Improving Maggot Therapy: Mass Rearing And Molecular Identication of Lucilia Sericata (Meigen, 1826) Larvae In Maggotarium, School of Health, Shiraz

Background: Lucilia sericata as a member of the family Caliphoridae has a complete metamorphosis. They feed on on necrotic and livingtissues as necrophage species. Sterile larvae of this species has been utilized to heal wounds for decades. The aim of this study were to establish the breeding and identifying of the L.sericata species based on morphological and molecular techniques. Freshly harvested grown under standard conditions in the maggotarium of Health School , Shiraz of. They were screened using conventional morphology, then Primarily, different parameters related to larvae were measured morphologically. Subsequently, DNA was extracted and molecular marker of cytochrome C oxidase (co1) was amplied using PCR assay and sequence data were used for molecular and phylogenetic analysis. Result: In this study, 50 samples which grown collected from maggotarium were identied as L. sericata using morphological and molecular methods. This species was placed in a separate clade of the phylogenetic tree based on COI nucleotide sequences of different species and has a phylogenetic similarity to Lucilia purpurascens species of ies. Conclusion: Larval therapy especially by Lucilia sericata is a promising strategy in wound healing. Due to the importance of larval species in this technique, having an accurate knowledge of aplicable species leads to a proper larval therapy. Increasing in resistance of wounds to antibiotics has led to the use of maggot therapy in the past. Larval therapy is a low-cost, non-surgical way to remove dead tissue (predecessors and necrosis) in chronic wounds and prevents excessive soft tissue damage and infection from reaching the underlying tissues and bones (osteomyelitis). Maggot therapy is a promising way to prevent amputation, especially in people with diabetes.


Introduction
The Caliphoridae family is one of the largest and most diverse families of ies. Up to 1500 species in 97 genera of this family have been identi ed so far (1)(2)(3)(4), which are distributed in neotropical regions and a large number of them in Africa and southern Europe (5). From Iran, 20 species of three subspecies, Calliphorinae, Chrysomyinae, and Luciliinae have been reported so far (6).
This family have been considered by many researchers in the eld of medical sciences, forensic medicine, veterinary medicine, pharmaceutical and biomedicine due to the possibility of easy and fast breeding, ability and growth of this insect on cheap materials, the rapid output of the target product, the non-toxicity of the target product, and the stability of the physiological parameters of the insect during the biotechnology process (7). Also, many studies are still being done on this family (8-10).
Lucilia sericata belongs to the Calliphoridae family (8). This species is mostly distributed in hot and humid regions of the world, although they are also present in areas with dry climates (11). It is a domesticated species and has a high adaptability (12). Lucilia sericata has a complete metamorphosis (Holometaboluse). Life cycle of this species includes egg stages, three larval stages (1, 2, and 3), pupa, and adult. The eggs are placed in clusters of about 2000-3000 on animal carcasses and excrement.
About 18 to 24 hours later, they will be opened and entered the larval stage. The larvae are milky, conical in shape with anterior and posterior spirals, and after 4 to 5 days, they turn into pupae. Pupae usually have a hard, brown and black coating, and nally, after 6 to 14 days, they turn into adult ies (13). These ies are capable of mass production in the standard environment and conditions of the insectarium.
Adults are mostly metallic green. The antenna is tripoded and aristate, and the RS ri e is branched out twice. Frontal sutures are well visible and calypters have been grown well in them. The average lifespan of an adult is usually 7 days (14). One of the nutritional habits is that it feeds on corpse and dead tissues; therefore, it has a special place in medical sciences, veterinary medicine, and forensic medicine (9,12,15,16). One of the useful and practical uses of the larvae of L. sericata is in the eld of Maggot therapy (17). Maggot therapy is a useful, effective and controlled method using sterile larvae of L. sericata to treat a variety of acute and chronic wounds (18). This method was approved by the US FAD in 2004 with the approval of K033391 (19). This method is also used to treat wounds, which are caused by bed sores, traumatic skin injuries, and burns.
It is also effective in chronic diabetic ulcers, osteomyelitis, and ulcers created after cancer surgery (20,21). Chronic wounds, which are primarily associated with severe and prolonged in ammation, stop the proliferation of cells and cause the extracellular matrix to be regenerated incompletely. Excessive ECM overexpression temporarily and its ineffective removal from the wound surface usually stop the healing of chronic wounds (22). Necrotic tissue is also a good place for the accumulation of various pathogens and the production of bio lms, which in turn cause the infection of dead cells and wounds. (23,24).
Traditional treatments for chronic wounds include surgery, enzymatic debridement, and rinsing (25). The most important drawbacks of these methods are pain, mechanical damage to healthy tissues, and human error. Maggot therapy can be used as an alternative, classical and controlled method instead of the mentioned methods to treat wounds (26,27). Important advantages of this method include cleaning the wound surface, disinfecting and accelerating wound healing (28).The mechanical movement of larvae on the wound and the production and secretion of various digestive enzymes by them are two very important factors in accelerating wound healing (29). Maggot excretions / secretions contain a variety of digestive enzymes, including carboxypeptase A, B (carboxypeptidase A, B), collagenase-amino peptidase (aminopeptidases collagenases), aspirin and serine protease (aspart), trypsin-and chymotrypsin-like ones., and metalloproteases, which are active in a wide range of pH. In larval secretions, by breaking down bronectin and converting it into biological active components, broblasts are multiplied and migrated (12,30). Today, with the advancement of biotechnology, new research is being done on the use of ies products in the pharmaceutical industry, and the production of recombinant proteins from some Lucilia members is being developed and evaluated (31,32). As brie y mentioned above, due to the importance of this species in various elds and sciences, accurate identi cation of members of this subfamily requires the necessary accuracy and precision. Blow ies have a wide variety of species and their morphological diagnosis is confusing and di cult (31), and due to the similarity of the species, errors in morphological diagnosis may occur. However, the accurate identi cation of this species of insect can be done by experts and experienced people (33). Due to the importance of accurate identi cation of species, the use of molecular methods to accurately identify and con rm morphological methods is recommended (34). In general, for molecular identi cation, a variety of nucleotide and mitochondrial loci and different gene markers are used to accurately identify species. The CO1 Cytochrome c oxidase has a high degree of nucleotide diversity and it is considered by many specialists to identify species. It was rst proposed by Harbor and it is well established that this barcode can be used as a suitable marker for identi cation. Different species of humans, birds, and insects has been considered by many biotechnologists and molecular entomologists as a marker that has been successful in differentiating two species (31). We also decided to use this method to identify the species of Lucilia sericata in the maggotarium of Shiraz School of Health due to the speci city of this marker.

Material And Methods
The y species The rst L. sericata was bought from Shiraz Wound healing and Biotherapy center.

Breading:
The larvae were transferred to cages (45×45×45) in the Shiraz health school Maggotarium. There were chicken liver and water-sugar inside the cages for ies feeding. The larvae were placed on chicken liver as well as a special container for laying eggs. The eggs were transferred to a new cage. They were exposed to a standard condition; 12-h light/dark cycle with humidity of 40-60% at 16-25°C.
Collection and identi cation: 50 samples of adult larvae were collected from the cage. ( Fig:1), identi ed using the morphological identi cation key and then, they were con rmed by molecular methods.

Dna Extraction
The genomic DNA of samples was extracted using the instructions of the AccuPrep® Genomic DNA Extraction Kit -Bioneer.

Primer Design
The mRNA sequences of cytochrome oxidase subunit 1 (COI) gene, partial cds, and mitochondrial genes of ies were obtained from the NCBI base gene bank. Then, obtained sequences were entered into the Gene Runner software separately and copied in the MEGA 6.0 software. In this application, they were aligned and based on the conserve points between the compared genes, forward and reverse primers were designed using Oligo7.0 software program and Gene Runner version 4.0 .
Gene speci c forward and reverse primers (GSP) were blasted using NCBI (https://blast.ncbi.nlm.nih.gov/Blast.cgi). The sequence of designed primers is shown in Table 1.

Polymerase Chain Reaction
Using Gene runner and Mega 6 software, forward and reverse primers were designed for CO1 barcode. Primer sequences can be seen in Table 1. Co1 fragment was ampli ed using pair primer. The PCR reaction volume was 20 µl and contained 1 µl cDNA, 1 µl of pair primer, 10 µl master mix and 7 µl water.
The ampli cation program included initial denaturation at 95°C for 5 min followed by 35 cycles, including denaturation at 95°C for 30 s, annealing at 59°C for 30 s, extension at 72°C for 80s, and an additional nal extension at 72°C for 10 min.

Sequencing
The products of PCR were puri ed using Thermo Scienti c Gene JET Gel Extraction Kit. The samples were sent to Phishgam Biotech Company in Tehran for sequencing.

Phylogenetic Tree
Phylogenetic tree based on COI nucleotide sequences of ies from several worldwide geographical areas, including available data from Gene Bank was constructed by neighbor joining (NJ) method with the Kimura's 2-parmeter model implemented in the MEGA_ version 6.1, and the trees were tested by 1000 bootstrap replicates.

Morphological Identi cation
In the present study, samples were collected from Maggotarium and identi ed as Lucilia sericata using morphological identi cation key.

Molecular Identi cation
The expected band of 300 bp appeared on the gel (Fig:2). The results of the sequencing (Gel extraction) were blasted on NCBI site, and all samples were identi ed as Lucilia Sericata.

Phylogenetic Analysis
In order to analyze and compare, the phylogenetic tree based on COI nucleotide sequences of different species of ies, their information was registered in the Gene Bank, which was constructed by neighbor joining (NJ) method with the Kimura's 2-parmeter model implemented in the MEGA_ version 6.1, and they were tested by 1000 bootstrap replicates (Fig. 3). In the present study, different species such as Lucilia purpurascens, L. papuensis, L. porphyrina, L. illustris, L.sericata, chrysomya ru facies, Tabanus taeniola, Musca domistica, and Calliphora vicina were compared with our study. Sarcophaga utilis was used as the outgroup of the phylogenetic tree. According to the tree, current study and Lucilia purpurascens the same group and there was a closer relationship between the two species (73 nucliotide). L. papuensis and chrysomya ru facies are closely related and Tabanus taeniola Will be placed in the next close relationship. L. illustris, L. porphyrina, and Calliphora vicina are also similar and they were placed in a separate subgroup.
L. sericata was clearly separated from them.

Discussion
In the present study, Lucilia sericata was identi ed using morphological and molecular methods.
The aim of this study was to identify the morphological and molecular method for identifying Lucilia sericata, and this species was reared on a mass scale in maggotarium at Shiraz School of Health. This species is considered as a suitable model for medical and pharmaceutical research, including the discussion of Maggot therapy. The proteins in larval secretions are now being used both chemically and also in the production of recombinant proteins. Due to the importance of this issue, it is important to accurately identify this species in medical discussions and maggot therapy, which uses sterile larvae of this species to heal wounds.
Temperature and humidity are two important factors in the breeding and growth of insects, including Lucilia, which were raised in the present study with the optimum temperature and humidity of larvae. The larvae were fed using fresh chicken liver and sugar water. In a study, Tachibana ea al. tested arti cial diet primarily composed of whole milk powder, dried yeast, and wheat germ in order to compare the lucilia sericata larval diet and its effect on growth and development. They did not observe a signi cant difference between them on beef liver, although there was a small difference between arti cial feeding and longer growth period. Otherwise, there was no difference between the mortality rate and the pupal weight between the arti cial meal and the beef liver (35). Today, due to the development of societies and the increase in accidents and burns, as well as not following a proper diet among people and suffering from diabetes, the wounds, which are caused by these diseases and accidents are spreading. Also, due to the resistance of various bacteria on the wounds to antibiotics, larval therapy is used as a classic and modern method and a supplement to surgical methods. Therefore, accurate knowledge of Lucilia sericata and its use in larval therapy is very important. Sometimes, due to the similarity of some diagnostic characters, this species may be misidenti ed and the larval therapy may be failed.

Declarations Acknowledgments
In this study, all those who helped in the implementation of this project should be thanked and appreciated. The respected staff of Shiraz Larval Therapy Center, the respected experts of the Insectarium and the laboratory of Shiraz School of Health are thanked. This article is extracted from the rst part of the thesis of Ms. Masoumeh Bagheri, PhD student in Vector Biology and Control of Diseases, School of Health, Shiraz University of Medical Sciences, Shiraz, Iran.
Authors' contributions MB and HA wrote the main manuscript. MB and MSh performed the experiment. SM and AR reviewed the nal revision of the manuscript. All Authors read and approved the manuscript.

Funding
This experiment was funded by Shiraz University of Medical Sciences.

Availability of data and materials
The datasets used and analyzed during the present study are available from the corresponding author.   Phylogenetic tree based on CO1 sequences (Flies)