## 5 Harvesting of Antimicrobial Peptides from _Hermentia illucens_  
_Hermentia illucens_ are present ubiquitously, and since it survives in highly contaminated environment, it is capable of producing a huge spectrum of antimicrobial peptides. _H. illucens_ consume cheaper feed and also grow faster, which is a highly recommended attribute for AMP isolation [50,51]. Defensins, cecropin, attacin and lysosomes are considered as representative antimicrobial peptides of _H. illucens_[10]. The mechanism of harvesting AMP from insects involves some important fundamentals, as discussed below. General steps in AMP harvesting are mentioned in the flow chart (Figure 6).  
1. Larva collection and rearing:  
In the process of insect formation from larva, several growth and development stages occur, which are usually known as instar. There is no specification for considering any specific instar larva for the AMP collection. However, in a study, the lowest transcription level was observed during first-instar larval stages and the highest transcription level during the fifth-instar larval stage [52]. At the fifth stage, growth is completed, and metamorphosis is initiated [53]. Therefore, considering fifth-instar larva will be beneficial in a higher rate of synthesis of peptides. If the larva is to be used as a feed, prepupa is better than larva because of its high chitin content [54]. Rearing of larva varies from study to study, depending on the criteria of the research. In an investigation, researchers observed that aqueous extracts of black soldier fly larvae fed a cellulose diet and protein-rich diet showed antimicrobial activity against Gram-negative bacteria, while larvae fed chitin, cellulose, bacteria and plant oil had higher inhibitory activity against Gram-positive bacteria [33]. Chloroform extract of larva showed the highest inhibitory activity when fed lignin, bacteria and plant oil diets. In contrast, in a report by [55], larvae were starved after inoculating the _Lactobacillus sp._ to study the antimicrobial activity of _H. illucens_ extract. At the industrial level, cottonseed press cake is a good choice as feed for _H. illucens_ due to its sustainability and cheaper cost [56].  
2. Incorporation of microorganism into larvae:  
Microorganisms are injected into the body of a larva or haemolymph using a needle. Inoculated microorganisms should be in the stationary phase because natural microbial infection in insects occurs in the stationary phase [12]. The microorganism is injected either to isolate and analyse the characteristics of the AMP formed for that specific microorganism  
Figure 6: The general methodology for harvesting of AMP from _H. illucens._or to examine the influence of immunisation on the antimicrobial activity towards different microorganisms. Several reports have claimed the antimicrobial activity of AMPs harvested from _H. illucens_, among which a few are mentioned in Table 3. In a study, AMPs from _H. illucens_ were purified and investigated. AMPs were harvested from three samples: larvae that were immunised during the last instar stage, unimmunised larvae and mutilated larvae. From all the samples, haemolymph was collected and tested against _E.coli_ and methicillin-resistant Staphylococcus aureus. Among all the samples, larvae that were immunised during the last instar stage showed the highest antimicrobial activity, while the mutilated group showed the least [51]. [PERSON] observed that _M. luteus_-immunised haemolymph of _H. illucens_ larvae showed inhibitory activity against Gram-positive bacteria, whereas _E.coli_-immunised larvae showed activity against Gram-positive and Gram-negative bacteria. Additionally, it was concluded that synthesis of AMPs vary depending on the bacteria used for the immune challenge [57]. Usually, when AMPs are treated with enzymes, such as trypsin or chymotrypsin, it results in a loss of antimicrobial activity. However, when _H. illucens_ are immunised with L. casei and given the same treatment, they remain unaffected [55].