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Evolution of insect flight muscle structure as revealed by the high-flux X-ray of SPring-8

Release Date
14 Dec, 2005
  • BL40XU (High Flux)
Dr. Hiroyuki Iwamoto of JASRI/SPring-8 (Director General, Akira Kira) and his research team clarified the process of evolution of the structure of insect flight muscle, by using the high-flux X-ray microbeam of the High Flux Beamline BL40XU and the technique of diffraction recording from frozen-hydrated biological specimens.

December 14, 2005

 Dr. Hiroyuki Iwamoto of JASRI/SPring-8 (Director General, Akira Kira) and his research team clarified the process of evolution of the structure of insect flight muscle, by using the high-flux X-ray microbeam of the High Flux Beamline BL40XU and the technique of diffraction recording from frozen-hydrated biological specimens.
The wingbeat frequency of advanced insects like mosquitoes sometimes reaches 500 Hz, a frequency that cannot be achieved by repeating ordinary contraction-relaxation cycles. The flight muscles of these insects are capable of high-frequency vibrations while staying activated. Structurally, the arrangement of the contractile proteins in flight muscle cells is so regular that their contractile machinery is often compared to a crystal. Previously, the same research team has shown that, in the flight muscle of a bumblebee, the regularity is not confined to a single sarcomere but extends throughout the entire length of the muscle cell (millimeters in length) (see the SPring-8 topic of Aug. 6, 2002, entitled "X-ray diffraction recordings from a single myofibril"). To examine how this long-range regularity has developed in the process of evolution, the research group moved on to record and analyze >4600 diffraction patterns from 50 insect species, covering all the major winged-insect orders. The results show that the occurrence of the long-range regularity is generally restricted to advanced insect groups. However, the results also show that some extent of structural ordering has developed sporadically among well-flying lower insects.
This research article was published online ahead of print in Proceedings of the Royal Society Series B. Dec. 13, 2005.

"Evolution of long-range myofibrillar crystallinity in insect flight muscle as examined by X-ray cryomicrodiffraction."
H. Iwamoto, K. Inoue and N. Yagi.
Proc. R. Soc. B, First Cite Early Online (13 December, 2005); 0962-8452 (Paper) 1471-2954 (Online) 

   Insects are the most prosperous group of animals on the earth, and the keys to the prosperity are their small body sizes and their ability of flight. The muscle used for flight, or flight muscle, is classified as a cross-striated muscle, like vertebrate skeletal muscle (Fig. 1). Functionally, the flight muscles of insects are classified into two groups, based on their modes of operation. One is called "synchronous", and found in relatively primitive insects like locusts. In this mode of operation, a single nerve impulse elicits a single contraction-relaxation cycle, causing a single wingbeat (Fig. 2a). The flight muscle can usually shorten 10% of its length or more. These features are shared by vertebrate skeletal muscles. The maximal wingbeat frequency achievable in this operation is believed to be ~100 Hz. However, insects smaller than a locust very often need wingbeat frequencies much higher than this.
   The other mode of operation is called "asynchronous", and is distributed among advanced insects such as flies and bees. In this mode, the flight muscle is kept activated by low-frequency nerve impulses, and the muscle undergoes high-frequency vibrations by utilizing the resonation of the thoracic exoskeleton. Thus, the nerve impulses are much less frequent than the wingbeats (Fig. 2b). The length change of the flight muscle is only ~3% during flight. To be able to precisely control the contractile force by such a small length change, a regular arrangement of contractile proteins would be required within a sarcomere1). In fact, the arrangement of the contractile proteins within a sarcomere of "asynchronous" flight muscle is known to be so regular that it can be regarded as a protein crystal. These features are not observed in vertebrate skeletal muscle, and it is clear that the "asynchronous" muscle is specialized for the purpose of flight.

Progress leading to current results
   A further surprise came from a study using the flight muscle of a bumblebee, a typical insect with asynchronous operation. When a single myofibril2) within its flight muscle cell was irradiated by an X-ray microbeam (diameter, ~2 µm), it generated a diffraction pattern that came from a single hexagonal lattice of contractile proteins (see the topic of Aug 6, 2002). This means that the lattice plane of the protein crystal is strictly preserved along the entire length of the myofibril (~3 mm or 1000 sarcomeres in series). In other words, the whole myofibril can be regarded as a huge, single protein crystal. For comparison, the protein crystals that researchers grow to determine atomic structures are less than 1 mm. Insects grow much larger crystals in their own bodies. Crystals that can contract.
   In ordinary X-ray diffraction experiments, the incident X-ray beam is irradiated perpendicular to the muscle cell. On the other hand, in the experiment described above, the X-ray microbeam was irradiated along the axis of the muscle cell (end-on irradiation). By doing this, the research group was able to irradiate only one of the innumerable myofibrils in the cell without mechanically isolating it. At that time, however, beams were irradiated directly on raw hydrated materials, which were vulnerable to radiation damage and dehydration. To overcome this problem, the research group has developed a technique to quick-freeze hydrated biological specimens and record diffraction patterns by irradiating X-ray microbeams while the specimens are kept at liquid nitrogen temperature (-199°C) (see the SPring-8 topic of June 29, 2005, entitled "X-ray diffraction recordings from a single sarcomere within an isolated myofibril"). This technique makes specimens much more resistant to radiation damage and dehydration, enabling long-time recordings under stable conditions.

Summary of current results
   As mentioned above, the previous study revealed that the myofibrils of bumblebee flight muscle are giant single protein crystals. Then a question arises as to how this giant protein crystal arose in the process of insect evolution. To address this question, the research group recorded myofibrillar X-ray diffraction patterns from the flight muscles of as many insect species as possible, from the most primitive to the advanced. The methods for recording were the same as described above --- to quick freeze the flight muscle cells and irradiate end-on with X-ray microbeams while keeping the specimens at the liquid nitrogen temperature.
   Figure 3 shows examples of myofibrillar diffraction patterns recorded in this way. The diffraction patterns recorded from insects belonging to Hymenoptera (bees), Diptera (flies), Coleoptera (beetles) and Heteroptera (true bugs) consist of spot-like reflections arranged in a hexagonal array, indicating that these insects have the single-crystal type myofibrils. All of these insects are asynchronous. Large coleopterans like longhorn beetles may beat their wings slowly, and they would not need the asynchronous operation. However, all of the examined insects belonging to these orders had the single-crystal type myofibrils. It is believed that the ancestral asynchronous insects were very small in body size and had to beat their wings at high frequencies. Some of their descendants became large, but they kept the original design of their ancestors.
   By contrast, in Lepidoptera (butterflies and moths), Orthoptera (grasshoppers) and other ‘synchronous’ orders, the diffraction pattern consists of blurred concentric circles, meaning that the lattice planes are not in register. However, the synchronous cicadas (Homoptera) show partial register of lattices, and in rare examples the patterns show signs of hexagonal lattices. Cicadas may be in the middle of evolution towards asynchronous insects, or alternatively, are losing the features of asynchronous muscle because of their enlarged body sizes. The flight muscles of dragonflies (Odonata) are worth noting, because they are among the most primitive winged insects but their diffraction patterns show clear signs of hexagonal lattices. Odonates have a life style which heavily depends on flight. Lepidopterans show poorly registered lattices with a notable exception of Sphingids (hawkmoths). Their diffraction patterns consists of numerous spots, which turn out to have come from several overlapped hexagonal lattices upon analysis. Hawkmoths feed on nectar of flowers while hovering in the air.
   To summarize, the occurrence of the single crystal-type myofibrils largely coincides with asynchronous insects, but some of the synchronous insects show partially crystallized myofibrils, especially when they have a lifestyle which depends heavily on flight. Possibly the single crystal-type myofibrils can transmit force efficiently, and this type of myofibrils may arise many times independently in the process of evolution, whenever they are needed. Even in asynchronous insects, their leg muscles show poor crystallinity, meaning that the highly crystalline myofibrils are structures specific to flight muscle. Finally, the extents of myofibrillar crystallinity in various insects are summarized in Table 1.

   The significance of the present achievement is that they are the first major scientific results obtained by using the SPring-8-developed technique of high-flux X-ray microbeam in combination with quick freezing: Clarification of one of the processes of evolution of the most flourishing animals on the earth. By reducing their body sizes, the existing insect species have increased the efficiency of body functions to a maximum level. One example of this is the adoption of asynchronous flight muscle, which have eliminated the wasteful use of energy due to repetitive contraction-relaxation cycles. Its structure must also be optimized for this purpose, and one of the ways to achieve this would be to make each myofibril a giant single protein crystal. Humans should have a lot to learn from insects, which achieved a maximum degree of efficiency in the long history of evolution. This work was supported by Grant-in-Aid, Grant No. 15500294, Ministry of Education, Culture, Sports, Science and Technology and Special Coordination Funds of the Ministry of Education, Culture, Sports, Science and Technology, Japan.


Figures and Table

Fig. 1 Structure of vertebrate skeletal muscle (striated muscle)
Fig. 2 Mode of operation of insect flight muscle
Fig. 3 Myofibrillar diffraction patterns recorded from the flight muscles of various insects
Table 1 Mode of operation of flight muscle and myofibrillar structure in various insects


(1) Sarcomere:
the minimal functional unit of muscle contraction. Its size is 2 - 3 µm in both diameter and length. Within the sarcomere, the myosin and actin filaments slide past each other to generate force.

(2) Myofibril:
a long fibril made of a large number of sarcomeres (see above) connected in series. A single muscle cell (muscle fiber, 100 - 300 µm in diameter) contains a large number of myofibrils.


For further information, please contact:
for research,
   Dr. Hiroyuki Iwamoto
   Research & Utilization Division

 phone: +81-(0)791-58-2506
fax: +81-(0)791-58-2512
e-mail: iwamoto@spring8.or.jp
for SPring-8,
   Public Relations Office, JASRI

 phone: +81-(0)791-2785
fax: +81-(0)791-2786
e-mail: hara@spring8.or.jp

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