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The earth­worm in new light

Jun 29, 2021

Even if they seem very common for us – earthworms are special because they keep our soil healthy, all over the world. From the outside they appear simple and inconspicuous. But what the earthworm looks like from the inside, from its organs to the microbes and parasites that colonize it, has been difficult to grasp. Scientists from the Max Planck Institute for Marine Microbiology have now developed a method to visualize these anatomical structures including the products of an earthworm's metabolism. It opens up new paths in microbiology, parasitology and immunology and has now been published in the US-American Proceedings of the National Academy of Sciences (PNAS).

 

3D CHEMHIST atlas or the posterior end of an earthworm, used in this study. The atlas combines data of mass spectrometry imaging (MSI), fluorescence in situ hybridization (FISH) and microtomography (micro-CT). (© Max Planck Institute for Marine Microbiology/PNAS ref.)
3D CHEMHIST atlas or the posterior end of an earthworm, used in this study. The atlas combines data of mass spectrometry imaging (MSI), fluorescence in situ hybridization (FISH) and microtomography (micro-CT). (© Max Planck Institute for Marine Microbiology/PNAS ref.)

Earth­worms ex­per­i­ence con­stant chem­ical in­ter­ac­tions with bac­teria, fungi, plants and small in­ver­teb­rates across soil eco­sys­tems. Even within their tis­sues, earth­worms har­bor sym­bi­otic mi­crobes and small an­imal para­sites that trig­ger in­ternal meta­bolic re­sponses such as in­nate im­munity. To re­veal the fun­da­mental pro­cesses that en­able an­imal–mi­crobe sym­bi­oses to form and per­sist, we have to study their meta­bolic in­ter­ac­tions in situ. By com­bin­ing novel ima­ging tech­niques, a team of re­search­ers around Be­ne­dikt Geier from the Max Planck In­sti­tute for Mar­ine Mi­cro­bi­o­logy (MPIMM) in Bre­men, Ger­many, has now suc­ceeded in ima­ging the ex­cit­ing vari­ety of in­ter­ac­tions that take place in­side the earth­worm. This al­lows us to ob­serve them in a com­pletely new light. 

The chem­istry of the worm in 3D

Depicted is a microtomography-based 3D model of the anterior end of an earthworm that schematically shows how CHEMHIST enables to link between anatomic structure and metabolic function. The 3D data of the worm’s microanatomy allows a virtual dissection of the animal as shown here. For example, making the skin of the animal transparent or removing it as shown in the image reveals the internal structures. With CHEMHIST one can not only visualize the anatomy, but also the associated molecules each organ consists of by using intensity maps shown in the figure with as blue to yellow colored overlay. (© Max Planck Institute for Marine Microbiology/open access microCT data by Lenihan et al.)
Depicted is a microtomography-based 3D model of the anterior end of an earthworm that schematically shows how CHEMHIST enables to link between anatomic structure and metabolic function. The 3D data of the worm’s microanatomy allows a virtual dissection of the animal as shown here. For example, making the skin of the animal transparent or removing it as shown in the image reveals the internal structures. With CHEMHIST one can not only visualize the anatomy, but also the associated molecules each organ consists of by using intensity maps shown in the figure with as blue to yellow colored overlay. (© Max Planck Institute for Marine Microbiology/open access microCT data by Lenihan et al.)

Our un­der­stand­ing of the chem­ical in­ter­ac­tions between small an­im­als and the mi­croor­gan­isms that live in­side their bod­ies is ex­tremely lim­ited. This gap of know­ledge has its ori­gin in a meth­od­ical chal­lenge: To study the meta­bolic in­ter­ac­tions in sym­bi­osis we have to know who is pro­du­cing which meta­bolic product where in the body. The core of this prob­lem, however, is not only to im­age how mo­lecules are dis­trib­uted at the mi­cro­meter scale. Also, the chem­ical im­ages are al­most im­possible to in­ter­pret without know­ing if a tis­sue sample is healthy, dis­eased or in­fec­ted with be­ne­fi­cial or patho­genic mi­crobes or even an­imal para­sites.

The new com­bin­a­tion of high-res­ol­u­tion ima­ging tech­niques can of­fer a solu­tion to this prob­lem, as the re­search team now shows. “In our study, we in­tro­duce chemo-histo-tomo­graphy, a spe­cial three-di­men­sional ima­ging ap­proach of the chem­istry and ana­tomy of mil­li­meter-sized an­im­als and their para­sites at a cel­lu­lar level,” says Be­ne­dikt Geier, first au­thor of the pa­per. “This method of­fers a new strategy to visu­al­ize the most fun­da­mental pro­cesses – meta­bolic in­ter­ac­tions – in small an­imal sym­bi­oses. It al­lows us to spa­tially as­sign meta­bolic pro­ducs to the an­imal host and its mi­cro­bial part­ners at the mi­cro­meter scale.“

Chemo-histo-tomo­graphy (CHEM­HIST) com­bines chem­ical ima­ging of meta­bol­ites based on MALDI mass spec­tro­metry ima­ging with the mi­croana­tomy of the same an­imal that is re­cor­ded with mi­cro-com­puted X-ray tomo­graphy. The lat­ter is a non-in­vas­ive ap­proach al­low­ing X-ray ima­ging of the 3D his­to­logy, and can reach sub­cel­lu­lar res­ol­u­tion. For meta­bol­ite ima­ging, MALDI mass spec­tro­metry tech­niques have be­come a state-of-the-art tool to visu­al­ize mi­cro­meter-scale, nat­ural dis­tri­bu­tions of meta­bol­ites. This en­ables a spa­tial as­sign­ment between chem­ical pro­files and their pro­duc­tion site and pos­sibly to their pro­du­cer.

“This ad­vance al­lowed us to take an earth­worm from the en­vir­on­ment and cre­ate a 3D at­las of its chem­ical and phys­ical in­ter­ac­tions with the mi­croor­gan­isms nat­ur­ally oc­cur­ring in­side its tis­sues,” says Manuel Liebeke, leader of the Re­search Group Meta­bolic In­ter­ac­tions at the MPIMM and senior au­thor of the study. “However, we were in­ter­ested in more than just the bio­logy of the earth­worm. Our ob­ject­ive was to also make CHEM­HIST ap­plic­able to an­im­als dir­ectly sampled from their nat­ural hab­itat, which can be es­sen­tial for sym­bi­osis re­search.” The res­ol­u­tion of CHEM­HIST out­per­forms ex­ist­ing sim­ilar meth­ods de­veloped for med­ical re­search in mice by up to two or­ders of mag­nitude. This may also lead into new av­en­ues for re­search on in­sects or cor­als, which are key mod­els for sym­bi­osis re­search, both on land and in wa­ter.

The whole is more than the sum of the parts

Us­ing the com­bin­a­tion of dif­fer­ent in situ ima­ging tech­niques through CHEM­HIST, the re­search­ers at the MPIMM in Bre­men un­covered meta­bol­ites – products of the meta­bol­ism – in the earth­worm that could shed light on how it chem­ic­ally de­fends it­self against para­sites and how these, in turn, pro­tect them­selves against the earth­worm's im­mune re­sponse. However, to even re­cog­nize that the chem­ical im­age data were meta­bol­ites of para­sites in earth­worm tis­sue, the 3D ana­tom­ical model was in­dis­pens­able. A high-res­ol­u­tion mi­cro-com­puter tomo­graph at the Ger­man Elec­tron Syn­chro­tron in Ham­burg first made it pos­sible to identify the para­sites in the tis­sue.

Not­ably, meta­bolic in­ter­ac­tions between an­im­als and their mi­crobes are not re­stric­ted to sym­bi­otic tis­sues. Along the gut-brain axis, mi­cro­bial meta­bol­ites pro­duced in the gut can af­fect tis­sues across the host, even reach­ing the brain. There­fore, ex­tend­ing cor­rel­at­ive chem­ical ima­ging into 3D ap­proaches can be cru­cial for cap­tur­ing the dis­tri­bu­tion of meta­bol­ites in­volved in sym­bi­otic in­ter­ac­tions and thus show how chem­ical sig­nals from mi­crobes pos­sibly af­fect cru­cial pro­cesses in their host. Also, the method can be used in many ways: The re­search team around Liebeke and Geier is cur­rently ap­ply­ing it to deep-sea mus­sels.

“Since I have spe­cial­ized in the 3D visu­al­iz­a­tion of in­ver­teb­rate ana­tomy for years, it was par­tic­u­larly in­ter­est­ing for me to see the mo­lecules hid­den be­hind the mor­pho­lo­gical struc­tures,” says Bernhard Ruthen­steiner, leader of the sec­tion In­ver­teb­rate Varia at the Bav­arian State Col­lec­tion of Zo­ology. The res­ults of the study were made pos­sible by an in­ter­dis­cip­lin­ary col­lab­or­a­tion that brought to­gether sci­ent­ists from the fields of mi­cro­bi­o­logy, zo­ology, chem­istry and phys­ics. It quickly be­came clear that visu­al­iz­a­tion such as the cor­rel­at­ive 3D at­las of the worm fa­cil­it­ate sci­ence com­mu­nic­a­tion of the data.

The image shows a cross-section of an entire earthworm, images with two techniques: Mass spectrometry imaging (left) and fluorescence microscopy (right). The different colors of the mass spectrometry imaging data show the distribution of different metabolites in the tissue, whereas the fluorescence microscopy shows the tissue structures underlying the chemical distributions (© Max Planck Institute for Marine Microbiology/B. Geier)
The image shows a cross-section of an entire earthworm, images with two techniques: Mass spectrometry imaging (left) and fluorescence microscopy (right). The different colors of the mass spectrometry imaging data show the distribution of different metabolites in the tissue, whereas the fluorescence microscopy shows the tissue structures underlying the chemical distributions (© Max Planck Institute for Marine Microbiology/B. Geier)
 

Ori­ginal pub­lic­a­tion

Be­ne­dikt Geier; Jan­ina Oetjen; Bernhard Ruthen­steiner; Maxim Po­likar­pov; Har­ald Gruber-Vodicka; Manuel Liebeke: Connecting structure and function from organisms to molecules in small animal symbioses through chemo-histo-tomography, PNAS, Juni 2021

 

DOI: 10.1073/pnas.2023773118

Par­ti­cip­at­ing in­sti­tu­tions

  • Max Planck Institute for Marine Microbiology, Bremen, Germany
  • Bavarian State Collection of Zoology, Munich, Germany
  • European Molecular Biology Laboratory, Hamburg Unit c/o German Electron Synchrotron in Hamburg, Germany
  • MALDI Imaging Lab, University of Bremen, Germany

Please dir­ect your quer­ies to:

Group leader

Research Group Metabolic Interactions

Dr. Manuel Liebeke

MPI for Marine Microbiology
Celsiusstr. 1
D-28359 Bremen
Germany

Room: 

3244

Phone: 

+49 421 2028-8220

Dr. Manuel Liebeke
 
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