Life-Long Mosquito Marking: Are Stable Isotopes the Key?

Post provided by ROY FAIMAN

Importance of Marking (Wild) Mosquitoes

Dr. Dao (crouching on right) and team with Dr. Tovi Lehmann (with sandals), Dr. Yaro (with white cap), and Moussa Diallo (front).

The fact that mosquitoes are insects of massive importance is of little dispute. With malaria still killing almost half a million people annually and after recent outbreaks of Zika, dengue and West-Nile viruses the threat of mosquito-borne diseases is becoming common knowledge. The meme of ‘Mosquitoes are the No.1 killer of all time,’ is also growing more popular (I even heard it from my 8-year-old kid one day after he returned from school!). Yet, with all we think we know about the little bug(ger)s, it’s probably only the tip of the iceberg.

Much work was done over the past century to try to answer basic questions about mosquitoes like:

  • How big are their populations?
  • How long do they live?
  • Where do they go when we don’t see or feel them?

Different methods have been developed to provide insights and notions on the mosquitoes’ movements, survival, and populations estimates; but the limitations and conditions of these methods mean that our knowledge is still incomplete.

One of the gold-standard tools for answering questions like those above is Mark-Release-Recapture (MRR). It was developed almost a century ago and has been modified and remodified through the years, as different marking technologies became available.

Mark-Release-Recapture Bottle-Necks and Challenges

MRR techniques are diverse, but all share the same idea: mark an animal, release it, and try to find it later. From this simple concept we can get a lot of information on entire populations (if we marked and captured enough individuals).

Dr. Yaro (left), Moussa Diallo (back) and Zana (right) Collecting adult mosquitoes from a larval site covered with an emergence trap.

When studying mosquitoes, the problem(s) begin right at the start: what do we mark them with? Mosquitoes are so small (usually under 5 mm long and weighing less than 0.5 mg), we can’t tag them with a transmitter (yet…). So, what can we use? Paint? Dust? Dye? Food colouring? Radioactive isotopes? Yes. All these have been used in the past; some are still in use today. The problem is that to date, most, if not all marking methods either alter the behaviour of the insect, shorten its life, or wear off after a short while; and, due to the large dilution in typically large populations, they all involve a huge amount of work. These factors limit the abilities of MRR studies, both temporally and spatially, leaving questions of seasonal aestivation (summer diapause) or long-distance migration beyond our reach.

Stable Isotope Marking: The Case of Mosquitoes

Zana Lamissa Sanogo adding deuterium-oxide to mosquito larval site.

This is where stable isotopes may shine. Stable isotopes are found naturally in the environment (in background levels). Isotopes of carbon (13C), nitrogen (15N), oxygen (18O), sulfur (34S) or hydrogen (2H) are either heavier or lighter substitutes of these atoms. When used for marking (generally in low concentrations), they negligibly alter the mosquitoes’ biology and behaviour (if at all). Being non-radioactive (stable), they emit no particles or radiation and are harmless to the environment.

Increasing the ratio of these isotopes in mosquito breeding sites to levels significantly above background is straight-forward but requires maintenance of enrichment levels following rain events. Mosquitoes that develop in the isotope-enriched environment carry the isotopic signature within their hard, chitinous tissues for life. Stable isotopes are detected by isotope-ratio mass-spectrometry (IRMS), yielding robust results from dry samples as small as single a mosquito (or parts thereof) (<0.1 mg).

Dr. Adama Dao (with hat) Assessing larav site volume with the support of Torodo village community.

The first to use stable isotopes for marking mosquitoes were Drs. Gabriel Hamer (currently collaborating with us) and Ed Walker from Michigan State University (back in 2012). Using 13C and 15N in urban breeding sites of Culex pipiens, they were able to track their dispersal in suburban Chicago. By doing so, they’ve opened the door to life-long marking of mosquitoes.

Stable Isotopes and the Question of Aestivation in A. coluzzii

Studying malaria mosquito ecology in Mali for over a decade, we’ve noticed that among the three species inhabiting the edge of the Sahel, A. coluzzii somehow sticks around through the 6-8 month long dry season. Its other two sibling species (Anopheles gambiae s.s and A. arabiensis) are absent during this time and migrate in during the wet season.

The natural Sahelian larval site during the mid-wet season (Left. ©R. Faiman) and the transition into the dry season (Right. ©A.S. Yaro).

During the dry season, no surface water can be found anywhere (mosquitoes need water to breed). To make things worse, hot, dry, dusty winds from the Sahara blow fiercely through the region. Not your usual mosquito paradise in any way. Yet, somehow, A. coluzzii seem to show up during this period. We’ve actually shown their numbers even spike to levels surpassing the wet season levels at times! Once the rains return, A. coluzzii populations start building up fast, preceding their sibling species by 6-8 weeks; another indication they’ve never left but have only been in hiding.

Seeing this phenomenon year after year, never able to ascertain much beyond their presence or absence (despite amassing indirect evidence of their presence) is very frustrating. Solving this mystery seemingly demands heavy guns. As we are generally of a peaceful nature, we chose stable isotopes (Big thanks to our co-author Dr. Keith Hobson who put us on this track!). Specifically, heavy isotopes of hydrogen (2H-deuterium-oxide, i.e. ‘Heavy water’). Using 2H we are basically adding water to water, giving our emerging mosquitoes the signature we’re after. This water (much like normal water) eventually evaporates without leaving a trace. This means we can come back and repeat our work year after year without the fear of ‘burning’ our sites. Neat!

The Fieldwork

Dr. Dao’s team with Dr. Tovi Lehmann adding water to larval site through a sieve to prevent aquatic predator transfer into the site.

In ‘Marking mosquitoes in their natural larval sites using 2H‐enriched water’ we describe the process of marking mosquitoes through 2H-enrichment of wild larval sites. The theory behind the technique makes it sound simple to pull off. But this work involved a crew of dedicated, hard-working  researchers from ICER Mali , led by Dr. Adama Dao. Without their efforts, this work would have been a far greater challenge. From selecting the village and seeking approval for the study, through the setting up of the larval site embankments, their systematic enrichment, microbiota culturing, predator removal, larval counts, water level measurement and maintenance, mosquito collection and rearing, all the way through to shipping the mosquito samples desiccated and ready for IRMS to the US. No small feat! We are so grateful to him, Dr. Alpha S. Yaro, Zana L. Sanogo, Dr. Djibril Samake, Moussa Diallo, and Yossi Ousmane, who we continue to work with on a daily basis.

Doorway to the Future

Marking mosquitoes with 2H was specifically developed with the goal of providing a definitive answer to the question of aestivation of A. coluzzii through the dry season (and to show the contribution of aestivators to the buildup of the population at the onset of the wet season). This question has engaged malariologists for nearly a century. Now, having tested it in the field, we feel we might finally have a robust tool to answer this question definitively. Stay tuned!

Team Members Reminisce…

Dr. Adama Dao, Moussa Diallo, Yossi Ousmane and Djibril Samake Conducting stable isotope compensation calculations (front). Drs. Lehmann and Yaro removing predators from larval site with yellow water tracer (behind, left).

“Marking mosquitoes in their natural larval sites using deuterium-oxide was a very exciting protocol. The larval separation was the tedious part of the protocol execution. Hundreds of bags, filled with sand were transported to the larval sites and so, real dikes were constructed. A large quantity of sand and manpower were used. It was the most laborious work and critical part of this protocol. But my satisfaction was the fact that this was a successful way to naturally isolate a larval site because when using fluorescent yellow water tracer dye, water was not passing the dikes. In this case, the quantity of water and deuterium dosages were manageable.” (Adama Dao)

“It was very impressive to see under microscope when checking the microbiota culture for mosquito larvae food, that there are highly heterogenic and the microbes composition and organisation is following the law of the strongest, so the biggest were eating the smallest, and then grow and spread very fast in the culture solution by generating many other microbes with smaller size” (Alpha S. YARO)

Children of Doneguebougou on a curiosity break. Thanks guys!

Working like pig farmers for the marked mosquitoes… The game was well worth it… (Zana L. Sanogo)

“One of my favourite moments after a long day of work in the village was chatting with our local helpers! The incredible kids of Doneguebougou posing joyfully in the shade. Fun times!” (Roy Faiman)

 

To find out more about using stable isotopes for MRR for mosquitoes, read our Methods in Ecology and Evolution article ‘Marking mosquitoes in their natural larval sites using 2H‐enriched water: A promising approach for tracking over extended temporal and spatial scales’.

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