste (1000g) were taken individually in plastic containers of standard size having length, width, and depth of 15 inches, 10 inches, and 5 inches respectively (Fig. 5). A similar method was used for all four sites. After 3 days, eggs were successfully hatched in each waste source.

Each larva was provided with 5 g feed daily i.e., fruit waste, chicken waste, cow dung, and kitchen waste while after hatching the feed quantity was increased by 1 gm per larva each day up to the pre-pupal stage.

Fig. 4.

a) Eggies from cardboard, b) Eggs clutch and channeled in cardboard.

Fig. 5.

Hermetia illucens eggs harvesting on different sources.

Determination of Larval Instar

After 2 days of egg hatchlings, the larvae were transferred to Petri dishes to examine the larval stages in detail. Development of all larval instar stages was examined microscopically by using an Olympus SD-30 microscope with an attached camera (6 megapixels).

Morphological Identification of Black Soldier Fly

The H. illucens was observed under a microscope and the body parts were examined in detail with the help of an attached camera. The H. illucens were identified by using identification keys [13].

Chemical Composition of H. Illucens

The H. illucens products such as Pre pupa 21 g, Pupa 28 g, Shell 24 g, and die Adult H. illucens 53 g were washed in tap water and then dried in a micro-oven at 90ºC for 8 minutes. These four products were powdered in a grinding machine and stored in a sterilized plastic box at room temperature for further analysis.

Climatic Conditions in Nature

There were no significant differences in both temperature and relative humidity on the microhabitats during H. illucens collection and egg clutch harvest as determined by One-way ANOVA for temperature (df = 3, F = 1.126, P = .353) as shown in Fig. 7, and relative humidity during egg trapping (df = 3, F = 1.754, P = .233) as shown in Fig. 8. The attractants are used to attract female flies towards themselves, whereby the adult female H. illucens lays eggs over the waste. Sripontan et al. have reported similar results to our study, the presence of H. illucens in a humid and warm area in nearby locations to its natural habitat, which provides a more suitable habitat for the survival and offspring feeding of the H. illucens [26]. Lewylle et al. have reported that certain environmental conditions like humidity, temperature, and feeding substrate should be taken into consideration during H. illucens rearing [27]. The current study is in accordance with the above statement, which indicates that the habitat and life cycle of H. illucens is completely dependent upon these conditions (i.e., humidity, temperature, and feeding substrate).

Fig. 6.

Collection of Hermetia illucens eggs clutch from the target dumping site.

Fig. 7.

Mean temperature during Hermetia illucens eggs clutch collections.

Fig. 8.

Mean relative humidity during Hermetia illucens egg clutch collections.

Larval Rearing

The larva was reared in fruit waste, cow dung, kitchen waste, and chicken waste in separate larval boxes under standard conditions in a controlled environment. To evaluate morphological differentiation, survival, and growth response of each instar to different feed i.e. fruit waste, cow dung, chicken waste, and kitchen waste were recorded. It has been reported in certain research papers that H. illucens larvae are used to manage municipal organic wastes, chicken, swine, and cattle manure with its descendent initiation from the wild. [28, 29]. In the current research investigation, it has been documented that H. illucens are attracted by organic wastes, fruit waste, chicken waste, cow dung, and kitchen waste in the wild.

Fig. 9.

1: Head, 2: Thorax, 3: Abdomen. Of Hermetia illucens.

Fig. 10.

A: Head region, B: Antenna, C: Transparent region, D: Legs.

Fig. 11.

Sexual dimorphism of Hermetia illucens.

Adult

The H. illucens body has three regions: head, thorax, and abdomen (Fig. 9). The head consists of a pair of antennae, compound eyes that are bluish and yellowish green, a spongy mouth, and one pair of maxillary palp, spherical, and clypeus. The antenna is differentiated into three parts; scape, pedicle, and flagellum (long terminal segment) abdominal region of H. illucens is transparent, with three pairs of legs, and a pair of wings. Legs have visible and distinct three parts; the femur dorsal is black and the upper part is covered with tiny hairs while the lower part bears no hair, ventral is white. The tibia is black with white spots while the tarsus is white (Fig. 10).

There is no obvious external sexual dimorphism except genitalia. The male genitalia organ is short, looks like a flower, and is protractible, with two pairs of posterior lobes (Table 1). Female genitalia are fork-shaped and protractible and the genitalia are entirely covered by microscopic hairs in both males and females (Fig. 11).

Fig. 12.

Wings of Hermetia illucens.

Wings are shiny and clustered, bearing a thin and delicate vein that provides passage of hemolymph, which is essential for the proper functioning of wings and various sense organs found on the wings. Wing venation plays an important role in the morphological identification of insects and is an important characteristic in taxonomy (Fig. 12). A mature adult of black soldier fly survives only for about 7 days.

Eggs

A mature H. illucens adult lays about 870 eggs individually during its lifetime (about 7 days) Hatching period is 3 days and is dependent on the source. The eggs are laid in colonial clusters, shape structure having a length and width of about 0.9 mm and 0.2 mm respectively bear pale yellow or creamy white color with a black spot over the egg. After hatching, the egg clutch should be transferred to a clean and moist surface immediately to prevent microbial contamination (Fig. 13).

Larva: (1^st instar)

1^st instar larvae were kept under observation after 3 days of hatching. Special morphometric characteristics are distinguished, color yellow-white, time 4 days, average length 2.10 mm, average width 0.5 mm, average weight 0.03 g, segment 11, and brisks of hair are not present over the body (Fig. 14).

Fig. 14.

Morphology of instar 1.

Fig. 15.

Morphology of instar 2.

Fig. 13.

A (eggs colonial cluster), B (Black spot over egg), C (length of egg).

Instar 2^nd

Morphometric character; time 4 days, color light brown, average length 4.04 mm, average width 1.06 mm, average weight 0.06 g, segments 11 and brick of hair are visible over the body (Fig. 15).

Instar 3^rd

Recorded gradual changes occurred in coloration. The color was recorded as dark brown, average length of 8 mm, average width of 2.77 mm, average weight of 0.09 grams, time of 5 days. The number of segment 11 and bricks of hair are visible (Fig. 16).

Instar 4^th

Morphometric was recorded; Average length 16mm, Average width 2.96mm, average weight 0.1 grams, time 2 days, color light brown, Segments 11 and hair are visible (Fig. 17).

Instar 5^th

The Color is dark brown, the Average length is 2 cm, the average width is 0.4cm, the average weight is 0.20 grams, Segment 11, the time is 7 days, and the hair size of each segment is large and visible (Fig. 18).

Instar 6^th

Pre-pupae are similar and look like mature larvae, the color is blackish, average length 2.5 cm, average width 0.4 cm, average weight of 0.30 grams, and segments 11 with hair-like structure known as bent hooks used for locomotion (Fig. 19).

Fig. 16.

Morphology of instar 3.

Fig. 18.

Morphology of instar 5.

Fig. 20.

Shown pupa morphology.

Fig. 17.

Morphology of instar 4.

Fig. 19.

Morphology of instar 6.

Pupa 7^th

Pre-pupa changed to pupa, the color is fully black, and the remaining character was similar to pre-pupa. It starts movements in a dark, dry place and stops feeding (Fig. 20).

After seven days the pupa starts molting in a dark and suitable environment. The shell remains and raises a complete fly. Fly starts flying toward light for mating and starts the next cycle.

Proximate Chemical Composition of (H. Illucens) Products

The four products of (H. illucens) were analyzed at (Feed & Water Testing Laboratory Poultry Research Institute, Murree Road Rawalpindi) (Table 2).

The highest moisture was recorded at 38.5% in dead H. illucens, crude protein at 55.13%, crude fat at 28.8% in pre-pupa, crude fiber at 21.5%, and total ash at 15.5% in H. illucens larvae shells.

In another study, [30] showed suitable temperature (24-40ºC) for mating of the adult H. illucens which is in accordance with our study during the morning, afternoon, and night time which was 31.925ºC, 37.05ºC and 27.975°C, respectively. Average humidity recorded in the wild was 50.5%, 42%, and 44.5% during morning, noon, and evening, respectively. Temperature was maintained between 23ºC and 33°C during the harvesting period in the control environment but 25ºC was found to be more suitable for H. illucens mating and oviposit along with other standard conditions. Our results show the highest recorded humidity (48%) and lowest humidity level (42%), which is in accordance with the humidity level (44-68%) as reported by [8, 31]. although the humidity was maintained in the range of 60%-96% during indoor rearing which showed the best results. Kim et al. reported in a subsequent study, the Larval stages up to 5 have and 6 [32, 33].

The life cycle of H. illucens is 45 days in total. They spend two stages of their life in the growing media eggs (4 days) and larva stage (18 days). Only when they are transforming from pre-pupa (14 days) to adults (9 days), they complete the metamorphosis [34]. In this study, we found the life cycle of H. illucens a total 39 days. Egg hatching time is 2 days, 1^st instar 4 days, 2^nd instar 4 days, 3^rd instar 5 days, 4^th instar 2 days, 5^th instar 5 days, 6^th instar 6 days, 7 instar 3 days and adluts live for 8 days to complete their cycle.