**7. Factors contributing to suitable infection model**

It was previously reported that in order to replicate the outcome of future studies it is important to optimize the lethal dosage selection and the route of inoculum [63]. It is suggested that the selection of microbial strain and gender of flies are two important factors which could potentially impact the findings of future research.

## **7.1 Route of inoculum**

*Animal Models in Medicine and Biology*

**5. Markers of infectious diseases**

**6.1 Negative geotaxis assay**

**6.2 Circadian rhythm**

the control/uninfected flies [60].

screening anti-microbial drugs in flies [59].

flies upon stress response generated due to DNA damage, P53 mediated apoptotic genes are regulated by Jak–STAT-MAPK [49]. The dP38 stimulation in flies triggers Unpraired gene (upd protein) a mammalian IL-6 homolog further activates Jak–STAT-*Turandots* pathway which increases tolerance towards the viral invasion [50]. The intrinsic to cell the Dicer2 a viral sensor protein mediates silencing through RNA-induced silencing complex (RISC) dependent RNAi production which inhibits viral components transcription and vago gene activation finally controls viral growth [51, 52]. The anti-viral RNAi are transported from one cell to another through canonical nano-tubes structures [53]. dERK pathway regulates viral infection of flies gut epithelial infection during orally challenge of arbovirus, Sindbis virus and vesicular stomatitis virus [54]. Despite of dynamic immune response against the viral infection viruses like Nora virus, Sigma virus (DmelSV), Drosophila C virus

(DCV), and Drosophila X virus (DXV) can cause fatality in flies [55].

further reveal the effect of drugs during an ongoing pathogenesis [58].

**6. Behavioral and physiological characterization of infected flies**

Negative geotaxis assay serves the purpose to manifest ongoing pathogenesis inside the live model. It was demonstrated previously that infected flies display significantly lesser motility than healthy flies when exposed under bright light. It could be considered as an important parameter to explain drug efficacy while

Circadian rhythm in flies was studied, the genes timeless or period controls the circadian rhythm of activity-sleep cycle during day-night respectively. It has been observed that infected flies exhibit interrupted circadian control of locomotion thus flies with this deficit shows restlessness at the same time gets lesser sleep than normal flies. The behavioral changes could also be studied in infected flies beside

Wasting is commonly seen physiological changes associated with prolonged diseased condition in humans. Wasting is a common symptom in HIV/AIDS,

In the recent decades extensive research has been conducted to understand the regulation of immune system in *Drosophila melanogaster*. Using techniques like genome wide screens, *Drosophila* S2 cell line in-vitro models and tissue specific loss of function mutation in transgenic GAL4/UAS fly allows studying selective pathways of immune response [56]. Up-regulation of antimicrobial peptides (AMP) in flies during bacterial and fungal infection was frequently observed, these six AMP genes expression level could be analyzed in flies [42, 57]. ROS level in flies trigger several pathways responsible for tolerance (cell survival) and apoptosis (cell death) could be assayed in virally infected flies [49, 50]. Rescue of diseased transgenic flies upon feeding of desired drug could reveal drug efficacy [56]. Survival of flies would

**112**

**6.3 Wasting**

There are two prime techniques for inducing infection in flies, primarily by feeding the flies with the microbes secondly by pricking micro needles dipped in bacterial liquid (inoculum) into fly's abdomen or thorax [62, 63]. Flies could be pricked in the abdomen with micro-needle dipped in the microbial solution, known amount can be useful in pharmacodynamic as well as pharmacokinetic studies [64].

#### **7.2 Flies gender selection**

Selecting gender should be considered strictly, few studies do not prefer to report the reason behind choosing the gender male/female type. In a study with *Vibrio cholera* infection narrated that female flies survived approximately 24 h longer than male flies [65].
