Drosophila - Science topic

My apologies for not noticing that you are using cDNA. It’s a large size transcript so you may need to optimize the cDNA synthesis parameters (longer synthesis time).

Or, try for a tissue or time points predicted to have enriched expression of your gene.

do your primers work in gDNA? Any possible chance of alternative splicing?

Hi, what exactly do you need? A matrix from excel sheet. Then, simply read excel file using

as.matrix(readxl::read_excel(your_file_location))

function. You need to remove few columns and then matched the columns to meta data.

and IF degPatterns() function is not working properly, then you may need to clean and re-transform your data.

Hey Melville B Vaughan That is a good insight. Although, I did not freeze the cell and I only had to use that frozen culture. But I will keep your points in mind when I make frozen stock!

A previous labmate suggested that DMSO removal is not required for s2 cells and hence I am letting them grow. Let's see how the culture proceeds. If it is successful, I will update it here on the thread!

Hi Roshima, thank you for your response! I'm actually using MAKER since Drosophila is eukaryotic. And you're right, MAKER takes a very long time to run. I got 1 species down and many more to go. I'm happy to answer any questions you might have for MAKER as it's a very complicated process.

You can get cell culture flasks that have filters built into the caps (eg. Corning). You can also place the plates in a sealed box that has a filter inserted and sealed into the lid. Overall try and keep the work area as clean as you can and give the bottle a good spray with disinfectant before handling in a laminar flow hood.

Neuronal silencing, also known as gene knockdown, is a technique used to inactivate the expression of specific genes in order to study their function. In Drosophila melanogaster, one of the most common techniques for neuronal silencing is the use of RNA interference (RNAi) through the injection of double-stranded RNA (dsRNA) into pupae. Here is a general overview of how to test neuronal silencing in Drosophila pupae:

Hello,

When performing the APEX2-Gold method on animals, there are a few things to consider:

Overall, the APEX2-Gold method is a powerful tool for detecting and analyzing APEX2-tagged proteins in electron microscopy. With proper technique and high-quality reagents, you should be able to detect a strong signal in your samples.

Regards/

Hi Máté Nászai ;

Thank you for sharing your ideas. It is very annoying that Excel has uncorrected issues. I've tried hard to change something in Excel to get clear results. I suppose I have found the reason for my corrupted data.

My issue was much simpler than I thought. It was all about language settings! Seems funny; however, region-dependent date/time/number formatting changes my data because I use Excel in Turkish language and with its settings. What I did was change the decimal and thousand separators to the EN version. It worked!

I appreciate your help and your other suggestions. I am happy not to do analysis in R or other since I haven't used them before :)

Thank you for your time.

Best,

Dilara

Sodium hydroxide is a potent CO2 absorbent and there are commercial products based upon alkaline materials with large surface areas. To manage the levels you may need an external circulation that you control through using a flow controller

w1118 and w1 are loss of function alleles (different mutations). mini-white is a short version of the gene that is used as a marker to detect if a transformation of a sequence (vector) into the genome was successful (in white mutant background). The w mutant fly has the no more white eyes (w1118; mini-white+ = orange or red)

Working with log-transformed intensity (or concentration) data is very convenient. Protein expression is approximately log-normal, so it's approximately normal at the log scale, where you can use the standard toolbox for analysis (i.e. linear models like t-tests, ANOVA, regression, ANCOVA etc).

I think visualization is also better done with log concentrations or log fold-changes. The resulting picture (and possibly the interpretation) should not depend on the perspective, that is, which group is chose as the "reference" and wich as the "experimental" condition. To give an example: you study the expression of protein X in males and in females. The fold-change male/female is 5 (wow what a drastic increase!), so the fold change female/male is 0.2 (eh, well, yes, some kind of reduction). The numbers look very differently impressive althought the express the very same difference (effect, ratio if you like). The (base-2-)logarithms are +2.3 and -2.3: same number, opposite sign. This is (imho) easier to understand and faithfully reflects the symmetry of the problem.

Using z-scores serves a different purpose. Here, z-scores are calculed from the log intensities, for the reasons explained above. The z-scores more closely resemble what a t-test or ANOVA "sees" from the data, as here the differences (from the mean) are related to the variability (the standard deviation). What gets lost is the information about the base intensity (expression level), what is often less interesting than the difference in expession levels between samples and groups.

try this one, it would work for Target explorer software, alternatively you may want to try CT finder and DNA star.

If the genome has been annotated, you can likely find at least predicted genes/transcripts using NCBI Gene.

For example, this is the transcript predicted to be PKA https://www.ncbi.nlm.nih.gov/gene/?term=XM_030165592.2

You can then find the linked protein sequence in the transcript entry:

The M. sexta genome is also available on the UCSC genome browser, which helps visualize homology with other organisms (i.e. D. melanogaster): https://genome.ucsc.edu/cgi-bin/hgTracks?db=hub_2790993_GCF_014839805.1&lastVirtModeType=default&lastVirtModeExtraState=&virtModeType=default&virtMode=0&nonVirtPosition=&position=NC_051137.1%3A10146384%2D10157377&hgsid=1339887779_hUFhAZiDsqmTWUDn8AVpWwXAnFFx

If you need to find something that isn't already annotated/predicted you can use Protein BLAST to look for similar proteins in a specific organism using a known/presumed homolog from a better annotated organism.

@jagdishpaithankar thank you bhaiyya

Dear Thomas: I have used both MT and Sart. balances, and MT is so accurate, however, sartoruis is so good, and you can use it for these small and minute masses. You need a well levelled plane or bench and a quite room to get excellent results. Regards.

Hello Jillian Porter

RNase treatment is not required when using DAPI. DAPI is fairly specific to DNA. But for your information DAPI does bind to RNA but with lower affinity and with less intense fluorescence. When bound to RNA evidently through AU-selective intercalation, the emission is about 5-fold weaker, and the excitation maximum is shifted into the green range.

Best.

Hi, Josef,

take an article about yeast.

Before staining with phalloidin fixed embryos for 30 min at room temperature in a 1:1 mix of heptane and 8% formaldehyde.

After fixation, hand-devitellinize embryos in PBS, block in 1% BSA/0.1% Triton in PBS for 30 min, incubate in 1 µgml−1 Alexa594–phalloidin for 30 min, wash three times for 15 min in PBS containing 0.1% Triton and analyse on the confocal microscope.

Thank you guys for your answer. I am actually looking for program that will do the initial recording not the analysis. I have already the camera and the software to analyse the behaviour. What I don't have is a freeware which allow me to do initial recording. Does CTrax do the recording too?

Thank you again,

C

Thank you, Arkajyoti, I was curious, whether they mate with Drosophila and give offspring. Because, they show mating behaviour to Drosophila, it is worth for searching, but I do not think they will give fertile offspring. Must search cytogenetic backround first. best wishes. Selcuk.

Neurons are often not very active (in particular ex vivo) under resting condition. I guess you don't ask why the cells don't fire at a potential of -50-55 mV (most likely negative of threshold potential), but why you never observed depolarisations to more positive potentials than threshold potential (i.e. causing action potentials). I am no expert in Drosophila fly brain recording, but you would expect to see many neurons without spontaneous action potentials in mouse brain slices (e.g. hippocampal neurons). If you need to see spontaneous action potentials you might want to change the extracellular solution (e.g. higher potassium concentration). Best, Jakob

I have only really seen the dichloride salt in both Drosophila and mouse experiements and this also tends to be the cheapest formulation. You'll find a lot of labs order their compounds from Sigma-Aldrich. You can find paraquat here: https://www.sigmaaldrich.com/GB/en/product/sial/36541?context=product

Hi, Celine, thank you. I have no answer, from anybody so far. Can you identfy the specimen?

If you are looking at an ensemble rather than individuals, that would work. Imaging allows you to see individual cells, while using a spectrometer allows measurement of a collection of flies or cells. Although many plate readers are simply spectrometers. Depending on your needs, you may get better statistics since you are measuring an average response to whatever you are measuring in your assay due to a large number of cells responding compared to those imaged.

Subhash C. Juneja Thank you for your kind!!

tomorrow we will try this method

Insect pest monitoring is typically performed in agriculture and forestry to assess the pest status in given locations (i.e., greenhouse, field, orchard/vineyard, forest) by collecting information about the target pest presence, abundance, and distribution. Within the integrated pest management programs in agriculture, the final goal of insect pest monitoring is to provide growers with a practical decision-making tool. Typically, an automatic trap equipped with a camera involves two modules: the hardware and the software. The hardware is typically composed of the trap structure containing the bait and retaining the trapped insects, an electronic box including the camera, a data transmission modem, a battery, and eventually an external power supply, such as a solar panel. The software is composed of the online repository in which the capture data pictures are stored and accessed plus optional image analysis algorithms to automatically identify and count the captures. Trap design may vary according to the target pest to be monitored, as detailed in this section.

Some to be considered: An automatic trap prototype modifying a commercial trap (Pomotrap®, currently Carpo® by Isagro S.p.A., Milan, Italy) with data acquisition and data transfer systems to monitor the codling moth Cydia pomonella L. (Lepidoptera: Tortricidae) in apple orchards;

‘Jackson trap’ was equipped with a camera device for the automatic monitoring of the Mediterranean fruit fly Ceratitis capitata Wiedemann (Diptera: Tephritidae)

Bucket traps are typically adopted to monitor fruit flies where a camera-based electronic McPhail trap was used to monitor by remote the olive fruit fly Bactrocera oleae Gmelin (Diptera: Tephritidae).

Agriculture operators are now facing the ‘Big Data Analysis’ prospect: organize, aggregate and interpret the massive sample size of available digital data with sophisticated algorithms to drive decisions based on data interpretation, prediction, and inference potentially on a global scale.

Camera-based insect monitoring can be exploited not only for pest monitoring but also for early detection and survey, allowing a prompt reaction especially for invasive species. There is a potential perspective to interconnect traps among sites and create a network at local, regional, country, continental, and global scales.

Ok, you're using very, very mild detergents, and then combining them "with or without SDS", which is sort of like saying "I'm scratching at this delicate glass sculpture with several different tissue papers, with or without a giant brick".

SDS will solubilise everything: it will not care whether it's a membrane protein or not. That is why we normally use SDS: to solubilise everything.

In this instance, this makes it convenient, because frankly I think you first need to establish whether you can detect your protein at all.

For membrane proteins, the key thing to be careful about is heat. You can get a membrane protein to solubilise in SDS just fine (coz it's SDS: it solubilises everything), but if you then boil it, which is the usual approach for protein samples, you'll probably force your TM protein out of solution.

So I would first use a conventional lysis buffer and conventional loading buffer, but I would keep everything at room temperature. Not, incidentally, on ice, because SDS precipitates out if you chill it, and you don't want that.

Use this to determine whether you can detect your POI at all in bulk lysate conditions, because if you cannot detect it when you're effectively loading everything, then any downstream studies are pretty much up in the air.

If all works as hoped (the protein is there, and you can see it), I would investigate replacing the SDS in the lysis buffer with milder detergents, like dodecyl maltoside (DDM), which in my experience is really quite membrane protein friendly.

There are a variety of online resources that can help optimise the workflow:

Lots. Probably 200-300. What are you using the tissue for? Most researchers that need that much tissue will use isolated heads, which is extremely simple.

It seems to me that trying different rounds of fixing (after the primary antibody) might help to stabilize the interactions therefore improving the quality of your experiment after incubation with secondary antibody.

On the other hand, using primary antibodies (nanobodies also) chemically labeled with fluorophores algo might help As you don’t need to use secondary antibodies, eliminating un cesar washing/incubation steps.

Are your drosophila still alive when you start fixation? If not, it is possible that air drying (and collapsing) of specimens already took place. I never worked with drosophila, but I would try to place a piece of wet filter paper on top of floating specimens. By the way, for insects proper dehydration could be even more important than fixation. Slow dehydration (like 24 hours for each step) may help. You may want to try HMDS instead of CPD.

Check https://pubmed.ncbi.nlm.nih.gov/16388142/

Craig Silver by any chance do you have a drosophila protein extraction protocol I can use? Thank you so much!

Yes- your anti-FLAG antibody strategy may work at 40 degrees. However the comments on biology are also relevant. You could consider doing this in a thermophile that tolerates different temperatures. You could also use something like Green Fluorescent Protein (GFP) instead of flag as your tag and then use a GFP nanobody for pulldowns as then the GFP color will allow you to see your pulldowns and follow possible unfolding.

You can use a Stereoscope device, which equipped with a digital camera.

As rightly pointed out, the Gal4 and UAS are heterologous elements. Therefore, it must be verified whether their insertion into the fly genome is not causing any mutation that may bear any impact on the observed phenotypes. This is simply verified by analyzing their motor neurons independently.

Hi Sayantan Datta, were you able to find a good antibody for dTor? If so, I would be interested in the reference. Thank you!

I have similar problem now, and I'm afraid that this is contamination. Have you managed your problem?

Try this Ibidi 8-well glass bottom slide "https://ibidi.com/glass-bottom/183--slide-8-well-glass-bottom.html" and you can coat the well with either ConA or Poly-L-Lysine with different concentration yourself.

Dear Marine Dehecq ,

we all would benefit from information on (at least a bit about "head lysate") and - most appreciated, processing of the spec's - especially resin type and STAINING the ultrathin sections.

My first guess would be (since there are - as you certainly know) at least two typical tangential section images of primary(?) cilia.... as one can find in some web-sources:

"The organelle is membrane-bound and contains multiple microtubules running along its length"

(cf. also - only ONE reference out of many):

"Cell Science at a Glance The primary cilium at a glance" Peter Satir, Lotte B. Pedersen, Søren T. Christensen Journal of Cell Science 2010 123: 499-503; doi: 10.1242/jcs.050377 to be found at:

or:

By: Mike Adams, Ph.D. (Dept. of Biology, Eastern Connecticut State University) © 2010 Nature Education  Citation: Adams, M. (2010) The Primary Cilium: An Orphan Organelle Finds a Home. Nature Education 3(9):54

https://www.nature.com/scitable/topicpage/the-primary-cilium-an-orphan-organelle-finds-14228249/

The electron dense structures/textures I would characterize as either (as usual!(:-)) ) a staining artifact (poor washing after staining) OR ((yes I know: NOT ALL artifacts are artifacts! (:-)) ) might reflect the presence of intensely stainable (TZ-Transition Zone)-Proteins in the basal bodies of cilia.

But unfortunately we see only 2 images out of your collection, not knowing how the images were achieved....in terms of practical handling of stains (guessing double staining UO2ac/lead citrate or eventually triple staining with e.g. Tannic acid 'ante' Uranyl acetate and Pb-citrate...)

Also cf.:

Drosophila sensory cilia lacking MKS proteins exhibit striking defects in development but only subtle defects in adults

Metta B. Pratt, Joshua S. Titlow, Ilan Davis, Amy R. Barker, Helen R. Dawe, Jordan W. Raff, Helio Roque

Journal of Cell Science 2016 129: 3732-3743; doi: 10.1242/jcs.194621

= https: @//ciliajournal.biomedcentral.com/articles/10.1186/s13630-015-0018-9

There are some commercially available products which are not fluorescent:

- https://www.thermofisher.com/order/catalog/product/A14291DJ#/A14291DJ

- https://www.thermofisher.com/us/en/home/life-science/cell-culture/mammalian-cell-culture/classical-media/fluorobrite-media.html

- Live-cell microscopy – tips and tools

Melanie M. Frigault, Judith Lacoste, Jody L. Swift, Claire M. Brown. Journal of Cell Science 2009 122: 753-767 (good read for live cell imaging)

Good luck!

@Sathya Srinivasan

This one worked very well for me.

https://www.emerginginvestigators.org/articles/development-of-diet-induced-insulin-resistance-in-drosophila-melanogaster-and-characterization-of-the-anti-diabetic-effects-of-resveratrol-and-pterostilbene

Hi,

Please find the attached link...

I agree with your point that it is difficult to rationalize the application of one drosophila read-derived correction factor genome wide. Our biggest problem is that Active motif suggests 50ng of spike in chromatin is sufficient to achieve ~2-5% drosophila reads, and we rarely do better than 0.05% drosophila reads for most ChIPs. It seems that I would need to make my own drosophila chromatin, because I cannot afford the amount of spike-in chromatin that I think I would need to get to 2%.

I also agree with your point that for a case like H3K27me3 chip in the presence of EZH2i, there should be very little H3K27me3 available to IP anyways. A case such as this is really the only time we see the drosophila spike in working. This is largely due to how we make the libraries. We balance the ng of DNA going in, even though we know there should be less from the EZH2i sample. Perhaps it is valid to just library prep the same volume of DMSO vs EZH2i rather than the same mass? Then I doubt we would need downstream normalization.

Our real trouble is this : We have a situation where in condition A we see many superenhancer-like sites of H3K27Ac that are lost in condition B. In condition B we also see a massive gain of smaller enhancer and promoter-like sites of H3K27Ac that are not present in A. We know by western that total H3K27Ac is higher in B than in A. When we sequence these ChIPs we have trouble calling the small gains in H3K27Ac in B, and we have trouble calling decreased H3K27Ac at superenhancers in condition A. Drosophila spike in fails miserably here because the H3K27Ac is abundant in both samples, and the antibody is great. The H3K27Ac is locally redistributed and globally increased.

So out of 100M reads, we only get like 200k drosophila reads, which is not statistically reliable. So we try this computational method for normalization and we see exactly what we would expect and what all our other data (ChIP-qPCR, western, etc) suggest we should see.

I have spent quite some time trying to understand this ChIPseqSpikeInFree method. It is true that it falls back on your point that the assumptions you make about the data must be orthogonally validated by other experiments. I think that you must have a hypothesis driven reason to use this computational method or the drosophila spike-in method. For the EZH2i H3K27me3 ChIP, I suppose a western blot is really sufficient to say that H3K@7me3 is depleted, but for H3K27Ac, we need to know where it is depleted and enriched, and we need to account for it being globally increased in abundance.

Our worry is that although this computational normalization idea has been published by bioinformaticists, it has not been vetted by biologists for real world application. I am just worried that even though this seems to work for my experiment, I will get killed by reviewers whether this method is valid or not, because drosophila spike-in is a lot easier to understand and explain.

thank you sir@Giovane R Sousa@

Solubility of rotenone in DMSO is 0.5mg/ml only and maximum concentration of DMSO I can use on flies medium is not more than 0.5-2%. But according to the rotenone solubility in DMSO and required weight of rotenone to be used to make 500uM, the concentration of DMSO to be used in food is large which is lethal to flies. Is there any other way or alternative, please suggest.

Xinlu Han Thank you for your recommendation. I will quantify with BCA.

Thanks Walid, that is indeed a good identification key, however it adressed adults only, and not pupae.

Does anyone have experience with drosophilid pupae identification?

Dear @Reaz Uddin,

Given the data sheet and probe design, this laser power meter is clearly designed to provide you with total laser power received at the detector, not power denisity. This will work as long as the projected light beam will not exceed the maximum area of the detector. Laser beams typically ar way smaller in diameter then 9mm. The meter is calibrated against a HeNe reference laser, which has a wavelength of 633nm. To measure the power of lasers at other wavelengths, you need to use the correction factors in the data sheet.

To get some more understanding of light measurements. look for The Light Measurement Handbook from Ales Ryer. This can be found on the web as a PDF. Very useful as a start.

There is an extensive literature on circadian rhythms in Drosophila and I am certain those labs have extensively characterized a variety of appropriate control genes. I would follow the leads of those labs so that when you try to publish you won't get criticism that you used an improper control.

Hello ,,Nicholas

Maybe this PDF helpful for you..

Hello everyone. I just faced the same problem that using beta actin, I didn't find any band. I used 1 ml of ripa buffer for extracting protein from 50 whole flies. Unfortunately I didn't use any protease inhibitors. Can anybody share his or her protocol of western blot with drosophila protein sample. Thank you.

Alternatively, you can use Microfluidics systems to immobilize the whole larva for CNS imaging, while the larva is fully intact.

For in vitro transcirption of dsRNA, try the MegaScript RNAi kit.

For in vivo transcription of dsRNA, try the E. coli HT115 (DE3) and L4440 vector.

If you simply want to buy ready dsRNA (large amounts but not as clear as the one you got by in vitro transcription), contact these companies:

RNagri

AgroRNA

GreenLight Biosciences

Nanosur

I dont know

Personally i use ACT (ARTEMIS comparaison tool ) easy to use with graphical interface

https://www.sanger.ac.uk/science/tools/artemis-comparison-tool-act

End resection of the double-strand break strands is the determining factor for the efficiency of HDR. If your modification is very near to the cut site, the efficiency of HDR will be very high and the efficiency decreases if it is away from the cut site. In cpf1, the end resection happens till ~25 nucleotides, indicating that the modifications can be made with high efficiency within 25nt of cut site. I haven't seen much of the studies on resection coverage in spCAS9. In our lab, we also tried with spcas9 and modification till 20nt from cut site works fine with pretty good HDR rates.

Please go through the following RG links.

Hey,

How about using just the water? The water solubility for picrotoxin is around 3-4 g/L; 3-4 mg/ml. Also, increasing the temperature of water improves its solubility in water.

Is this concentration enough for your experiment?

"Picrotoxin (Sigma-Aldrich, St. Louis, MO) was prepared in distilled water at a concentration of 0.4 mg/ml for intraperitoneal administration and 1 mg/ml for intravenous administration."

There's also the list of others solvents:

The propionic acid is in the fly food as a general microbial inhibitor, I have been able to get away without it as long as some other preservative is present (e.g. methylparaben/Tegosept, but this might not be cheaper).

If you really want to save on cost, you might be better off going with some oldschool way, like culturing flies on a banana/yeast mix (see attachment). I would keep an eye out for mold and bacteria though.

Thank yo so much Thomas Kidd and

Are you looking for 3D information like volume and surface area? If so, you need to analyze them as a volume with 3D Objects Counter. If you are looking for 2D information, then you don't need the 3D Objects Counter plugin and you can do standard 2D measurements. If the projection isn't too filled with structures (which can happen in large z stacks filled with dense structures), then I prefer to measure in the sum intensity projection rather than the maximum intensity projection. A max projection is only selectively displaying some brightest pixels from the volume and skews the measurements. The Sum projection shows the integrated value of all pixels, so especially if you are quantifying intensity this is important. or, if the information you need is contained within individual planes then you can measure a representative plane or planes.

HI Sarooj,

TM6B generally carries the Tb1 dominant mutation, which is responsible for the Tubby body (short body phenotype).

I can suggest two options for you.

If PTPmegKD/ + is not pupal lethal, you can collect non-tubbies at the pupal stage. It is easy to differentiate at the pupal stage on the 4th day of the cross.

If you must get at the larval stage. I will suggest the following steps.

i) cross eight virgin females (PTPmegKD/TM6B) with four males ( WIII8 +/+ ) or vise verse in single food vial only for 2-3 hours.

ii) So that flies mate and lay eggs on food within a window period of 2-hour. most of the larvae at same instar stage. (if you dont narrow the window for egg lay, you may confused with 2nd instar for tubby larvae)

iii) Remove parental flies. Leave only eggs on vials at 25C incubator. On the third day, you may see third instar larvae.

iv) on the third day, add 35% glucose solution on food vial, and gently scrub the food with a brush. All the larvae started to float on glucose solution (due to solution density).

v) Pour the solution into a clean Petri dish and collect longer larvae (not tubby) by picking one by one with brush under the microscope into a new Petri dish with 1XPBS.

Note (keeping in PBS will help to get rid of glucose solution, if you don't wash larvae with PBS, larvae may be stuck and die due to stickiness of the glucose).

vi) Collected long larvae can be kept in separate new food vials for them to grow.

Good luck with experiments.

Thank you, Laurina Manning and Matthias R. Schaefer

Came across this while having breakfast with my younger daughter and thought I'd add a bit of history to Jazz mix. I formulated this some years ago for Grier Eubank's company (I don't recall its name), which was subsequently bought by Fisher. Our purpose was to get micronutrients into the food, which helps weak stocks. Of note, the name of my daughter is...Jazz.