Introduction

In terms of nutrient cycling in the ecosystem, carbon fixation by photosynthesis has been paid much attention (Blodau, 2002; Nielsen et al ., 2011; Zhi et al ., 2015). However, when trophic level is considered, there are a lot more actors involved. For example, decomposers such as microbes have an essential role in the cycle (Moorhead et al ., 2014). In this regard, insects also play a pivotal role in degrading ingredients by simply consuming organic materials. In this way, organic materials are incorporated into nutrient cycling. Most notably, black soldier fly (BSF), Hermetia illucens (Diptera; Stratiomyidae), has been utilized for processing manures and other organic wastes such as human food waste (Kim et al ., 2008; Lord et al ., 1994; Park et al ., 2017; Sheppard et al ., 1994). In Korea, this insect is in mass production since its accidental introduction in early 2000s (Kim et al., 2008). This insect is also easily noticeable with its metallic black color. However, due to its sensitiveness to temperature and light availability, it is somewhat limited both in annual and diurnal activities.

Family Stratiomyidae is composed of at least 150 species worldwide (Rozkošný & Kovac & Kovac, 2000). In Korea, 15 species belonging to this family have been identified (Paek et al ., 2010)). Among them, Ptecticus tenebrifer is native to Korea. It seems to have remarkably similar ability to decompose organic materials as H. illucens does. Larvae of this insect consume wastes until they molt into pupae. Its adults usually hover over wastes. With this regard, we hypothesized potential competition between these two species in their adult stage to keep others from laying eggs in wastes. Thus, the objective of this study was to investigate their distribution and adult fighting to see their potential competition in the natural field.

Materials and Methods

Locations of the insect

In 2019, we surveyed 22 sites based on administrative district nationwide. Both adult insects were marked as presence/absence. Types of the wastes were also described (Table 1). These insects were also collected using insect nets for future genetic analyses.

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Table 1.
Observation sites and presence of H. illucens
Region Location Organic materials Lat. Long. H. illucens
1 Anseong AW 37.02 127.29
Seongnam AW 37.38 127.13
Yangpyeong AW 37.42 127.24
Gwangju AW 37.42 127.24
2 Seocheon FW 36.03 126.73
Gunsan AW 35.97 126.74
Cheongyang M 36.45 126.70 Yes
3 Boeun CC 36.59 127.71
Goesan AW 36.66 127.74
Cheongwon CC 36.58 127.51
4 Hampyeong AW 35.08 126.52 Yes
Jangseong FW 35.42 126.85 Yes
Damyang FW 35.31 126.92
5 Haman AW/FW 35.27 128.40
Goseong FW 35.01 128.31
Changwon FW 35.25 128.58
6 Bongwha AW 35.27 128.40 Yes
Youngju AW 36.95 128.62 Yes
Andong FW 36.77 128.81 Yes
7 Inje FW 38.03 128.47
Pyeongchang AW 37.64 128.50 Yes
Yangyang FW 38.08 128.67

Region 1: Gyeonggi, 2: Chungnam, 3: Chungbuk, 4: Jeonnam, 5: Gyeongnam, 6: Gyeongbuk, 7. Gangwon.

Mitochondrial (Mt) CO1 gene sequence and phylogenetic analysis of Stratiomyidae

Genomic DNA (gDNA) was extracted from the posterior end of each individual using a commercial kit (ExGen Tissue SV, Geneall, Korea, Cat No. 104-152) according to the manufacturer’s instructions with slight modifications. Extracted gDNA was kept at -20°C until further analyses. PCR was performed on a thermocycler (Peqlab, Germany). LCO-1490 (GGT CAA CAA ATC ATA AAG ATA TTG G) and HCO-2148 (TAA ACT TCA GGG TGA CCA AAA AAT CA) were used to obtain partial sequence of Mt-CO1 gene. PCR conditions were: pre-denaturing for 3 min at 94°C; 35 cycles of 1 min at 94°C, 1 min at 55°C and 1 min at 72°C and a final extension step for 4 min at 72°C PCR reaction had a total volume of 20 μℓ containing 0.5 mM of each primer and 1 μℓ of gDNA in the premix (Intron, Korea). A total OF 196 individuals of P. tenebrifer were tested for this genetic study.

Data analyses

Resultant CO1 sequences were aligned and analyzed initially on MEGA (Tamura et al ., 2013; Thompson et al ., 1994). Haplotype frequency was determined using DnaSP version 5 (Librado & Rozas, 2009). Genetic distance and diversity among haplotypes were calculated after selecting the best fit model for haplotypes on MEGA. Maximum likelihood was adopted to infer phylogenetic relationship of insects within the family using 1000 boot strapping on MEGA (Tamura et al ., 2013). Analysis of molecular variance was conducted to determine the population genetic structure with 1000 permutations among regions including Fst statistics on Arlequin (ver. 3.5.1.2) (Excoffier & Lischer, 2010). Additionally, neutrality tests were performed to determine whether the gene was subjected to a neutral evolution model Arlequin (ver. 3.5.1.2) (Excoffier & Lischer, 2010; Tajima, 1989). All other sequences were retrieved from GenBank.

Competition over resources

To test whether there was a potential conflict between the two insects, we observed contact interactions between adults of both insects. In this study, we were unable to examine larval stage competition.

Results and Discussion

Distribution

Results showed that these insects were distributed nationwide. Both insects were located on or near food wastes, organic wastes from various farm lands, and manures from cattle sheds (Fig. 1). Contrary to P. tenebrifer , BSF was found in eight localities (Table 1). This might mean that the introduced BSF is still in its expansion process. We were unable to estimate local population sizes of the two in this study.

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Fig. 1.
Typical types of habitats of P. tenebrifer A: Food waste trap (Seocheon), B: Agro waste (Seongnam), C: Chicken coop (Cheongju), D: Manure (Cheongyang).
PNIE3-f1.jpg
Genetic analysis of partial CO1 gene

Results showed that there was no genetic isolation among seven regions (Fst: 0.01935, p > 0.05). The genetic diversity of the insect can be explained by the diversity within populations (Table 2). In fact, the insect has very robust wings and flies actively. Therefore, it may fly or spread long distance and exchange genes in a frequent manner. Two neutrality tests showed negative values with opposite statistical values (Table 3). It is too early to conclude that the population has expanded recently. Further study is needed to reach conclusion on its population size change. Compared to P. tenebrifer , BSF showed a remarkably lower genetic diversity (Table 4). This indicates that the insect might have experienced a genetic bottleneck in the introduction period, although there is no conclusive evidence (Park et al ., 2017).

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Table 2.
Genetic differences among regions (Fst)
Source of variation d.f. Sum of squares Variance components Percentage of variation
Among 7 groups 6 5.721 0.00071Va 0.09
Among populations within groups 15 13.94 0.01502Vb 1.85
Within populations 174 138.681 0.79702Vc 98.07
Total 195 158.342 0.81274
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Table 3.
Neutrality test for population expansion
Neutrality Mean±s.d.
Tajima's D No. samples 8.9±1.6
S 5.59±2.19
Pi 1.59±0.51
Tajima's D -0.91147±0.60203
Tajima's D p-value <0.1
Fu's Fs No. alleles 5.5±1.3
Theta pi 1.59308±0.50839
Exp. No. of alleles 3.40424±0.57807
FS -2.2768 ± 1.21631
FS p-value <0.05
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Table 4.
Comparison between P. tenebrifer and BSF
P. tenebrifer H. illucens
No. tested 245 196
No. haplotypes 10 46
Genetic diversity low high
Phylogenetic status of P. tenebrifer

Results of phylogenetic analysis showed that P. tenebrifer was grouped with P. trivittatus , a Stratyiomidae sp. and H. illucens (Fig. 2). This was unexpected since H. illucens was placed as an outgroup. This result may reflect that partial gene sequence is not appropriate to resolve the phylogenetic status of this taxa. More sequence data are needed to further understand their evolutionary relationship.

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Fig. 2.
Phylogenetic relationships of P. tenebrifer with other Stratiomyid insects
PNIE3-f2.jpg
Competition over the resources

We initially hypothesized that the exotic BSF might encounter severe competition with the endemic P. tenebrifer over resources (Park et al ., 2017). Although both insects frequently flied over organic matters, there was no detectable competition between the two. Instead, intraspecific interaction, flight interruption, was routinely observed, especially for P. tenebrifer adults. This indicates that the introduced BSF might not have experienced any barrier in its settling period. Whether BSF competitively exclude the endemic P. tenebrifer in the future remains unknown.

In the field, P. tenebrifer should play a pivotal role in decomposing organic wastes. In the future, its ability to decomposition should be assessed. Recently, larvae of the insect have been tested as a food source for pigs (Ao & Kim, 2019). Therefore, this insect should be taken care of for future insect industry. This also will help protect invaluable genetic resources. In conclusion, this study shows that P. tenebrifer inhabits nationwide and that its genetic diversity is higher than that of BSF. There may be no interruption between these two insects. However, this needs to be elaborately examined in the future.

Acknowledgments

The authors declare that they have no competing interests.

Acknowledgement

Conflict of Interest

The authors declare that they have no competing interests.

References

1 

AoX., KimI.H. (2019). Effects of dietary dried mealworm (Ptecticus tenebrifer) larvae on growth performance and nutrient digestibility in weaning pigs. Livestock Science, doi:10.1016/j.livs-ci.2019.09.031, 230, 103815.

2 

BlodauC. (2002). Carbon cycling in peatlands – A review of processes and controls. Environmental Reviews, doi:10.1139/a02-004, 10, 111-134.

3 

ExcoffierL., LischerH.E.L. (2010). Arlequin suite ver 3.5: A new series of programs to perform population genetics analyses under Linux and Windows. Molecular Ecology Resources , doi: 10.1111/j.1755-0998.2010.02847.x , 10, 564-567.

4 

KimJ.G., ChoiY.C., ChoiJ.Y., KimW.T., JeongG., ParkK.H. (2008). Ecology of the black soldier fly, Hermetia illucens (Diptera, Stratiomyidae) in Korea. Korean Journal of Applied Entomology, doi:10.5656/KSAE.2008.47.4.337, 47, 337-343.

5 

LibradoP., RozasJ. (2009). DnaSP v5: A software for comprehensive analysis of DNA polymorphism data. Bioinformatics, doi:10.1093/bioinfor-matics/btp187, 25, 1451-1452.

6 

LordW.D., GoffM.L., AdkinsT.R., HaskellN.H. (1994). The black soldier fly Hermetia illucens (Diptera, Stratiomyidae) as a potential measure of human postmortem interval, observations and case histories. Journal of Forensic Sciences, 39, 215-222.

7 

MoorheadD., LashermesG., BertrandI. (2014). Interacting microbe and litter quality controls on litter decomposition: a modeling analysis . PloS One, doi:10.1371/journal.pone.0108769, 9, e108769.

8 

NielsenU.N., AyresE., WallD.H., BardgettR.D. (2011). Soil biodiversity and carbon cycling: a review and synthesis of studies examining diversity–function relationships. European Journal of Soil Science, doi:10.1111/j.1365-2389.2010.01314.x, 62, 105-116.

9 

PaekR.B., HwangJ.M., JungK.S., KimT.W., Kim M.C., LeeY.J. (Eds.). (2010). Seoul: Nature & Ecology. Checklist of Korean insects.

10 

ParkS., ChoiH., ChoiJ.Y., JeongG. (2017). Popula-tion structure of the exotic black soldier fly, Hermetia illucens (Diptera: Stratiomyidae) in Korea . Korean Journal of Environmental Ecology , doi:10.13047/KJEE.2017.31.6.520, 31, 520-528.

11 

RozkošnýR., KovacD. (2000). A revision of the Ptecticus tenebrifer species group (Insecta: Diptera: Stratiomyidae) . Raffles Bulletin of Zoology, 48, 103-110.

12 

SheppardD.C., NewtonG.L., ThompsonS.A., SavageS. (1994). A value added manure management system using the black soldier fly. Bioresource Technology, doi:10.1016/0960-8524(94)90102-3, 50, 275-279.

13 

Tajima F. Statistical method for testing the neutral mutation hypothesis by DNA polyhomrphism . Genetics123, 585-595 , - .

14 

Tamura K , Stecher G , Peterson D , Filipski A , Kumar S. MEGA6: Molecular evolutionary genetics analysis Version 6 . 0. Molecular Biology and Evolution30, 2725-2729 , https://doi.org/ 10.1093/molbev/mst197 .

15 

Thompson J.D , Higgins D.G , Gibson T.J. CLUSTAL W: improving the sensitivity of progres-sive multiple sequence alignments through sequence weighting, position specific gap penalties and weight matrix choice . Nucleic Acids Research22, 4673-4680 , https://doi.org/ 10.1093/nar/22.22.4673 .