## 8 Concluding Remarks
\begin{table}
\begin{tabular}{c c c c c} \hline \hline
**Immunological Determinant** & & **Assay Characteristics** & **Comments** & **Reference** \\ \hline \multirow{4}{*}{Macrophage activation (ROS)} & \(\bullet\) & DNA Damage ELISA (8-OHdG) & Measures aspects of reactive oxygen & \multirow{4}{*}{[80]} \\  & \(\bullet\) & Lipid Peroxidation assay (MDA) & species (ROS) burst and regulatory & \multirow{4}{*}{[38]} \\  & \(\bullet\) & Catalase Fluorometric resorwin & enzymes (catalase and SOD) & \\  & assay & (MDA is biomarker of oxidative stress & \\  & & Cu/Zn-Superoxide Dismutase & and 8-OHdG is a biomarker of DNA & \\  & & (SOD) ELISA & oxidative damage) & \\ \hline \multirow{2}{*}{Coagulation assay (in vitro)} & \multirow{2}{*}{Fluorescent microscopy} & Haemolymph incubated with & \multirow{2}{*}{[38]} \\  & & & fluorescent-labelled bacteria & \\ \hline Cytoskeleton morphisms & \multirow{4}{*}{Fluorescent microscopy} & Examines haemocyte cytoskeleton with & \multirow{4}{*}{[60,80]} \\ (Phagocytosis mechanism and pseudopod formation) & & & fluorescent-labelled phalloidin & \\ \hline \multirow{4}{*}{Autophagy} & \(\bullet\) & LC3 immunofluorescence & \(\bullet\) & Measures the autophagosomal & \multirow{4}{*}{[80]} \\  & \(\bullet\) & cationic amphiphilic tracer & marker LC3 in haemocytes & \multirow{4}{*}{[80]} \\  & (CAT) assay & \(\bullet\) & CAT dye selectively labels & \\  & & & autophagic vacuoles & \\ \hline \multirow{4}{*}{Apoptosis} & \(\bullet\) & Caspase measurement & Caspase is measured using & \multirow{4}{*}{[81]} \\  & \(\bullet\) & Annexin V measurement by & carboxyfluorescein derivative of & \\  & & flow cytometry & VAD-FMK and annexin V is a marker of apoptosis & \\ \hline \multirow{2}{*}{Eicosanoid biosynthesis} & Measurement of eicosanoids (e.g., & \multirow{2}{*}{[81]} \\  & & prostaglandandins) by ELISA & & \\ \hline miRNA expression levels & Using an insect-specific miRNA & \multirow{2}{*}{Employs >2000 mRNA probes} & [66] \\  & Microarray & & \\ \hline \multirow{2}{*}{Epigenetic gene regulation} & Measures gene-associated methylation & \multirow{2}{*}{Extraction of genomic DNA, RNA and \multirow{2}{*}{[86]}} \\  & and global histone acetylation by & & histones. \\ \hline \hline \end{tabular} Abbreviations; MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolzoIUM bromide); RT-PCR (reverse transcription polymerase chain reaction); LC-MS (liquid chromatography–mass spectrometry); MS-MS (tandem mass spectrometry); 8-OHdG (8-hydroxy-2-deoxyguanosine); MDA (malondialdehydedehyde); VAD-FMK (valylalanyl aspartic acid fluoromethyl ketone).  
\end{table} 
Table 2: _Cont._be addressed is the lack of standardised larval growth conditions amongst suppliers [21], as differences in light source, temperature and diet could affect laboratory experimentation and variability of results [89; 90]. Another important feature is the observation that the haemocyte composition fluctuates during the larvae life cycle, highlighting the importance of the standardisation of the larvae stage in experiments to ensure the reproducibility of results. The mature or late larval stage (6 th or 7 th instar weighing 250-350 mg) lacking cuticle pigmentation is recommended, where plasmacytes and granulocytes predominate and a larvae number of 10-20 is recommended for each experimental treatment [21; 60]. Animal welfare and subsequent ethical considerations in biomedical research are constantly under revision. In April 2022, new legislation come into UK law with the Animal Welfare (Sentience) Act 2022, which extended new regulations to protect decapods (e.g., crabs) and cephalopods (e.g., octopus) [91]. The need for animal welfare and protection is evident to all those who work in research that necessitates animal models, but the restrictions mean that robust, reliable, and reproducible alternatives, such as \(G\). _mellonella_, must be established.