Research

Doctoral project:

Molecular characterization of the Drosophila responses towards nematodes

Supervisor: Prof. Ulrich Theopold, Stockholm University, Sweden.

Host-pathogen interactions with bacteria and fungi were relatively well characterized in Drosophila compared to other pathogens. However, how other pathogens, for instance, entomopathogenic nematodes interact with the host (Drosophila) has yet to be elucidated. Entomopathogenic nematodes (EPN) are parasitic worms, which can infect insects and kill them. Studies estimate that around 3 billion people are infected by nematodes worldwide, causing diseases which could lead to death. Besides human health, nematodes have devastating consequences on livestock and in agriculture.

To obtain a complete picture of Drosophila response to EPN, we performed a genome-wide transcriptional analysis of the Drosophila larvae infected by EPN. We compared the transcriptome profile between infected and non-infected larvae and later we did a functional analysis by using RNAi lines and mutants for candidate genes identified in the array (Arefin et al., 2014 Link). Later, cellular immunity was investigated towards nematodes and in danger situation (please see for details, Arefin et al., 2015 Link). In order to investigate the immune reactions in cancer, three Drosophila leukemia models were generated for studying immune and pathophysiological consequences (Arefin et al., 2017 Link). Based on the data from the Theopold lab and others, we further studied 3 clotting factors for their sub-cellular localisation. It shedded some light on clot formation on how and when these factors involve in it. Of note, two of the proteins follow non-classical secretion since they lack signal peptides (Schmid et al., 2019 Link).

The model system used during PhD studies

The following tripartite system was used during my PhD studies. For several reasons, this is a beautiful model system. For example, one can study host-pathogen interaction at cellular and molecular level. This is a natural infection model, which means you do not need any means to infect the Drosophila larvae. Nematodes find their way to enter the larvae if nematodes and Drosophila are put together, which is in contrast to many bacterial infection (they need injection to enter the larvae). In addition, pathogen (nematode) inflicted wound response and blood coagulation can be studied.

Furthermore, host defense can be investigated to explore for additional immune pathways.

This system consists of three elements, Drosophila larvae, nematodes (Heterorhabditis bacteriophora) and their mutualistic bacteria (Photorhabdus luminesence). Once nematodes entered into larvae by penetrating cuticle (skin), they regurgitate their gut bacteria which produce toxins to kill the larvae. Then nematodes feeds on bacteria and digested larval tissues, they replicates inside the cadaver for 2-3 generations. Once the food resources are finished, they come out of the cadaver and seek for new hosts. (Anti-clockwise): Right panel- nematode infected larva, arrow indicates nematodes inside the larva. Left upper panel: Nematodes and their gut bacteria. Left lower panel: Nematodes in higher magnification. Inset: GFP labeled bacteria. Lower middle panel: Nematode infected larval hemolymph (blood) under microscope where circular cells are larval blood cells, and GFP labeled bacteria (green).

Courtesy: Robert Markus, a former postdoc in Prof. Ulrich Theopold lab, who brilliantly captured the tripartite system and allowed me to use these images.

Some snapshots of my PhD research: From the published papers (below)

Hemocytes (blood cells) recruitment upon infection

We have reproducibly infected Drosophila larvae by nematodes which are indicated as hemocytes recruitment, melanized wound and caused septicaemia.

h, i Sessile hemocytes (red) in hml-GAL4/UAS-RFP larva; noninfected larva shows hemocyte clusters (h) and after infection the clusters disperse (j). The punctate signal corresponds to hemocytes. i Noninfected larva seen in the fluorescence channel. To trace the infection the larva was infected with nematodes harboring GFP-expressing bacteria (green). Arrows indicate the wound (j, k) and arrowheads show bacteria (k). Inset shows bacteria at a higher magnification (all infected samples were analyzed 16 h after infection with nematodes). Inf./Non-inf. = Infected/noninfected.

Arefin B*, Kucerova L* et al., 2014 Link

Apoptosis in plasmatocytes and crystal cells triggers lamellocyte differentiation

One of the unexpected phenotypes we observed when we depleted plasmatocytes and crystal cells, the non-existent lamellocyte appeared in huge numbers. Normally, plasmatocytes and crystal cells consitute the hemocyte population in naïve larvae.

Hemocyte preparations from 3rd instar larvae were analyzed under the epi-fluorescence microscope. (A-I) Both Hid- and Grim-expressing samples showed massive lamellocyte differentiation (F and I) whereas control samples showed none (C). In addition when apoptotic cell bodies were included in the counts, an increase in counts was observed (using DAPI staining) in Hid- and Grim-expressing samples (D and G) compared to controls (A). HFP- w; hml (Δ)-GAL4 UAS-eGFP

Arefin B et al., 2015 Link

Phagocytosis is not impaired in leukemic lines

(A and B) Ex-vivo phagocytosis of Texas Red–conjugated E. coli (K-12 strain). A representative image of the HFP/w and HRS-leukemia lines is shown after phagocytosis. The red arrow indicates phagocytosed E. coli. HFP- w; hml (Δ)-GAL4 UAS-eGFP, and HRS- HFP/UAS-Scrib.RNAi; UAS-Ras85Dv12.

Arefin B et al., 2017 Link

FIMTrack: A method to capture Drosophila larval locomotion

while they encounter nematodes

FIMTrack

Kunc M, Arefin B et al, 2017 Link

How the Drosophila larval trajectory look like after recording.

FIMTrack: During preliminary experiments, we observed that Drosophila larvae moved from a food source onto a water-soaked filter paper (without nematodes). In contrast, if the filter paper was soaked in a solution containing nematodes (H. bacteriophora), fewer larvae left the food. This observation inspired us to establish methods that allowed us to quantitatively assess food-searching behavior in the presence and absence of nematodes. (B) We placed larvae in the middle of an arena where they could choose to travel to the food source via either nematode-free or nematode-containing area. (C) In another approach, larvae were placed in a circular fashion on an agarose based crawling platform that was covered with water containing EPNs, and we assessed the chemotactic behavior that required larvae to reach a food source in the center of the platform. (D) How the Drosophila larval trajectory look like after recording.

Three clotting factors: How and when they function during clot formation

Schmid MR*, Dziedziech A*, Arefin B* et al., 2019 Link

Deposited crystals release from the Drosophila crystal cells, a type of blood cell present in Drosophila which contributes to clotting .

Scimid MR*, Dziedziech A*, Arefin B* et al., 2019 Link

Methodology (PhD studies):

Microarray data analysis, standard molecular biology techniques including gateway cloning technology, diverse Drosophila genetics, making transgenic lines for the candidates genes, nematode infections, Drosophila blood cell preparations, Ex-vivo phagocytosis, Drosophila cell culture, clotting methods, Drosophila tissue dissections, extensive microscopy (stereo, epi-fluorescence, Cell Observer, confocal and TIRF microscopy), bioinformatics, FIM imaging and FIMtrack (Drosophila larval locomotion behavior with or without entomopathogenic nematodes) Different radiations (UVA, UVC and gamma radiation on Drosophila larvae and on primary Drosophila blood cell culture).

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