Insects use lubricants to minimize friction and wear in leg joints

A protein-based lubricating substance is discovered in the femoro-tibial joint of the darkling beetle Zophobas morio (Insecta). The substance extrudes to the contacting areas within the joint and appears in a form of filiform flows and short cylindrical fragments. The extruded lubricating substance effectively reduces the coefficient of sliding friction to the value of 0.13 in the tribosystem glass/lubricant/glass. This value is significantly lower than 0.35 in the control tribosystem glass/glass and comparable to the value of 0.14 for the tribosystem glass/dry PTFE (polytetrafluoroethylene or Teflon). The study shows for the first time that the friction-reducing mechanism found in Z. morio femoro-tibial joints is based on the lubricant spreading over the contacting surfaces rolling or moving at low loads and deforming at higher loads, preventing direct contact of joint counterparts. Besides Z. morio, the lubricant has been found in the leg joints of the Argentinian wood roach Blaptica dubia.


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Comments to the Author This is a very interesting study and I recommend publication. However, I want the authors to add some more information about the friction experiment: Add to the main paper the sliding speed and the nominal contact pressure (the information is now only in the supplementary materials). Also add information about what these quantities are for the insect. Some insects (and the tree frog) use fluids for adhesion and in those cases the fluid is likely pulled spontaneously into the contact area by capillary effects. But if the lubricant in the present case is in a solid state how is it injected into the leg joints in the narrow pore channels? It appears to me that this cannot occur spontaneously by capillary effects but may require a big force or pressure which is generated how?

04-Jun-2021
Dear Dr Nadein I am pleased to inform you that your manuscript RSPB-2021-1065 entitled "Insects use lubricants to minimize friction and wear in leg joints" has been accepted for publication in Proceedings B.
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Sincerely, Dr Locke Rowe mailto: proceedingsb@royalsociety.org Associate Editor Comments to Author: The discovery that some insects produce lubricants analogous to human synovial fluid is fascinating. Reviewer 1 asks for clarification of the generality of the results to insects and a longer more contextual discussion on epicuticular grease and insect secretions. Reviewer 2 also asks about the generality of this work, considering the cuticle of beetles may be different from other insects. Reviewer 3 asks for more details on the lubrication experiment and discussion of how semi-solid materials can be ejected through the holes. Note that Reviewer 1 and 2 have word and pdf files with additional comments. Overall, some toning down of the scope of the results may be needed and some greater context should be given with regards to other insect secretions. All reviewer comments should be addressed.
Reviewer(s)' Comments to Author: Referee: 1 Comments to the Author(s) Please see the attached file.
Referee: 2 Comments to the Author(s) Briefly speaking, the article in its short main text is novel and interesting, but the reviewer asks them to understand that the femoral cuticle in beetles is an other construction then in other insects, but they can not repeat in the manuscript any novel details described by the reviewer.
Referee: 3 Comments to the Author(s) This is a very interesting study and I recommend publication. However, I want the authors to add some more information about the friction experiment: Add to the main paper the sliding speed and the nominal contact pressure (the information is now only in the supplementary materials). Also add information about what these quantities are for the insect. Some insects (and the tree frog) use fluids for adhesion and in those cases the fluid is likely pulled spontaneously into the contact area by capillary effects. But if the lubricant in the present case is in a solid state how is it injected into the leg joints in the narrow pore channels? It appears to me that this cannot occur spontaneously by capillary effects but may require a big force or pressure which is generated how?

09-Jun-2021
Dear Dr Nadein I am pleased to inform you that your manuscript entitled "Insects use lubricants to minimize friction and wear in leg joints" has been accepted for publication in Proceedings B.
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Insects use lubricants to minimize friction and wear in leg joints
by Konstantin Nadein, Alexander Kovalev, Jan Thøgersen, Tobias Weidner & Stanislav Gorb A long-term mystery uncoveredthe lubrication of beetle leg joints! Congratulations to the authors on a meaningful, well-illustrated manuscript! Several minor issues to be raised … The Title "Insects use lubricants to minimize friction and wear in leg joints" does not fit the provided study and obtained results. This was done, and which is shown: "Darkling beetles use lubricants to minimize friction and wear in leg joints". -In this context, the 6 other beetle and a cockroach species mentioned in the Supplementary Material are rather confusing and not referred to in the main body of the manuscript.
The Introduction is succinct, however, quite short-cut. In particular, related to arthropod joints and cuticle one to three further statements could be included, such as about epicuticular grease and insect secretion in general. This aspect in comparison with the present results would still emphasize the significance of the author's findings. Also, the special structures covering the joint and/or joint edge surfaces could be considered, discussing the "multifunctionality" of the joints unifying lubrication on the one hand and friction enhancement on the other handthis role of the structures on the joint cuticle counterparts is also worth to be considered in illustrations and discussion.
The question arises about the state, condition, and age of the tested animals. Do aged ones secrete a similar lubricant and similar amounts?
The structure of secretion, in particular in Fig. 1d, f, l looks unusual compared to other viscoelastic fluids found in insects and plants so far. They remember bacteriashaped particles; maybe characteristic for proteinaceous material as detected for the joint lubrication? -Here some comparison with the state of literature about shapes of proteinaceous structures would be value-adding and supporting the innovation of the present study.

Response to Referees
Associate Editor Comments to Author: The discovery that some insects produce lubricants analogous to human synovial fluid is fascinating. Reviewer 1 asks for clarification of the generality of the results to insects and a longer more contextual discussion on epicuticular grease and insect secretions.
Reviewer 2 also asks about the generality of this work, considering the cuticle of beetles may be different from other insects.
Reviewer 3 asks for more details on the lubrication experiment and discussion of how semi-solid materials can be ejected through the holes.
Note that Reviewer 1 and 2 have word and pdf files with additional comments. Overall, some toning down of the scope of the results may be needed and some greater context should be given with regards to other insect secretions. All reviewer comments should be addressed.

Reviewer(s)' Comments to Author:
Referee: 1 Comments to the Author(s) Please see the attached file. Authors: The corrections have been made.
Referee: 2 Comments to the Author(s) Briefly speaking, the article in its short main text is novel and interesting, but the reviewer asks them to understand that the femoral cuticle in beetles is an other construction then in other insects, but they can not repeat in the manuscript any novel details described by the reviewer. Authors: The femoral cuticle in beetles can be of different construction. However the study of the femoral cuticle in beetles and its comparison with that of other insects was out of the scope of the present paper. In this study we aimed to describe the newly discovered lubrication-based friction-reducing mechanism and check its effectiveness experimentally. We believe that the possible difference in the structure of the femoral cuticle cannot affect the generality of the work since the lubricant is found in the cockroach species as well.
Referee: 3 Comments to the Author(s) This is a very interesting study and I recommend publication. However, I want the authors to add some more information about the friction experiment: Add to the main paper the sliding speed and the nominal contact pressure (the information is now only in the supplementary materials). Authors: The information is added.
Also add information about what these quantities are for the insect. Authors: The information is added.
Some insects (and the tree frog) use fluids for adhesion and in those cases the fluid is likely pulled spontaneously into the contact area by capillary effects. But if the lubricant in the present case is in a solid state how is it injected into the leg joints in the narrow pore channels? It appears to me that this cannot occur spontaneously by capillary effects but may require a big force or pressure which is generated how? Authors: This is a very interesting question. At the moment the mechanism of delivery the viscous or semisolid lubricant to the cuticular surface in unclear. In this first paper about lubrication-based friction-reducing mechanism we tried to avoid any speculations on this but focused on the experimental checking of the lubricative properties of the extruded substance. Undoubtedly this question will be addressed in our further studies of the lubricant in the insects' joints.

Appendix B
Review on RSPB-2021-1065 Insects use lubricants to minimize friction and wear in leg joints by Konstantin Nadein, Alexander Kovalev, Jan Thøgersen, Tobias Weidner & Stanislav Gorb A long-term mystery uncoveredthe lubrication of beetle leg joints! Congratulations to the authors on a meaningful, well-illustrated manuscript! Several minor issues to be raised … The Title "Insects use lubricants to minimize friction and wear in leg joints" does not fit the provided study and obtained results. This was done, and which is shown: "Darkling beetles use lubricants to minimize friction and wear in leg joints". -In this context, the 6 other beetle and a cockroach species mentioned in the Supplementary Material are rather confusing and not referred to in the main body of the manuscript. Authors: Thank you very much! Indeed, the experimental part of the manuscript deals with the darkling beetle Zophobas. However, the lubricant was found in cockroaches and other beetles as well, not in the darkling beetle only. The presence of the lubricant in the leg joints of other insect species studied is a fact that we found significant to publish. Beetles and cockroaches are insects (actually from two major groups -Hemimetabola and Holometabola) and therefore to the large extent can represent Insecta. In our opinion, restriction the title to the only darkling beetle may inevitably (and wrongly) suggest that only this insect species possesses lubricant. There are also other reasons in favour of the chosen title. First, the reason, why only the lubricant from legs of darkling beetle Zophobas was tested, is that it was the only one possible to collected in a sufficient amount. The lubricant of other insects is produced in smaller amounts that were impossible to collect and test. Perhaps in the future, with involvement of other equipment, it will be possible to collect and test the lubricants from the other insects. Second, the electron microscopy data on 6 other beetles and a cockroach support the hypothesis of the broad (or even universal) distribution of the lubricating-based friction-reducing mechanism in the insects' leg joints. It means it is not an occasional, unique or deviant phenomenon found in the darkling beetle Zophobas only. Third, the title initiates and promotes the further interest in studying lubricant-based mechanism of friction-minimisation in joints of insects that for a long time has been overlooked.
The Introduction is succinct, however, quite short-cut. In particular, related to arthropod joints and cuticle one to three further statements could be included, such as about epicuticular grease and insect secretion in general. This aspect in comparison with the present results would still emphasize the significance of the author's findings. Authors: We provided short general statements about cuticle, epicuticular grease and insect secretion in general.
Also, the special structures covering the joint and/or joint edge surfaces could be considered, discussing the "multifunctionality" of the joints unifying lubrication on the one hand and friction enhancement on the other handthis role of the structures on the joint cuticle counterparts is also worth to be considered in illustrations and discussion. Authors: We did not mention 'multifunctionality' but 'multi-adaptability' which is discussed in the manuscript. Indeed, the contacting surfaces in joints are of different structure from the smooth to variously textured. In fact, the variety of textured surfaces is huge from species to species (or at least from genus to genus). Therefore, it seems not possible to characterise all this in detail in such a short paper. The 'speciality' and role of these structures in leg joints is out of the scope of the present paper. Nevertheless, the tribological properties of the joints' surfaces are currently in the focus of our current project.
The question arises about the state, condition, and age of the tested animals. Do aged ones secrete a similar lubricant and similar amounts? Authors: We did not test the lubricant production or properties regarding to the age, state and/or condition of animals since it was out of the scope of our study. The tested adults were mature enough (at least two or three weeks or elder). Lubricant production varies greatly from individual to individual and even from leg to leg of the same individual. It may supposedly be connected with the physiological state of an individual, its physical activity, and many other reasons which are awaiting for study.
The structure of secretion, in particular in Fig. 1d, f, l looks unusual compared to other viscoelastic fluids found in insects and plants so far. They remember bacteria-shaped particles; maybe characteristic for proteinaceous material as detected for the joint lubrication? -Here some comparison with the state of literature about shapes of proteinaceous structures would be value-adding and supporting the innovation of the present study. Authors: Here in this paper we aimed to report on the lubricating-based mechanism in insect joints for the first time. The structures under consideration are definitely not bacteria: one of the co-authors has some experience in SEM studies on different kinds of bacteria. We are not sure, which literature about which proteinaceous structures is suggested.

Detailed comments:
Authors: All the corrections highlighted blue by the Reviewer are made. Here further are the comments requiring the responses. page 4, Results Please provide the distinct values for "At the room temperature" and "at the room temperature". Authors: The value of the room temperature is added.
is (substantially/remarkably) protein-based -Mostly doesn´t fit the rather qualitative results. Authors: The ATR-FTIR analysis is indeed qualitative and we believe is an appropriate way to understand the principal chemical composition of the lubricant at the present state of knowledge and study. Quantitative analysis at the moment is hard to accomplish since the amount of lubricant is quite small for the most of the available methods. Nevertheless, based on the intensity of the resonances it is possible to suggest on the relative abundance of this or that types of molecules. According to the obtained spectra one can conclude that the set of peaks is a clear 'fingerprint' of proteins and the other compounds (if any) are in the significantly smaller amount undetectable by this very sensitive method.
Please specify "it": "It allows" -What allows? Authors: Changed to 'This property'.
page 5, Discussion, (b) Here, several examples are given for the presence of a lubricant in insect leg joints. -This fact should be mentioned in the Introduction to avoid the implication of a new finding which has been already previously supported. Authors: Yes, there are reports on the presence of some amorphous substances in the insect joints, but these have never been structurally and experimentally studied. There were only suppositions (as ideas or hypotheses) without factual observational evidences or experiments. The insertion 'such as beetles and cockroaches' is added to the Introduction.  Here, all the compounds should be mentioned and indicated to underline the chemical complexity of the secretion mixture. Authors: All the components that were possible to detect by the ATR-FTIR are mentioned in the text. They are presented by the peaks of different intensity and assigned to resonances that were identified as different components of proteins and, presumably, lipids. Such a set of peaks at the specific resonances bands is a 'fingerprint' of the specific type of substance as proteins in this case.   Figure 5 could benefit from some more distinct labels, e.g., relating the images in a, c, and e to the schemes in b, d, and f (arrows, colors, or similar …)