Feb 04, 2020
Version 3
Infecting and mosquitoes with Dirofilaria immitis and Brugia malayi V.3
- 1University of Pennsylvania School of Veterinary Medicine;
- 2University of Pennsylvania
- povelabTech. support email: mpove@vet.upenn.edu
Protocol Citation: Michael Povelones, Abigail Mccrea 2020. Infecting and mosquitoes with Dirofilaria immitis and Brugia malayi. protocols.io https://dx.doi.org/10.17504/protocols.io.bb7birin
Manuscript citation:
Edgerton EB, McCrea AR, Berry CT, Kwok JY, Thompson LK, Watson B, et al. Activation of mosquito immunity blocks the development of transmission-stage filarial nematodes. Proc Natl Acad Sci U S A. 2020. Epub 2020/02/06. doi: 10.1073/pnas.1909369117. PubMed PMID: 32015105.
McCrea AR, Edgerton EB, Oliver GT, O’Neill FM, Nolan TJ, Lok JB, et al. A novel assay to measure the emergence of third-stage filarial nematodes in individual mosquitoes. bioRxiv. 2020. doi: https://doi.org/10.1101/2020.02.04.934653.
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol in our group with both parasites and different mosquito strains and it is working.
Created: February 04, 2020
Last Modified: March 22, 2023
Protocol Integer ID: 32707
Keywords: Dirofilaria immitis, heartworm, Brugia malayi, lymphatic filariasis, elephantiasis, Aedes aegypti, mosquito, microfilaria, Malpighian tubule, L3i, infectious L3, host-parasite interaction
Abstract
This protocol is for infecting Aedes aegypti and Aedes albopictus mosquitoes with the filarial nematodes Dirofilaria immitis or Brugia malayi. Although it is not necessary, it is strongly recommended to measure the number of ingested parasites, which indicates the exposure level for the mosquito and allows for better comparison between replicates. We use a post-feed treatment protocol, but if mosquito groups are treated prior to infection, then it is not just strongly recommended, but critical to measure ingested microfilariae in a group of mosquitoes immediately following the feed to confirm that treatment does not change the extent of blood feeding. Note that microfilariae are large (approximately 300 µm) and will quickly settle in blood samples. This should be considered throughout the protocol and this fact is pointed out in places in this protocol.
Guidelines
Filariae are human and animal pathogens so all mosquito husbandry and infections should be performed in accordance to local safety and containment guidelines that meet or exceed published guidelines. We have performed this assay with Brugia malayi (a human pathogen) and Dirofilaria immitis (an animal pathogen) under the appropriate containment level.
American Committee Of Medical Entomology American Society Of Tropical Medicine And Hygiene. Arthropod Containment Guidelines, Version 3.2. Vector Borne Zoonotic Dis. 2019 Mar;19(3):152-173. doi: 10.1089/vbz.2018.2431. Epub 2019 Jan 29. PMID: 30694736; PMCID: PMC6396570.
Materials
MATERIALS
Sheep bloodHemostatCatalog #SBH100
STEP MATERIALS
Sheep bloodHemostatCatalog #SBH100
Protocol materials
Sheep bloodHemostatCatalog #SBH100
Materials
Sheep bloodHemostatCatalog #SBH100
Materials, Step 3
Safety warnings
Brugia malayi is a human pathogen
Dirofilaria immitis is an animal pathogen
Safe blood handling procedures and a laboratory specific Exposure Control Plan in place.
Before start
Secure all approvals. Establish standard protocols. Assemble all materials.
Counting microfilariae
Counting microfilariae
Disperse 5 µL or 10 µL microfilaremic blood in a 50 µL spot of water on a microscope slide. This will lyse red blood cells and make the microfilariae more conspicuous and easy to count.
Note
- Water type is not important. We have used deionized water or ultrapure molecular biology grade water.
- Before pipetting microfilaremic blood from the source container, mix evenly by gently inverting 10-15 times. Microfilariae are large and settle in the blood quickly.
D. immitis microfilaria in lysed blood sample
Immediately count the entire drop by scanning through the sample. Determine the concentration and express it as mf/ml by multiplying by the dilution factor. For example, if 5 µL was used, multiply by 200. If 10 µL was used, multiply by 100. Replicate counts are performed and averaged.
Scanning strategy for reading total microfilariae in an entire sample.
Note
For best accuracy replicates counts are made. When counting more diluted blood, count 10 µL . If needed the water spot size could be increased and more blood can be dispersed. For example 40 µL of blood can be counted in a 200 µL spot of water.
Expected result
From our infected dog, we typically count 5 µL of freshly drawn blood and count 200-300 microfilariae resulting in a concentration of between 40,000 - 60,000 mf/ml.
Blood feeding mosquitoes
Blood feeding mosquitoes
We typically set up infections at 4000 microfilariae/mL to 5000 microfilariae/mL . We use heparinized sheep blood to dilute. The infected blood and diluent blood are aliquoted under sterile conditions in a tissue culture hood. Once the desired dilution is created, recount the diluted to sample to confirm and then keep the sample warm at 37 °C in a heat block or circulating water bath. For recounts we use 10 µL of the dilution in 50 µL drop of water. Replicate counts are performed and averaged.
Sheep bloodHemostatCatalog #SBH100
Note
Because the microfilariae will settle during the course of feeding, the volume of blood and geometry and speed of blood feeding will all contribute to the number of microfilariae ingested per mosquito. Under the conditioned described here, mosquitoes typically have 10-15 microfilariae ingested.
Once the water required for the feeder and the diluted blood are prewarmed to 37 °C then fill a plastic baby bottle with a solid cap (see picture below) with 37 °C water excluding as much air as possible without spillage. Then, invert the bottle and add 3.5 mL of diluted blood to the depression in the bottom of the bottle and cover with stretched Parafilm.
Note
Take care to not slosh water from the water bath onto the feeder as it might contaminate the sample and may also inhibit the mosquitoes from feeding. You better attract the mosquitoes for feeding, rub the side of the Parafilm that will be facing the mosquito cup/cage on a part of your body that does not have lotion or fragrance.
Equipment
Baby bottle
NAME
300 mL bottle
TYPE
Avent Phillips
BRAND
SCF693/59
SKU
LINK
Invert the bottle and place on top of a cup/cage of mosquitoes. If required for safety reasons, perform this step in a secondary container in the incubator, such as a Large Bugdorm cage. To encourage mosquito blood feeding, exhale a warm breath into the cup or cage before placing the feeder on top. Feed for 00:15:00 . We feed at 27 °C and 80 % relative humidity (our standard incubator conditions). If practical, turn off the light in the incubator.
Filled baby bottles ready to feed. They will be gently shaken before putting them on top of mosquito cups/cages parafilm side down.
Mosquitoes in a cup
Baby bottle inverted on a mosquito cup
Note
For important comparisons, dilute enough blood, so that each cup gets the same volume of the same dilution and is fed for the same time. It is ok to reuse the feeders as described below, but it is best not to compare groups that are fed serially since the conditions will not be identical.
Equipment
Large Bugdorm Cage
NAME
Large mosquito cage
TYPE
Bugdorm
BRAND
BD4M3074
SKU
LINK
If required, you can refresh the water in the feeder to feed more mosquitoes. Take care not to spill water onto the Parafilm during this process. Hold the feeder, and remove the cap. Do not put it down on the Parafilm side or it will get damaged and leak. Quickly pour out the water. While the feeder is empty and inverted, give it a firm shake to disperse the blood again. Replace with fresh 37 °C water, cap, and put back on mosquito cup/cage for another round of feeding.
Sort blood fed mosquitoes. Mosquitoes are anesthetized using carbon dioxide and sorted on Flypad using a paint brush.
Blood fed Aedes aegypti (left) separated from unfed mosquitoes
Note
Alternatively, blood fed mosquitoes can be separated on ice.
Equipment
Flypad
NAME
CO2 anesthetizing apparatus
TYPE
Flystuff
BRAND
59-114
SKU
LINK
Equipment
Paint brush
NAME
Paint brush
TYPE
Generic
BRAND
Size 00
SKU
Checking uptake of microfilariae
Checking uptake of microfilariae
Remove blood meal from some of the blood fed mosquitoes and count the microfilariae uptake. The objective is to count all microfilariae in the entire blood meal. Place the mosquito in a drop of water on a depression slide. Tear the abdominal cuticle where it meets the thorax with fine forceps. Tear in several places to facilitate gut removal without damaging it. Perform the dissections in ultrapure or deionized water in a depression slide. Discard any damaged guts that are leaking blood. Transfer the gut to a new slide with a 50 µL drop of ultrapure water. After transferring, remove the gut tissue by peeling it away from the blood bolus with fine forceps and "wash" through the water to remove residual blood, then discard the gut tissue. The peritrophic matrix typically stays intact. Disrupt the blood meal with the fine forceps and make sure to remove as much residual blood from the tines as possible. Disperse the blood meal fully using a P20 pipette.
Removing blood fed gut from Aedes aegypti mosquito. (A) Female mosquito in a drop of water. (B) Blood-filled gut removed. (C) Gut after dissection and transfer to new slide. (D) Blood meal following disruption with forceps. Pieces of peritrophic matrix are visible near the top of the image. (E) Blood meal dispersed in water using a P20 pipette.
Blood fed midguts on glass slides. Midguts were placed in a 50 µL drop of ultrapure water and the gut tissue was removed from around the blood bolus. Several guts can be placed on slides prior to dispersing.
Equipment
Fine Forceps
NAME
Forceps
TYPE
Dumont
BRAND
11251-10
SKU
LINK
Count the entire drop as in step 2. go to step #2 . Ideally, count 10 mosquitoes for all groups being compared.