US 2010.0025527A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2010/0025527 A1 Lal et al. (43) Pub. Date: Feb. 4, 2010

(54) SURGICALLY IMPLANTED Related U.S. Application Data MICRO-PLATFORMS AND MICROSYSTEMS IN ARTHROPODS AND METHODS BASED (60) Provisional application No. 60/874,433, filed on Dec. THEREON 12, 2006. Publication Classification (76) Inventors: Amit Lal, Ithaca, NY (US); John Ewer, Valparaiso (CL): Ayesa Paul, (51) Int. Cl. Ithaca, NY (US); Alper Bozkurt, B64C 33/00 (2006.01) Ithaca, NY (US) B64C39/02 (2006.01) B25.J II/OO (2006.01) Correspondence Address: (52) U.S. Cl...... 244/1 R: 244/22; 901/50 MARJAMA MULDOON BLASAK & SULL VAN LLP (57) ABSTRACT 250 SOUTHCLINTON STREET, SUITE 300 A method is provided for producing an arthropod comprising SYRACUSE, NY 13202 (US) introducing a microsystem such as a MEMS device into an (21) Appl. No.: 12/518,847 immature arthropod under conditions that result in producing an adult arthropod with a functional microSystem perma (22) PCT Filed: Dec. 11, 2007 nently attached to its body. A method is also provided for producing a robotic apparatus. The method can comprise (86). PCT No.: PCT/US07/25437 introducing a microsystem such as a MEMS device into an immature arthropod under conditions that result in producing S371 (c)(1), a robotic apparatus with the microsystem permanently (2), (4) Date: Sep. 24, 2009 attached to the body of the adult arthropod.

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SURGICALLY MPLANTED mately been unsuccessful (Ellington C P 1999. The novel MICRO-PLATFORMS AND MICROSYSTEMS aerodynamics of insect flight: applications to micro-air IN ARTHROPODS AND METHODS BASED vehicles. The Journal of Experimental Biology. 202:3439 THEREON 348), however, because the power sources needed for power ing flight have not been Sufficiently miniaturized, even CROSS-REFERENCE TO RELATED though micro-scale actuators such as MEMS (Micro-Electro APPLICATIONS Mechanical Systems), have existed for decades. 0001. This application claims priority to and the benefit of 0006 Insects can provide models for robotics as “biobots' co-pending U.S. provisional patent application Ser. No. (Crary, S. B., Moore, T. E., Conklin, T. A., Sukardi, F., and 60/874,433, filed Dec. 12, 2006, which is incorporated herein Kotiditschek, D. E. 1996. Insect Biorobotics: Electro-neural by reference in its entirety. control of cockroach walking, Abstr. IEEE Int. Conf. Robot STATEMENT REGARDING FEDERALLY ics and Automation, Workshop WT3, Bio-Mechanics, pp. SPONSORED RESEARCH ORDEVELOPMENT 42-54; Louden, C. 1995. Insect morphology above the molecular level: biomechanics. Ann. entomol. Soc. Am..., 88: 0002 The disclosed invention was made with government 1-4), in which an intact (or nearly intact) biological system is Support under grants from the National Science Foundation incorporated into the design of a microdevice Such as a and the Defense Advanced Research Projects Agency MEMS device (Cary 1996 supra; Louden 1995 supra). (DARPA), Microsystems Technology Office. The govern Insects are autonomous flying machines and have aerody ment has rights in this invention. namic performance Superior to that of existing unmanned mechanical flying machines. Insects also exist in diverse REFERENCE TO APPENDIX shapes and sizes. 0003) Not applicable 0007. One important challenge that has not been solved, however, has been the permanent attachment of devices to 1. TECHNICAL FIELD adult insects. Several research groups have glued telemetric and electronic devices onto adult insects to track the insects 0004. The present invention relates to methods for produc movements and migratory paths (Riley J.R. 2005. The flight ing arthropods bearing functional and permanently attached paths of honeybees recruited by waggle dance. Nature 435(12 Microsystems such as MEMS devices. The invention also May 2005): 205-207: Riley J. R. Valeur P. Smith A D, Rey relates to methods for permanently attaching devices to adult nolds DR, Poppy G. M. 1998. Harmonic radar as a means of insects. The invention also relates to robotic apparatuses or tracking the pheromone finding and pheromone—following biobots comprising biological and non-biological system flight in male moths. Journal of Insect Behavior 11(2): 287 components. The invention further relates to methods for 296), and have attached miniature “backpacks' for environ producing robotic apparatuses or biobots comprising biologi mental monitoring, wireless communication, or biobotic cal and non-biological system components. The invention manipulation of behavior (Crary 1996 Supra; Mohseni P. also relates to microprobe microSystem platforms used with Nagarajan K. 2001. An ultralight biotelemetry backpack for robotic apparatuses or biobots. recording EMG signals in Moths. IEEE Transactions on Bio medical Engineering 48(6): 734-737; Kuwana Y. Ando N., 2. BACKGROUND OF THE INVENTION Kanzaki R., and Shimoyama 1. 1999. A radio telemetry sys 0005 Over the past four decades, many attempts have tem for muscle potential recordings from freely flying insects. been made to develop Small (centimeter-scale) autonomous Proceedings of IEEEBEMS/EMBS conf, Atlanta, Ga., Oct. flying machines (micro-aerial vehicles “MAV' or unmanned 1999: 846; Kutsch W. Schwarz G, Fischer H, and Kautz H. aerial vehicles “UAV) for applications ranging from recon 1993. Wireless transmission of muscle potentials during free naissance in the battlefield to environmental monitoring. Sig flight of locust. The Journal of Experimental Biology 185: nificant advances in microfabrication technology have made 367-373). it possible to realize mm-scale low-power microSystems 0008 Attachment of devices to insects as adults, however, capable of carrying out a wide array of sensing and actuation leads to reduced flying agility, presumably because the insect tasks. These attempts to create mechanical MAVs have ulti perceives the load as a foreign weight (Table 1).

TABLE 1. List of advantages and disadvantages of using robotic MAV and insects as payload carriers. Advantage Disadvantage MAV manual robotics 1. Manipulation of flight is 1. Expensive to design possible. and manufacture 2. Loss of MAV not due to versatile models. external natural stimuli 2. Useful load (pheromone, ultrasound) disappears with decrease in MAV size. Insect as Superficial 1. Versatile MAV models. 1. Lack of control of payload Attachment 2. Can carry a load of half its flight direction. carriers muscle weight 2. Unreliable and prone to dislodge. US 2010/0025527 A1 Feb. 4, 2010

TABLE 1-continued List of advantages and disadvantages of using robotic MAV and insects as payload carriers. Advantage Disadvantage 3. Lightweight power Source not available. Surgical 1. Permanent structure for 1. Lack of control of Attachment attachment of payload. flight direction. 2. Versatile MAV models. 2. Lightweight power 3. Can carry a load of half its Source not available muscle weight. 4. Acclimatization to the extra load is easier when load is attached at a pre-adult stage.

0009 Telemetric devices glued onto insects are temporary 0023. A method is also provided for producing a robotic attachments, and have been reported to fall off when the apparatus comprising: insect is in a confined space or foraging in vegetation. Han 0024 a. introducing a microSystem into an immature dling a mobile and active adult insect to attach a payload is arthropod (or portion thereof) under conditions that result in also difficult for mass production. Furthermore, with typical production of a robotic apparatus, adult insect life spans of 2-3 weeks, delivery to the site of 0025 wherein the robotic apparatus comprises: deployment at the correct time is challenging. 0026 i. the microsystem, and 0027 ii. a biological system, the biological system com 0010. There is therefore a need in the art for methods for prising the adult arthropod (or portion thereof), permanently attaching devices to adult insects. There is also a 0028 and wherein the microsystem is attached to the body need in the art for unmanned, micro-aerial vehicles with of the adult arthropod (or portion thereof). permanently attached telemetric or electronic devices. 0029. In one embodiment, the microsystem can be perma 0011 Citation or identification of any reference in Section nently attached to the body of the adult arthropod (or portion 2, or in any other section of this application, shall not be thereof). considered an admission that such reference is available as 0030. In another embodiment, the biological system can prior art to the present invention. comprise a portion of the adult arthropod capable of generat ing or controlling a physiological function of the adult arthro pod, and the robotic apparatus is capable of carrying out the 3. SUMMARY OF THE INVENTION physiological function (or a portion or Subfunction of the physiological function). 0012. A method is provided for producing an arthropod 0031. In another embodiment, the physiological function comprising introducing a microsystem into an immature can be selected from the group consisting of locomotion, arthropod under conditions that result in production of an vision, hearing, chemosensation, temperature sensation, adult arthropod, wherein the microsystem is functional and is mechanoreception or pressure sensation, acceleration sensa attached to the body of the adult arthropod. In one embodi tion, gravity sensation, magnetic sensation, feeding, diges ment, the microSystem can be permanently attached. tion, mating and oviposition. 0013. In another embodiment, the immature arthropod can 0032. In another embodiment, the microsystem can acti be an insect, crustacean or arachnid. vate a muscle (or muscle group) that regulates the physiologi 0014. In another embodiment, the insect can be a holom cal function. etabolous insect. 0033. In another embodiment, the physiological function 0.015. In another embodiment, the insect can be a hemi can be locomotion. metabolous insect. 0034. In another embodiment, the muscle (or muscle 0016. In another embodiment, the immature arthropod can group) can be flight muscle and the locomotion can be flying. be a larva. 0035. In another embodiment, the muscle (or muscle 0017. In another embodiment, the immature arthropod can group) can be leg muscle and the locomotion can be walking. be a pupa. 0036. In another embodiment, the immature arthropod can 0018. In another embodiment, the immature arthropod can be an insect, crustacean or arachnid. be a nymph. 0037. In another embodiment, the insect can be a holom etabolous insect. 0019. In another embodiment, the introducing step can be 0038. In another embodiment, the insect can be a hemi Surgical implantation. metabolous insect. 0020. In another embodiment, the microsystem can be an 0039. In another embodiment, the immature arthropod can attachment apparatus. be a larva. 0021. In another embodiment, the microsystem can be a 0040. In another embodiment, the immature arthropod can microplatform. be a pupa. 0022. In another embodiment, the microsystem can be a 0041. In another embodiment, the immature arthropod can MEMS device. be a nymph. US 2010/0025527 A1 Feb. 4, 2010

0042. In another embodiment, the introducing step can be 0068. In one embodiment, the power layer can comprise a Surgical implantation. power source and a Switch. 0043. In another embodiment, the microsystem can be an 0069. In another embodiment, the power source can be a attachment apparatus. battery. 0044. In another embodiment, the microsystem can be a 0070. In another embodiment, the power layer can com microplatform. prise a printed circuit board (PCB). 0045. In another embodiment, the microsystem can be a 0071. In another embodiment, the control layer can com MEMS device. prise a PCB and a microcontroller functionally connected to 0046. An apparatus is also provided comprising: the PCB. 0047 a. a microsystem; and 0072. In another embodiment, the control layer can com 0048 b. a biological system, the biological system com prise an LED. prising an adult arthropod (or portion thereof), wherein the 0073. In another embodiment, the probe can be a micro microsystem is attached to the body of the adult arthropod (or fabricated silicon probe. portion thereof). 0074. In another embodiment, the probe can be positioned 0049. In one embodiment, the microsystem can be perma between the control layer and the power layer. nently attached to the body of the adult arthropod (or portion 0075. In another embodiment, the probe can comprise a thereof). plurality of tips, wherein a member of the plurality can be 0050. In another embodiment, the biological system can separated from a second member of the plurality by a distance comprise a portion of the adult arthropod capable of generat that corresponds to the distance between two cells, tissues or ing or controlling a physiological function of the adult arthro physiological systems of interest. pod, and the robotic apparatus can be capable of carrying out 0076. In another embodiment, the two physiological sys the physiological function (or a portion or Subfunction of the tems can be two muscles (or muscle groups). physiological function). 0077. In another embodiment, the two physiological sys tems can be two neurons (or neural systems, neural centers, or 0051. In another embodiment, the physiological function ganglia). can be selected from the group consisting of locomotion, 0078. In another embodiment, the probe can be activated vision, hearing, chemosensation, temperature sensation, by an algorithm whereby sequential electrical pulses are sent mechanoreception or pressure sensation, acceleration sensa to the probe tips. tion, gravity sensation, magnetic sensation, feeding, diges 0079. In another embodiment, the algorithm can comprise tion, mating and oviposition. outputting time-scheduled square current or Voltage pulses. 0052. In another embodiment, the microsystem can acti 0080. In another embodiment, the LED on the control vate a muscle (or muscle group) that regulates the physiologi layer can be used to monitor electrical excitation generated by cal function. the probe. 0053. In another embodiment, the physiological function can be locomotion. 4. BRIEF DESCRIPTION OF THE DRAWINGS 0054. In another embodiment, the muscle (or muscle group) can be flight muscle and the locomotion can be flying. I0081. The present invention is described herein with ref 0055. In another embodiment, muscle (or muscle group) erence to the accompanying drawings, in which similar ref can be leg muscle and the locomotion can be walking. erence characters denote similar elements throughout the sev 0056. In one embodiment, the immature arthropod can be eral views. It is to be understood that in Some instances, an insect, crustacean or arachnid. various aspects of the invention may be shown exaggerated or 0057. In another embodiment, the insect can be a holom enlarged to facilitate an understanding of the invention. etabolous insect. 0082 FIG.1. Schematic illustration of one embodiment of 0.058. In another embodiment, the insect can be a hemi the method of the invention. Arrows indicate the different metabolous insect. stages during the life cycle of a moth (a holometabolous 0059. In another embodiment, the immature arthropod can insect) at which a microSystem (a titanium or silk wire, a be a larva. silicon platform or a microSystem as a backpack) is Surgically 0060. In another embodiment, the immature arthropod can inserted. be a pupa. I0083 FIGS. 2a-d. Schematic diagrams of (a) a titanium balsa micro-cargo platform, (b) a Manduca sexta moth pupa 0061. In another embodiment, the immature arthropod can with a platform and wire inserted in the body, (c) an adult be a nymph. Manduca sexta moth with a platform, and a photograph of (d) 0062. In another embodiment, the introducing step can be an adult moth with a platform attached with titanium wires. Surgical implantation. See Example 6.1. 0063. In another embodiment, the microsystem can be an I0084 FIGS. 3a-d. Schematic diagram of (a) a silicon chip attachment apparatus. inserted in a Manduca sexta pupa and photographs of the 0064. In another embodiment, the microsystem can be a Ventral abdomen (b) the pupa and (c) the newly emerged microplatform. adult. (d) The chip bonded intimately with the moth's body 0065. In another embodiment, the microsystem can be a tissue when inserted at the pupal stage, and when the chip was MEMS device. removed from the body, tissue was torn away along with the 0066. In another embodiment, the physiological function chip. See Example 6.1. can be flying and the apparatus is a MAV. I0085 FIGS. 4a-d. Photographs of (a) a titanium and balsa 0067. A microprobe microsystem platform is also pro wood micro-cargo platform, (b) late stage Manduca sexta vided. The microprobe microSystem platform can comprise a pupa bearing a titanium and balsa wood micro-cargo platform power layer; a probe layer, and a control layer. attached to the abdomen, (c) newly emerged adult with the US 2010/0025527 A1 Feb. 4, 2010 titanium and balsa wood micro-cargo platform, and (d) scan (0098 FIGS. 17a-b. (A) Experimental setup of tethered ning electron micrograph (SEM) showing tissue growth on moth wing actuation using titanium wires. (B) Schematic the titanium wire inside the abdomen of the adult (wire was diagram of the setup. See Example 6.3. inserted at the late pupal stage). Such tissue growth produces 0099 FIG. 18. Measured muscle actuation current thresh a strong and reliable attachment of the microsystem due to old (log, mA) and power (log, mW) VS. Stimulus intervals tissue Support. See Example 6.1. (log, ms) of two different subject moths. Moth 1 (diamonds). I0086 FIGS. 5a-d. Photographs of a silicon chip (2x4 mm) Moth 2 (squares). See Example 6.3. inserted into (a) the Ventral abdominal segment of a late pupa, 0100 FIG. 19a-c. Assembled platforms using microfabri (b) schematic of a silicon insert in a late pupa, (c) the adult cated probes, microcontroller and batteries. A. Front view. B. after emergence with the silicon insert, and (d) a SEM pho Back view. C. Schematic diagram illustrating the assembly tograph of a silicon chip in a two-day old adult (silicon chip and its components. See Example 6.3. inserted at the late pupal stage). See Example 6.1. 0101 FIG. 20. Microprobe fabrication process. See 0087 FIGS. 6a-d. Schematic diagram of (a) silk thread Example 6.3. insert transplanted into a fifth instar Manduca sexta larva. 0102 FIG. 21. Time-scheme of one cycle of the flight Photographs of (b) silk thread inserted in a larva, (c) silk muscle excitation algorithm. See Example 6.3. thread inserted in a pupa and (d) silk thread in an adult after 0103 FIG. 22. Power consumption of each component of emergence. Such silk thread inserts can be used as tethers to the platform. See Example 6.3. attach a micro-cargo to the adult moth. See Example 6.1. 0104 FIG. 23a-c. A. X-ray image of adult moth thorax, 0088 FIGS. 7a-c. Schematic diagram of (a) insertion of a showing inserted silicon probes. B. Image processing of the titanium wire through the posterior end distal end of the image shown in (A), to show components of high absorbance, abdomen of a Manduca sexta pupa and running anterior indicating growing tissue. C. Photograph showing the orien posterior in the abdomen. (b) X-ray scanned image of the tation of the moth in (A) and (B). Arrows indicates the emerged adult moth with the wire, (c)MCT-scanned image of inserted silicon probes. See Example 6.3. a cross-section of the adult through the thorax showing the wire embedded within the flight muscle tissue. See Example 5. DETAILED DESCRIPTION OF THE 6.1. INVENTION 0089 FIG. 8. Circuit board with a miniature LED-based 0105. A method is provided for surgically implanting a device to regulate direction of flight in an adult moth. See microSystem into an arthropod at a developmental stage prior Example 6.1. to adulthood, without adversely affecting the arthropod's life 0090 FIG. 9. Schematic diagram of the Manduca sexta span, behavioral or locomotor capacity. To solve the problem life-cycle, indicating the site and stage for insertion of a associated with attaching devices to adult arthropods, the biocompatible microfabricated microcontroller-based elec method can take advantage of the arthropod's life cycle to trical probe for flight muscle actuation (shown with arrows). implant and attach (for example, permanently attach) one or See Example 6.2. more microSystems (or an attachment site or platform for a 0091 FIG. 10. Plot of current threshold (mA) for muscle microsystem, hereinafter also referred to as a “microsystem') activation (mA) versus various stimulus durations (ms). See into an immature stage of the arthropod for eventual deploy Example 6.2. ment at the adult stage. 0092 FIGS. 11a-c. Microfabricated platform. (A) front 0106. In one embodiment, a method is provided for pro views. (B) back view. (C) schematic of different layers of the ducing an arthropod comprising introducing a microSystem platform. See Example 6.2. (e.g., a MEMS device or an attachment for a MEMS device) into an immature arthropod under conditions that result in 0093 FIG. 12. Schematic diagram of the fabrication pro production of an adult arthropod. The microSystem can be cess of a microfabricated microcontroller-based electrical functional and attached (e.g., permanently attached) to the probe. See Example 6.2. body of the adult arthropod. 0094 FIG. 13. SEM image of the tip of a microfabricated 0107 Arthropods grow and develop by periodically molt microcontroller-based electrical probe, with an enlargement ing to replace their external exoskeleton. The replacement of of the ground and actuation pads. The asymmetry in obtaining the exoskeleton that takes place during a molt allows for the the sharp tip resulted from mask asymmetry. See Examples repair of wounds that are caused by the permanent implanta 6.2 and 6.3. tion of external (anchoring) devices or internal devices. A 0095 FIGS. 14a-d. Photographs of (A) transplanted plat method is provided that utilizes the process of arthropod form comprising a probe inserted in the pupa and (B) the development, which involves periodic molting, to enable the emerged adult bearing the platform. (C) Transplanted glass implantation of a microSystem without injuring or traumatiz capillary in the pupa and (D) the emerged adult with the glass ing the arthropod. Such trauma is commonly induced by capillary. See Examples 6.2 and 6.3. attempting to attach a microSystem or microdevice to an 0096 FIG. 15. Photograph of anterior transverse dissec adult-stage arthropod. tion of the thorax of an adult moth with transplanted platform 0108. A method is also provided for producing a robotic (left) and schematic view of a cross-section of the thorax apparatus comprising: through the dorsolongitudinal and dorsoventral flight 0109 introducing a microsystem into an immature arthro muscles (right). In the photograph on the left, the transplanted pod (or portion thereof) under conditions that result in pro probes are shown with arrows and the flight muscles are duction of a robotic apparatus, wherein the robotic apparatus encircled. See Examples 6.2 and 6.3. comprises: 0097 FIG.16. Schematic of lifespan of a moth, indicating 0110 i. the microsystem, and stage at which the microsystem and microcapillary was 0.111 ii. a biological system, the biological system inserted (indicated by the arrow). See Example 6.3. comprising the adult arthropod (or portion thereof), US 2010/0025527 A1 Feb. 4, 2010 and wherein the microSystem is attached (e.g., permanently butterflies and moths, have powerful flight muscles. Using the attached) to the body of the adult arthropod (or portion method provided, such insects can be equipped to carry a thereof). microsystem (e.g., a MEMS device, a telemetric device). In 0112 A robotic apparatus or “biobot' is also provided. one embodiment, the microsystem can weigh 0.001-1.0 g. In The robotic apparatus can comprise both a biological system another embodiment, the microsystem can weigh 1-5 g. In and non-biological components. another embodiment, the microsystem can weigh 5-10g. In 0113. A microprobe microsystem platform is also pro another embodiment, the microsystem can weigh at least 10 vided that comprises a power layer; a probe layer, and a 9. control layer that can be implanted into an arthropod and can I0127 5.2. Introduction of Foreign Objects into Arthro become functionally and permanently attached to the body of pods the adult arthropod. I0128 Methods are provided for introducing a biocompat 0114 For clarity of disclosure, and not by way of limita ible foreign object into an arthropod at a stage of its develop tion, the detailed description of the invention is divided into mental cycle prior to the adult stage. The biocompatible for the subsections set forth below. eign object can a microsystem (see Section 5.3 below). 0115 5.1 Biological Systems I0129. By introducing the biocompatible object into the 0116. The biological system can be an intact arthropod arthropod during post-embryonic development and prior to Such as an insect, crustacean or arachnid. adulthood, one can take advantage of the self-healing and 0117 The biological system can also be a nearly intact tissue restructuring that occurs during postembryonic devel arthropod that is capable of a specific motor or locomotor opment to form reliable electromechanical interfaces to the function associated with the arthropod's normal behavior, arthropod body. After the adult emerges or fully develops, the Such as wing flapping, walking, crawling, Swimming, etc. microSystem is perceived as natural weight and the arthro 0118. The biological system can be capable of carrying pod's behavior is not greatly altered. out, or can be a functional unit for, a normal physiological 0.130. A method is provided for introducing a microsystem function in an arthropod, Such as locomotion (e.g., flying, (or a substrate for the attachment of a microsystem) into an walking, Swimming), vision, hearing, chemosensation, tem arthropod by Surgical implantation. According to this perature sensation, mechanoreception or pressure sensation, method. Such Surgical implantation does not adversely affect acceleration sensation, gravity sensation, magnetic sensation, the arthropod's adult lifespan. As well known in the art, feeding, digestion, mating and oviposition. arthropods, including insects, crustaceans and arachnids, can 0119 The biological system can comprise a portion of the Survive extreme Surgery (e.g., parabiosis, decapitation, iso adult arthropod capable of generating, regulating or control lated development of imaginal disks within a host) and meth ling a specific physiological function. For example, the por ods for Such Surgeries are well known in the art (see, e.g., tion of the arthropod can be the portion of the arthropod Williams, C.M. 1946. Physiology of insect diapause: the role nervous system that controls a physiological function such as of the brain in the production and termination of pupal dor locomotion, vision, hearing, chemical sensing, temperature mancy in the giant silkworm Platysemia Cecropia. Biol. Bull. sensing, pressure sensing, acceleration sensing, gravity sens Mar. biol. Lab., Woods Hole 90:234-243; Williams, C. M. ing, magnetic sensing, feeding, digestion, Oviposition, etc. In 1952. Physiology of insect diapause. IV. The brain and pro a specific embodiment, the nervous system controls, regu thoracic glands as an endocrine system in the Cecropia silk lates or is associated with wing flapping, walking, crawling, worm. Biol. Bull. Mar. biol. Lab., Woods Hole 103: 120-38: Swimming, etc. In Such embodiments, the biological system Ephrussi, B. and Beadle, G. W. 1936. A technique of trans may or may not comprise an arthropod appendage controlled plantation for Drosophila. Am. Nat. 70, 218-25). by a portion of the nervous system. I0131. In one embodiment, a substrate, microplatform, or 0120 In another embodiment, the microsystem can acti microSystem can be surgically implanted prior to the adult vate a muscle (or muscle group) that regulates the physiologi stage of the arthropod during a larval, nymphal stage or other cal function. pre-adult stage, Subsequent to hatching from the egg but prior 0121. In another embodiment, the physiological function to the adult stage. can be locomotion. 0.132. In another embodiment, a microsystem can be sur 0122. In another embodiment, the muscle (or muscle gically implanted in a holometabolous insect during a larval group) can be flight muscle and the locomotion can be flying. stage or a pupal stage of post-embryonic development or 0123. In another embodiment, the muscle (or muscle metamorphosis (FIG. 1). In a one embodiment, Surgical group) can be leg muscle and the locomotion can be walking. implantation of the microSystem can be performed in a pupal 0.124. In one embodiment, the arthropod can be a holom stage, when the pupa is immobile. etabolous (metamorphic) insect, which has four distinct life I0133. In a preferred embodiment, a microsystem can be stages: egg, larva, pupa (imago) and adult (FIGS. 1, 9 and 16), introduced into a late-stage pupa or other late developmental Such as Lepidopeterans (butterflies or moths), Dipterans stage of the arthropod. Introduction at the late pupal stage (flies), or beetles. In a specific embodiment, the insect can be usually results in higher Survival and rate of adult emergence. the hawkmoth (or tobacco hornworm moth), Manduca sexta. I0134. In a specific embodiment, a microsystem can be 0.125. In another embodiment, the arthropod can be a introduced into a moth pupa one day before emergence. hemimetabolous insect, i.e., an insect that goes through a 0.135 Implantation of a microsystem during the pre-adult series of nymphal stages prior to the adult stage, such as a stage (e.g., during the larval or pupal stages, during nymphal cockroach or grasshopper. development) does not adversely affect the insect's survival 0126 The aerodynamic performance of insects is superior or normal locomotory capability (e.g., flying, walking, Swim to existing flying machines. A method is provided by which a ming). The provided method can be used to produce a “bio flying insect can be surgically modified to produce a highly bot’ or robotic apparatus comprising biological tissue (e.g., efficient MAV. Some large insects, such as certain species of an entire insect or portions thereof) and one or more non US 2010/0025527 A1 Feb. 4, 2010

biological components (e.g., a MEMS device). Such a robotic abdomen to the thorax. According to this embodiment, a apparatus can be adapted for a variety of applications (see microSystem can be Subsequently integrated into the body. Section 5.4 below). 0146 In a specific embodiment, the wire can be inserted at 0136. According to the provided method, a foreign object an early pupal stage through the posterior end of the abdo Such as a microSystem can be inserted in a region of the men, and can run the whole length of the abdomen to the arthropod body in which it will not interfere with normal thorax (FIG. 7a-c). According to this embodiment, a micro development of the adult structures. In one embodiment, the system can be subsequently integrated into the body of the foreign object can be inserted into the abdomen. Abdominal arthropod. In this embodiment, however, the arthropods implantation is preferable since the inserted object does not undergoing implantation generally exhibit lower rates of Sur interfere with proper leg and wing formation occurring in the vival than any of the above-mentioned inserts. thorax during metamorphosis or post-embryonic develop 0.147. After surgery is completed, the arthropods can be ment. kept under conditions well known in the art that promote 0.137 In another embodiment, the foreign object can be development, e.g., maintained in an incubator, until they inserted into the head or thorax of the arthropod, preferably in metamorphose, develop, emerge or molt into adults. a region that will not impede the development or eversion of 0148, 5.3. Microsystems adult structures Such as eyes, appendages (e.g., antennae, 0149. A method is provided for introducing a biocompat wings, or legs), etc. ible foreign object into the body of an arthropod at an imma 0.138. In another embodiment, an “attachment-type' ture stage, under conditions that allow it to be permanently microSystem, e.g., a silk thread insert, silicon insert, or a anchored to the body of the adult insect once the insect titanium wire, can be introduced into a larva (e.g., late instar matures and metamorphoses (e.g., holometabolous insect) or larva), nymph, or pupa. molts (e.g., hemimetabolous insect, crustacean, arachnid) 0.139. According to the method, the inserted microsystem into the adult stage. In one embodiment, the biocompatible (or any tether, attachment point, Substrate or platform for an foreign object can be a microSystem. attached microSystem, which are also referred to herein as a 0150. An apparatus is also provided comprising a non microSystem) can be permanently and securely anchored biologically derived microSystem and a biological system. onto the arthropod's body because its tissues grow, Surround 0151. Biocompatible materials are materials that are and attach to the microSystem during development. This designed to exist and perform specific functions within living growth, however, does not interfere with the normal function organisms. These include a broad range of Substances such as ing or performance of the microsystem. structural metallic orthopedic prosthetic implants, artificial 0140 Since in certain embodiments, the insertion of the blood and skin, and Surface coatings for implantable sensors microSystem can be performed on an immobile stage of for chronic (long-term) monitoring or electrodes for func arthropod development, the method can be used for the mass tional electrical stimulation. In arthropods, biocompatible production of arthropods with surgically implanted Micro materials include, but are not limited to, silicon, silk, tita systems. Furthermore, permanent attachment of the micro nium, microcapillary tubes, micro-nano beads and hydrogels. system can ensure secure delivery of the microSystem to a 0152 Methods for determining biocompatibility of a for target site. eign object in arthropods are well known in the art. While 0141. In one embodiment, arthropods at a pupal, nymphal implant durability is one concern, another concern is the or other pre-adult stage can be prepared for Surgery as fol body's ability to reject these materials as foreign objects lows. The Surgery can be performed on arthropods that are either through an adverse immune system response or by preferably anesthetized, for example, by chilling on ice (cold attempting to “wall them off by surrounding them with a anesthesia). The cuticle is removed from the section of the protein layer. Arthropods have limited immune and “walling body where the microsystem is to be inserted. off responses (when compared with vertebrates such as 0142. In a specific embodiment, a micro-cargo platform mammals), and are well known in the art to exhibit a far can be implanted in an arthropod that is constructed of two greater tolerance for implantation of a foreign object. biocompatible titanium wires (0.01" diameter, 0.25" length) 0153. In one embodiment, the microsystem can be an and a 0.2"x0.2" balsa wood platform (FIGS. 2a-d and 4a-d). attachmentapparatus Such as a thread, wire, tether, loop, post, Such a platform can be inserted into the dorsal abdominal screw or hook. The attachment point can be composed of any cavity of a pupa (FIGS. 2b-d) at the point where the cuticle biocompatible material known in the art, e.g., titanium, silk, removed. silicon, microcapillary tubes, micro-nano beads and hydro 0143. In another embodiment, a silicon chip can be gels. In specific embodiments, the attachment apparatus can implanted. FIGS. 3a-d and 5a-d show a specific embodiment be a titanium wire or a silk thread. in which a 2 mmx4 mmx0.5um silicon chip was inserted in 0154) In another embodiment, the microsystem can be an the ventral abdominal cavity of a pupa at the point where the attachment Substrate or microplatform, to which one or more cuticle is removed. additional microsystems or “micro-cargoes' can be added or 0144. In another embodiment, a silk thread can be attached. In a specific embodiment, the Substrate can be a implanted, which forms an attachment point for another microplatform for a microelectromechanical system microsystem (FIGS. 6a-d). A curved surgical needle threaded (MEMS) device. with silk thread can be used, for example, to make a Suture on 0.155. In another embodiment, the microsystem can be a the dorsal abdominal cuticle in an immature or adult stage of MEMS device, which can include, but is not limited to, a the arthropod. meso-sized device having feature sizes between 500 mm and 0145. In another embodiment, one or more titanium or 2 mm, a micro-sized device having feature sizes between 500 other biocompatible wires can be inserted at a pre-adult stage nm and 500 mm, and a nano-sized device having feature sizes of arthropod development into the posterior end of the abdo between 1 nm and 500 nm. The MEMS device can be any men up into the thorax, and run the whole length of the MEMS device known in the art, for example, an accelerom US 2010/0025527 A1 Feb. 4, 2010

eter, gyroscope, temperature sensor, chemical sensor, electro system. In another specific embodiment, the interface can be physiological stimulator, micro-lens, comb-drive actuator, or between insect leg muscle and the inserted microSystem. piezoelectric actuator. 0173. In another embodiment, the apparatus can be a 0156. A microprobe microsystem platform is also pro micro aerial vehicle (MAV) or unmanned aerial vehicle vided that can comprise a power layer; a layer comprising a (UAV) comprising an adult arthropod (or a functional or probe (“probe layer'); and a control layer (see, e.g., FIGS. partially functional portion thereof) and a permanently 11a-c and 19a-c). attached microSystem, e.g., a mm-scale low power microsys 0157. In one embodiment, the power layer can comprise a tem, such as a MEMS sensor or actuator. power source and a Switch. 0.174 Since, according to the provided method, the 0158. In another embodiment, the power source can be a implantation of a non-biological component, e.g., a microde battery. vice, can be performed in the earlier stages of the arthropods 0159. In another embodiment, the power layer can com development (e.g., in the pupa during post-embryonic meta prise a printed circuit board (PCB). morphosis, during a nymphal stage, during a larval stage), the 0160. In another embodiment, the control layer can com adult arthropod can have enhanced capacity to carry a useful prise a microcontroller (or meso-, micro- or nano-scale con payload, e.g. a microsystem such as a sensor or an actuator. troller). 0.175. In one embodiment, the robotic apparatus can be 0161 In another embodiment, the control layer can com constructed according to the provided method, wherein the prise a PCB and a microcontroller functionally connected to method can comprise: the PCB. 0176 a. introducing a microSystem into an immature 0162. In another embodiment, the control layer can com arthropod (or portion thereof) under conditions that result in prise an LED. production of a robotic apparatus, 0163. In another embodiment, the probe layer can com 0177 wherein the robotic apparatus can comprise: prise a probe that is a microfabricated silicon probe. 0.178 i. the microsystem, and 0164. In another embodiment, the probe layer can be posi 0179 ii. a biological system, wherein the biological sys tioned between the control layer and the power layer. tem can comprise the adult arthropod (or portion thereof), 0.165. In another embodiment, the probe can comprise a 0180 and wherein the microsystem can be attached (e.g., plurality of tips, wherein a member of the plurality can be permanently attached) to the body of the adult arthropod (or separated from a second member of the plurality by a distance portion thereof). that corresponds to the distance between two cells, tissues or 0181. In one embodiment, the biological system can com physiological systems of interest. prise a portion of the adult arthropod capable of generating or 0166 In another embodiment, the two physiological sys controlling a physiological function of the adult arthropod. tems can be two muscles (or muscle groups). In a specific The robotic apparatus can be capable of carrying out the embodiment, the muscles can be flight muscles. In another physiological function (or a portion or Subfunction of the specific embodiment, the muscles can be leg muscles. physiological function). 0167. In another embodiment, the two physiological sys 0182. In another embodiment, the physiological function tems can be two neurons (or neural systems, neural centers, or can be selected from the group consisting of locomotion, ganglia). vision, hearing, chemosensation, temperature sensation, 0.168. In another embodiment, the probe can be activated mechanoreception or pressure sensation, acceleration sensa by an algorithm whereby sequential electrical pulses are sent tion, gravity sensation, magnetic sensation, feeding, diges to the probe tip(s). tion, mating and oviposition. 0169. In another embodiment, the LED on the control 0183 In another embodiment, the provided apparatus can layer can be used to monitor electrical excitation generated by be employed to monitor arthropod migration or dispersion, the probe. for example, locust migration, butterfly migration, biocontrol (0170 5.4. Robotic Apparatus agent e.g. weevil migration, or lobster migration. 0171 A robotic apparatus or “biobot' is also provided. 0184. In another embodiment, the apparatus can be The robotic apparatus can comprise biological tissue (e.g., an employed for environmental monitoring, e.g., atmospheric arthropod or portion thereof) and one or more non-biological conditions, air quality, or water quality. Flying arthropods components. In one embodiment, the apparatus can comprise such as Lepidoptera or Diptera are particularly well suited for a microsystemand a biological system. The biological system use as the biological system in the apparatus, e.g., for moni can comprise an adult arthropod (or portion thereof). The toring air or atmospheric conditions. Aquatic arthropods such microSystem can be permanently attached to the body of the as crustaceans or aquatic beetles are particularly well Suited adult arthropod (or portion thereof). for use as the biological system in the apparatus for monitor 0172. In another embodiment, the apparatus can be an ing water conditions. intact (or nearly intact) biological system incorporated into 0185. In another embodiment, the apparatus can be used as the design of a microsystem (e.g., a MEMS device). In a source of energy that can be harvested using routine meth another embodiment, the apparatus can comprise a portion of ods known in the art. Energy sources provided by the appa a biological system incorporated into the design of the micro ratus that can be harvested are, for example: (a) internal heat system. For example, a biological interface can be created that is generated during flight, walking, Swimming or other between the inserted microsystem and e.g., cell, tissues or forms of arthropod locomotion, (b) mechanical energy of physiological systems of the arthropod, e.g., muscle tissue or locomotion, such as wing beat frequency during flight or neural tissue that controls a specific behavior or physiological Swimmeret frequency during Swimming, that can be har function. In a specific embodiment, the biological interface Vested, e.g., by applying the mechanical stress or deformation can be between insect flight muscle and the inserted micro generated by the locomotion onto piezoelectric crystals, and US 2010/0025527 A1 Feb. 4, 2010

(c) energy harvested from reserve fat bodies and ATP pumps, 0.194. A circuit board with a miniature LED-based device thereby using these energy resources from the insect to derive to regulate direction of flight in the adult moth was also power. constructed according to routine methods. (FIG. 8). 0186. In another embodiment, the apparatus can be used as a cognitive or behavioral interface. For example, an 0.195 Control larvae, pupae and adults were subjected to implanted microsystem that has a neural, muscular, or neu chilling (cold anesthesia) only and were not subjected to romuscular interface can be used to control intrinsic arthro surgery. The insects were then housed individually to reduce pod behavior. Implanted Microsystems can be used in the the chances of contamination or infection. Larvae took 20 brain or in muscles that control the movement of appendages days, and the late pupae 24 to 48 hours, to complete meta (such as wings or legs) to enable better control of arthropods morphosis and emerge as adults. The operated and the control during locomotion. moths were observed for differences in the number of days each required for emergence and their survival time period as 6. EXAMPLES adults. FIGS. 5a-d show the results of the implantation of a 2 6.1. Example 1 mmx4 mmx0.5 um silicon chip into the Ventral abdominal cavity of a pupa. Surgically Implanted Micro-Platforms in the Moth, Manduca sexta, at Intermediate Stages of Metamor 0.196 6.1.3. Results phosis 0197) The survival rates of insects bearing silk or titanium loops that were inserted at the late larval, pupal (early and late 0187 6.1.1. Introduction pupae) or adult stage were first evaluated (Table 2). Larvae 0188 This example demonstrates the surgical implanta with silk loops inserted longitudinally (LS) showed higher tion of biocompatible platforms and substrates for attachment survival rates than did larvae with loops inserted transversally of microsystems in insects without adverse effect to adult lifespan. According to the methods set forth in this example, (CS) as the latter insertion often punctured the foregut of the a biocompatible balsa-titanium anchor was inserted in the late larvae. Individuals with silk tethers inserted at the pupal stage pupal stage of the hawkmoth, Manduca sexta, resulting in a showed the same emergence time as the control unaltered permanently anchored base in the adult. Silicon chips were pupae. Both the pupae and the adults with inserted silk tethers inserted in the late pupal stage. The inserted Substrates showed normal Survival rates when compared to controls became permanently incorporated into the resulting adult (Table 2). Larvae with titanium wire posts did not survive the moth as the wound healed and the anchor site reinforced Surgery and failed to molt into a pupa, most likely because the during the process of metamorphosis. This technology opens rigid structure of the wire prevented normal metamorphosis. up a wide venue for development of microSystems or microdevices (e.g., MEMS devices) that can be attached on or 0198 Late pupae and adults bearing titanium wire loops inserted into insects. Modified insects can be used, for and balsa wood micro-cargo platforms showed Survival rates example, as hybrid systems for generating telemetric units to of 100% and 93%, respectively (N=30 and N=15 for pupae study biomonitoring, insect migration and dispersion patterns and adults, respectively) (Table 2). and various other applications. (0189 6.1.2. Materials and Methods TABLE 2 0190. Manduca sexta larvae and pupae (Carolina Biologi Survival rates of Mandica sexia with insertions made at different cal Supply Company, Burlington, N.C.) were reared at 26°C., stages of its life cycle with 80% relative humidity and a 12:12 h light:dark photo period. Upon emergence, adult moths were transferred to a 1 Number Surviving with mx0.5 m wire cage, which provided perches for proper inserts at expansion of the wings. Implanted implanting Number developmental stage % 0191 Prior to surgery, fifth (last) instar larvae, pupae, and Structure ife stage implanted Larva Pupa. Adult survival adult moths were chilled on ice for 10 minutes. Microsystems that were attachment systems, i.e., loops of Surgical braided Silk thread 8W86 2O O O O O (CS insert) Pupae 10 8 8 8O silk surgical sutures (Fine Science Tool, Foster City, Calif.) Adul 5 5 1OO (FIGS. 6a-d) or biocompatible titanium wire posts 0.01 inch Silk thread 8W86 2O 19 18 18 90 diameter (Small Parts Inc., Miami Lakes, Fla.) were threaded (LS insert) Pupae 10 10 9 90 through the insect’s abdomen (in the case of larvae) and Adul 5 5 1OO dorsal thorax (in the case of pupae and adults). In the larva, the Titanium 8W86 10 O O O O wound was sealed by applying a small drop of melted dental Wire Pupae 30 30 30 1OO Wax Adul 15 14 93.33 0.192 The titanium anchors were looped through a 5x3 Silicon chip Larvae 5 O O O O mm balsa wood block to create a platform on the outside of Pupae 15 12 7 8O the insect to which a microsystem or weight could then be Adul 15 10 66.6 securely attached (FIGS. 2a-d). Total = 153 0193 For insertion of a silicon chip (4 mmx2 mmx0.5 Survival to the adult when insertion was made at the late larval stage mm), the late pupal stage or the adult stage was chosen. To Survival to the adult when insertion was made at the late pupal stage insert the chip, an incision was made along the body wall of Survival of adults 48 h after insertion was made at the adult stage the ventral side of the abdomen and the chip was inserted into the body cavity. The wound was then sealed with dental wax 0199 Inspection via scanning electron microscopy (SEM) (FIGS. 3a-d). of the titanium wire (inserted in the late pupal stage) two days US 2010/0025527 A1 Feb. 4, 2010

after adult emergence revealed that there was tissue growth positioned on or in the insect. This can override the insect's around the wire posts (FIG. 4d). instinctive behavior and result in more accurate control of its 0200. The survival rates of moths bearing a silicon chip locomotion towards a target. were also evaluated. Moths bearing chips inserted at the late pupal stage showed 80% survival and emergence as adults 6.2. Example 2 (N=15) (FIG.3a-d). When the insertion was made at the adult Biocompatible Microfabricated Probe Platform stage (1 day old) the survival rate was 67%. (N=15). Inserted During Metamorphosis to Actuate Insect 0201 Integration of the silicon chip within the abdominal Flight Muscle cavity was demonstrated by tissue loss while prying the insert out of the body cavity of the adult. This was due to tissue 0209 6.2.1. Introduction growth to, and attachment around, the silicon chip (FIG.3d). 0210. This example demonstrates the surgical implanta 0202 6.1.4. Discussion tion of a biocompatible, microfabricated microcontroller based electrical probe for the actuation of flight muscle in the 0203 The results obtained indicate that an insect can be hawkmoth, Manduca sexta. utilized to carry Microsystems, either tethered to its body or 0211 Many species of insects, including moths, have two inserted within its body cavity. Insertion of silicon chips in the sets of flight muscles that are present on either side of the body cavity opens up a venue for formulating and manufac thorax (Eaton, J.L. 1971. Morphology of the head and thorax turing microdevices that can be incorporated into arthropods. of the adult tobacco hormworm, Manduca sexta (Lepi 0204 Inserting devices during the pupal stage did not doptera: Sphingidae). I. Skeleton and muscles. Ann.ent.Soc. significantly affect the insect's Survival to the adult stage, and Am. 64: 437-445). These two sets of flight muscles are dif had the advantage that the wounds caused by Surgery healed ferentially actuated by voltage pulses that directly influence during the process of molting and the tanning (hardening) that the individual wing beat frequency and amplitude, hence took place after emergence. In addition, the tissue that resulting in turning behavior during flight. As demonstrated appeared to grow around the insert yielded a more reliable in the previous example (Section 6.1, Example 1) insects bio-microsystem interface between the insect and the insert. Survive insertion of foreign objects. Such as silicon chips and 0205 Thus, implantation of microsystems during imma titanium wires, during metamorphosis, and incorporate the ture stages prior to adulthood overcomes the difficulties inserted foreign object into the body by tissue growth around encountered when attempting to attach Such devices in an it (see also, Paul, A., Bozkurt, A., Ewer, J., Blossey, B. and adult arthropod. Insertion of devices/platforms at the rela Lal, A. 2006. Surgically Implanted Micro-Platforms in tively immobile metamorphic stage is also advantageous for Manduca sexta, 2006 Solid State Sensor and Actuator Work the mass production of these hybrid or “biobot' systems shop, Hilton Head Island, June 2006, pp. 209-211.) comprising both biological tissue and non-biological compo 0212 6.2.2. Materials and Methods nentS. 0213. In the first set of experiments, flight muscles were 0206. The technology demonstrated here confirms that actuated by inserting titanium wires in the thorax of a tethered substrates or platforms for Microsystems such as MEMS moth and using a current to stimulate the flight muscles. sensors and actuators can be implanted at various post-hatch 0214) A silicon-chip based probe system was then created ing, post-embryonic or metamorphic stages in an arthropods to actuate the flight muscles autonomously in an untethered lifecycle prior to adulthood. The cm scale microsystems of insect. This microsystem had batteries on it to provide elec many MEMS devices known in the art are well suited for trical stimuli to the flight muscles. Microprobes designed to implantation into the cm-dimensions of large flying insects actuate flight muscles were constructed using routine meth Such as a Lepidoptera. ods known in the art The microprobes (FIGS. 11 a-c, 12, 13) 0207. Furthermore, if a platform is inserted and the load were inserted, using insect Surgical methods commonly attached during an immature stage, the foreign load can be known in the art, into the thorax of the metamorphosing moth perceived by the insect as part of its body weight upon emer during the early pupal stage (FIGS. 9 and 14a-d). gence. This results in higher payload carrying capacity of the 0215 Titanium wires were inserted into the dorsoventral insect and/or greater flight activity, compared to insects in flight muscles in the thorax using routine Surgical methods. which tethers are attached in the adult. Moreover, modifying The titanium wires were used to initiate artificial wing flap the duration of development, e.g., post-embryonic or meta ping Current pulses of different pulse-widths were applied to morphic development, by modifying temperature and/or the muscle fibers using routine electrophysiological methods. other environmental or hormonal stimuli, can enable the pro (FIG. 10). duction of adults at a desired time. Finally, the tissue growth (0216) An 8x13 mm, 0.5g stand-alone platform was con around the microSystem in the insect body presents a bio structed, according to standard methods known in the art, to electromechanical interface that can be used to study various apply controlled Voltage pulses to the flight muscles. The physiological responses of the insect to external stimuli that platform consisted of three layers: power, probe and control control locomotion or flight. layers (FIGS. 11a-c and 12). The power layer was formed by 0208. Additionally, the tissue growth around the Micro two serially connected 1.5V SR416SW-type batteries and a systems in the insect body presents a bio-electromechanical slide-switch. The control layer comprised an 8x8 mm PCB interface that can be used to control locomotion by stimulat holding the microcontroller (Atmel Tiny 13V) and an LED. ing nerves and/or muscles to bias insect behavior Such as The probe layer was a microfabricated silicon probe sand flight. Locomotion can also be controlled by manipulating the wiched between these two layers and had a thickness and sensory organs with external stimuli Such as ultrasound, width of 200 um at the tip (FIG. 12). The micro-platform with pheromones, and light wavelength and patterns. These exter probes for stimulating the muscles was thus a self-contained nal stimuli, if generated by a MEMS-sized actuator, can be unit fabricated onto the silicon chip. US 2010/0025527 A1 Feb. 4, 2010

0217 FIG. 12 shows a schematic diagram of the fabrica the thorax, were differentially electrically actuated, which tion process of the microfabricated microcontroller-based influenced the individual wing beat frequency and amplitude, electrical probe, which was fabricated according to routine resulting in controlled turning behavior during flight. methods in the art. 0218. Distance between the two probe tips was 5 mm and 6.3.2. Introduction was equal to the distance between the right and left flight 0225. There have been several technical approaches muscles of the moth. The metal lines that actuate these muscle explored to develop insect-like Small (cm) scale autonomous cells were only exposed close the end of the tip to avoid any flying machines. These attempts have been unsuccessful in short contact (FIG. 13). Electrical contact between layers was reaching long-mission duration and insect-like flight because provided by wire-bonding and soldering. neither the flight actuators are efficient enough, northe power 0219. The micro-platform was inserted on the thorax and energy density of power sources high enough, for Sus using the surgical methods described herein after the first tained reliable flight (Ellington CP, 1999. The novel aerody quarter of the pupal stage (typically 15 days long), when the namics of insect flight:applications to micro-air vehicles. The pupal cuticle became hard. A glass capillary with a diameter Journal of Experimental Biology. 202:3439-348). Nature of 250 microns, which served as a microfluidic port, was provides us with insects, which are very good flyers and inserted into the posterior end of the pupa and extended into which have much better aerodynamic performance due to the thorax of the pupa (FIG. 14c). their high efficiency (10-15%) flight actuators. Insects also 0220. When the switch was turned on, the microcontroller carry correspondingly dense and efficient energy sources in sent sequential 3V voltage pulses with a frequency of 30 HZ their chemical fat stores (J. L. Eaton, 1971. Morphology of (natural wing-beat frequency) to the right and left tips of the the head and thorax of the adult tobacco hornworm, Manduca probe. To elicit differential wing actuation, the right flight sexta (Lepidoptera: Sphingidae). I. Skeleton and muscles. muscle was actuated for 5 seconds, which was then followed Ann. ent. Soc. Am. 64:437-445). Large insects like the by a 10 seconds idle period. After this, left muscle was actu Manduca sexta moths have powerful flight muscles (Eaton, ated for 5 seconds with a consecutive 40 seconds idle period Supra), and have been shown to carry payloads weighing up to to avoid habituation. This was repeated until the switch on the 1 gram. power layer was turned off. To indicate the onset of each 0226 Insects make attractive models for robotics as “bio actuation, an LED blink was programmed, using routine bots.” in which an intact biological system is incorporated methods, into the microcontroller. into a MEMS design. Insects have already been used to carry 0221 6.2.3. Results and Discussion telemetric payloads, but the success rates of these flights have 0222 Current pulses applied to the muscle fibers set up a been low as the method of payload attachment was temporary propagated action potential, which lead to the initiation of a and unreliable (T. E. Moore, S. B. Crary, D. E. Koditschek, twitch contraction. Current pulses with different pulse and T. Conklin. 1998. Directed locomotion in cockroaches: widths were applied and current amplitude to initiate the Biobots. Acta Entomologica Slovenica 6(2):71-78; B. Crary, flapping was measured (FIG. 10). Differential excitation of T. E. Moore, T. A. Conklin, F. Sukardi, and D. E. Koditschek, left and right muscle groups was also observed (data not 1996. Insect biobotics: Electro-neural control of cockroach shown). walking. Abstr. IEEE Int. Conf. Robotics and Automation, 0223) The pupae survived the surgical implantations and Workshop WT3, Bio-Mechatronics, 1996: 42-54. P. Mohs emerged as full grown adults (FIGS. 14b, 14d. 15). Most eni, K. Nagarajan, B. Ziaie, K. Najafi, and S. B. Crary. 2001. pupae emerged normally as adults, although in Some cases, An ultralight biotelemetry backpack for recording EMG sig they were unable to inflate and spread their wings. Dissec nals in moths. IEEE Trans. Biomed. Eng. 48(6): 734-737: R. tions of the thorax were performed to observe the results and Holzer and I. Shimoyama. 1997. Locomotion control of a efficiency of the insertion (FIG. 15). bio-robotic system via electric stimulation. Proc. IEEE/RSJ 6.3. Example 3 Internat. Conf. on Intelligent Robots and Systems, Grenoble, France, September 1997, 1514-1519.Y. Kuwana, N. Ando, R. Microprobe Microsystem Platform Inserted During Kanzaki, and I. Shimoyama. 1999. A radiotelemetry system Early Metamorphosis to Actuate Insect Flight for muscle potential recordings from freely flying insects. Muscle Proc. IEEE BMES/EMBS Conf., Atlanta, Ga., October 1999, p. 846). These payloads were either strapped on the adult 6.3.1. Abstract insect as backpacks or were adhesively bonded onto the body 0224. This example demonstrates the construction of a Surface. These attachments were perceived as foreign microprobe-based microsystem platform, which was weights, since they were Superficially attached and the insect designed with respect to the position of the flight muscles in would try to rid itself of the payload. Also, handling a live a moth, Manduca sexta. The platform was roughly 8x7x1.5 adult is challenging due to constant motion and is also stress mm3 in size with probe thickness of 200 um, and weighed ful for the adult insect. 500 mg. The platform was inserted into the thorax at day 4 of 0227. In this example, we developed a protocol using the the pupal stage and was carried by the moth in the adult stage. tobacco hawkmoth, Manduca sexta, as our working model, in In addition to the microsystem, an anchor for use in manipu which we surgically inserted MicroSystems in the pupal stage lating the adults was formed in the moth by placing a glass (an early stage of metamorphosis) when the insect was immo capillary through the pupa. Pupae with implants emerged and bile (FIG. 16). This procedure led to emergence of an adult the microSystem was used to characterize the potential for moth with the platform or silicon insert permanently attached flight control. To determine the microplatform design, the onto the insect, since the adult exoskeleton had healed over strength-interval profiles of the pulses needed for direct the inserts. Since the insert had been introduced into the insect muscle actuation were determined. Two sets of flight body prior to its emergence as an adult, it was perceived as the muscles, which are symmetrically present on either side of insect's own weight rather than extra (foreign) weight that it US 2010/0025527 A1 Feb. 4, 2010 had to carry during flight. Also survival rates of the moths 0237 6.3.3.2.1 Microprobe Microsystem Platform with the insert inserted during its metamorphic stage were 0238. The required actuation waveforms were generated comparable to normal unaltered moths. by a microsystem as shown in FIGS. 19a-c. The microsystem 0228. Using Surgical implantation and wound repair tech consisted of three layers, a power layer, a probe layer and a niques (Early Metamorphosis Insertion Technology, control layer. “EMIT) (see materials and methods described herein, e.g., 0239. The power layer comprised two coin batteries and a in Sections 6.1 and 6.2), we developed Microsystems for slide-switch positioned on a printed circuit board (PCB). implantation in early metamorphosis stages of insects, to Each battery had an energy capacity of 8 mAh and weighed 120 mg. Conductive adhesive was used to attach the batteries create an insect “cyborg, “biobot' or robotic apparatus. The to the platform. microSystems were used to intrinsically stimulate the insect's 0240. The control layer was an 8x8 mm PCB holding a physiological response, such as flight. The microSystem pro microcontroller (Atmel Tiny 13V) and an LED. The micro vided sensor, stimulation and navigation capabilities, while controller was electrically connected to the PCB via flip-chip the insect provided a naturally occurring flight actuator and bonding. Wire-bonding was used to connect the PCB to the energy Source. probe layer. The microfabricated silicon probe was sand 0229. Different actuation mechanisms can be used to con wiched between these two layers. trol the flight. The insect can be influenced by indirect exter 0241. The overall system had dimensions of 8x7 mm and nal stimulation or direct internal excitation. External stimu total mass of 500 milligrams. The silicon microprobe plat lation methods can be targeted, inter alia, towards sensory form (500 um thick) comprised two tips (each 200 um wide, organs, for example, visual stimulation using light patterns, 200um thick and 5 mm long) separated by a distance of 5mm, or pheromonal stimulation as olfactory cues. For internal which was the approximate distance between the right and excitation, selected regions of interest in the neuromuscular left flight muscles of the moth (FIG. 13). system can be actuated using either electrical or chemical 0242 Platinum (electrode metal) was evaporated and pat stimulation. In this example, we aimed to stimulate dorsoven terned on a 4-inch silicon wafer with a layer of 900 nm tral flight muscles, which are believed to express directional LPCVD nitride. PECVD nitride was deposited on the metal response (Eaton 1971, supra). Using properly timed electrical and patterned to provide passivation. For muscle-specific stimuli from MEMS devices implanted in the insect's flight excitation, the metal lines running along the length of the muscles, insect flight and other forms of locomotion can be probe tip that actuate the muscle cells were only exposed monitored and controlled. close to the end of the tip (100x100 m) (FIG. 20). The 0230. In this example we demonstrate (a) flight control of desired probe tip thickness was achieved by deep reactive ion the insect "cyborg, biobot or robotic apparatus, using wires etching (DRIE). Backside-only KOH etching (with LPCVD inserted in early-metamorphosis stages of the moth, nitride mask) was done to release the device. Manduca sexta, and (b) the development of a battery-pow 0243 6.3.3.2.2 Microcontroller Algorithms ered microsystem that was also implanted using EMIT. 0244 As discussed above, the concept of differential wing actuation was demonstrated with titanium wire-based electri 6.3.3 Experimental Procedure and Results cal excitation on tethered moths (see also Section 6.2, Example 2). In the present example, an algorithm was imple 0231 6.3.3.1 Tethered Wing Actuation mented on the microcontroller of the microsystemplatform to send sequential electrical pulses to the right and left tips of the 0232 To determine the location of appropriate muscles probe to observe the effect of the differential actuation during and the electrical waveforms needed to actuate muscles, we untethered flight. first inserted titanium wire electrodes in the muscle groups of 0245. The algorithm comprised outputting time-sched tethered moths. (FIGS. 17a-b). uled 3V square pulses with a duty cycle of 6% and frequency 0233 Square current pulses applied to the muscle fibers of 30 Hz, which corresponds to the natural wing-beat fre set up an action potential wave, which initiated the twitch quency (B. Heinrich, G. A. Bartholomew. 1971. An Analysis contraction. Current pulses with different pulse-widths were Of Pre-Flight Warm-Up In The Sphinx Moth, Manduca applied and current amplitude threshold to initiate the flap Sexta, J. Exp. Biol. 55: 233-239). The timing-scheme of each ping of the wings was measured (FIG. 18). excitation cycle is described in FIG. 21. The second idle 0234. During these experiments, left and right muscle period was kept longer (40 sec) to avoid habituation. This groups were differentially or selectively excited (data not scheme repeated itself until it was stopped by the switch on shown). The charge transfer from the electrodes to the the power layer. The LED on the control layer was used to muscles was controlled by conduction through the muscle externally monitor the electrical excitations. tissue and diffusion of charged ions in the Helmholtz plane. 0246 The power breakdown of the different components As the pulse width was reduced, the effective amount of is shown in FIG. 22. current required for actuation increased exponentially, indi 0247 6.3.3.2.3 EMIT-Based Insertions cating diffusion was controlling the actuation. As the duration 0248. The manufactured platforms were inserted in the of pulses increased, the actuation was limited by tissue con thorax using the Surgical methods described hereinabove, at ductivity (see, e.g., Borkholder. D. A. 1998. Cell Based Bio an angle to avoid interference with wing motion. Insertions sensors Using Microelectrodes. Ph.D. Thesis, Stanford Uni were performed after the first quarter of the pupal stage (on versity, Stanford Calif.). the third day of a 15-day pupal stage), when the cuticle 0235 6.3.3.2 Untethered Wing Actuation hardens. Additionally, a glass capillary with a diameter of 250 0236. After determining the required pulse width and um was inserted from the posterior end of the abdomen of the amplitude, a microSystem platform was designed, assembled pupa and extended to the thorax, to serve as an easy tether by and inserted in moths using EMIT. which the insect could be mechanically anchored and US 2010/0025527 A1 Feb. 4, 2010 manipulated. The cuticle was punctured with a hypodermic 0256 The citation of any publication is for its disclosure needle at the appropriate insertion location and the probe or prior to the filing date and should not be construed as an capillary was inserted. The puncture was then immediately admission that the present invention is not entitled to antedate sealed with a drop of wax to prevent any oozing around the such publication by virtue of prior invention. inserts, due to still relatively high inner pressure in the body 1. A method for producing an arthropod comprising intro cavity. Pupae survived both insertions and emerged as full ducing a microSystem into an immature arthropod under con grown adults (FIGS. 14a-d). ditions that result in producing an adult arthropod wherein the 0249 Dissections of the thorax were performed to observe microsystem is functional and attached to the body of the the insertion of the probes near the target muscle groups (FIG. adult arthropod. 15). Muscle growth around the probe tips indicated adapta 2. The method of claim 1 wherein the microsystem is tion by the moth body. permanently attached. 0250 Micro-Computer Tomography (Micro-CT) imaging 3. The method of claim 1 wherein the immature arthropod of the moth thorax was also performed, in parallel with the is an insect, crustacean or arachnid. anatomical dissections, to evaluate the insertion efficacy 4. The method of claim 3 wherein the insect is a holom (FIGS. 23 A-C). Micro-CT images were obtained with a Sky etabolous insect. scan 1172 Micro CT scanner (resolution of 17.4 microns). As 5. The method of claim 3 wherein the insect is a hemime seen on the obtained CT image in FIG. 23A, the probes were tabolous insect inserted directly to the dorsoventral wing elevator muscle for 6. The method of claim 1 wherein the immature arthropod initiation of the wing strokes. FIG. 23C shows the orientation is a larva. of the moth in FIGS. 23A-B. Prior to imaging, the body of the 7. The method of claim 1 wherein the immature arthropod microSystem platform was broken, keeping the inserted is a pupa. probes in their original locations, so that the Subject insect 8. The method of claim 1 wherein the immature arthropod would fit into the scanner. The tissue growth around the is a nymph. probes (shown by arrows) indicated the acceptance of the 9. The method of claim 1 wherein the introducing step is probes by the moth body (FIG. 23B). Surgical implantation. 0251. The rate of emergence of moths from implanted 10. The method of claim 1 wherein the microsystem is an pupae was found to be 90%. However, 6 out of 7 adults were attachment apparatus. notable to spread their wings properly. During eclosion, the 11. The method of claim 1 wherein the microsystem is a newly emerged adult normally aligns itself vertically on a microplatform. Surface, so that the wings can inflate via gravity-based flow of 12. The method of claim 1 wherein the microsystem is a hemolymph. Since the fully functional probes were heavy, MEMS device. they interfered with the ability of the adult to align itself 13. A method for producing a robotic apparatus comprising Vertically upon emergence and form functional wings. How introducing a microSystem into an immature arthropod or ever, wing inflation can be optimized by tethering the insects portion thereof under conditions that result in producing a in a vertical orientation during eclosion forbetter hemolymph robotic apparatus, wherein the robotic apparatus comprises: flow. a. the microSystem, and b. a biological system, the biological system comprising an 6.3.4 CONCLUSIONS AND DISCUSSION adult arthropod or portion thereof, 0252 Insects are autonomous flying machines that Sur and wherein the microsystem is attached to the body of the pass any existing mechanical flying machine of comparable adult arthropod or portion thereof. size, both in aerodynamic performance and flight actuation 14. The method of claim 13 wherein the microsystem is machinery. The aim of this study was to employ insects, attached permanently. particularly moths, as a biological system in which to create a 15. The method of claim 13 wherein the biological system hybrid system with intimate electromechanical interfaces comprises a portion of the adult arthropod capable of gener with the body for control of muscles. The present example ating or controlling a physiological function of the adult demonstrated the use of EMIT to insert microsystems in the arthropod, and wherein the robotic apparatus is capable of early pupal stages to obtain reliable interfaces. carrying out the physiological function or a portion or Sub 0253) We have also demonstrated that flight can be con function thereof. trolled in the moth, Manduca sexta, using in-built electrical 16. The method of claim 15 wherein the physiological stimulation of muscle. Using this information, we designed a function is selected from the group consisting of locomotion, silicon-microprobe based platform that was inserted in pupae vision, hearing, chemosensation, temperature sensation, using EMIT. We demonstrated emergence of the moth with a mechanoreception or pressure sensation, acceleration sensa platform that weighed 500 mg. tion, gravity sensation, magnetic sensation, feeding, diges 0254 The present invention is not to be limited in scope by tion, mating and oviposition. the specific embodiments described herein. Indeed, various 17. The method of claim 15 wherein the microsystem acti modifications of the invention in addition to those described Vates a muscle that regulates the physiological function. herein will become apparent to those skilled in the art from 18. The method of claim 17 wherein the physiological the foregoing description. Such modifications are intended to function is locomotion. fall within the scope of the appended claims. 19. The method of claim 18 wherein the muscle is a flight 0255 All references cited herein are incorporated herein muscle and the locomotion is flying. by reference in their entirety and for all purposes to the same 20. The method of claim 18 wherein the muscle is a leg extent as if each individual publication, patent or patent appli muscle and the locomotion is walking. cation was specifically and individually indicated to be incor 21. The method of claim 13 wherein the immature arthro porated by reference in its entirety for all purposes. pod is an insect, crustacean or arachnid. US 2010/0025527 A1 Feb. 4, 2010

22. The method of claim 21 wherein the insect is a holom 44. The apparatus of claim 65 wherein the immature etabolous insect. arthropod is a pupa. 23. The method of claim 21 wherein the insect is a hemi 45. The apparatus of claim 65 wherein the immature metabolous insect. arthropod is a nymph. 24. The method of claim 13 wherein the immature arthro 46. The apparatus of claim 31 wherein the microsystem is pod is a larva. an attachment apparatus. 25. The method of claim 13 wherein the immature arthro 47. The apparatus of claim 31 wherein the microsystem is pod is a pupa. a microplatform. 26. The method of claim 13 wherein the immature arthro 48. The apparatus of claim 31 wherein the microsystem is a MEMS device. pod is a nymph. 49. The apparatus of claim 37 wherein the physiological 27. The method of claim 13 wherein the introducing step is function is flying and the apparatus is a MAV. Surgical implantation. 50. The apparatus of claim 31 wherein the microsystem 28. The method of claim 13 wherein the microsystem is an comprises: attachment apparatus. g. a power layer, 29. The method of claim 13 wherein the microsystem is a h. a probe layer comprising a probe; and microplatform. i.a control layer. 30. The method of claim 13 wherein the microsystem is a 51. The apparatus of claim 50 wherein the power layer MEMS device. comprises a power Source and a Switch. 31. An apparatus comprising: 52. The apparatus of claim 51 wherein the power source is c. a microsystem; and a battery. d. a biological system, the biological system comprising an 53. The apparatus of claim 50 wherein the power layer arthropod or portion thereof, comprises a printed circuit board (PCB). wherein the microsystem is incorporated into the body of the 54. The apparatus of claim 50 wherein the control layer arthropod or portion thereof. comprises a PCB and a microcontroller functionally con 32. The apparatus of claim 31 wherein the arthropod is an nected to the PCB. adult arthropod. 55. The apparatus of claim 50 wherein the control layer 33. The apparatus of claim 31 wherein the microsystem is comprises an LED. permanently attached. 56. The apparatus of claim 50 wherein the probe is a micro 34. The apparatus of claim 31 wherein fabricated silicon probe. e. the biological system comprises a portion of the arthro 57. The apparatus of claim 50 wherein the probe layer is pod capable of generating or controlling a physiological positioned between the control layer and the power layer. function of the arthropod, and 58. The apparatus of claim 50 wherein the probe layer f, the robotic apparatus is capable of carrying out the physi comprises a plurality of probes. 59. The apparatus of claim 58 wherein a tip of a member of ological function or portion or Subfunction thereof. the plurality of probes is separated from a tip of a second 35. The apparatus of claim 34 wherein the physiological member of the plurality of probes by a distance that corre function is selected from the group consisting of locomotion, sponds to the distance between two cells, tissues or physi vision, hearing, chemosensation, temperature sensation, ological systems of interest. mechanoreception or pressure sensation, acceleration sensa 60. The apparatus of claim 59 wherein the two physiologi tion, gravity sensation, magnetic sensation, feeding, diges cal systems are two muscles or muscle groups. tion, mating and oviposition. 61. The apparatus of claim 59 wherein the two physiologi 36. The apparatus of claim 34 wherein the microsystem cal systems are two neurons. activates a muscle that regulates the physiological function. 62. The apparatus of claim 50 wherein the probe is acti 37. The apparatus of claim 36 wherein the physiological vated by an algorithm whereby sequential electrical pulses function is locomotion. are sent to a tip of the probe. 38. The apparatus of claim 37 wherein the muscle is a flight 63. The apparatus of claim 62 wherein the algorithm com muscle and the locomotion is flying. prises outputting time-scheduled square current or Voltage 39. The apparatus of claim 37 wherein the muscle is a leg pulses. muscle and the locomotion is walking. 64. The apparatus of claim 55 wherein the LED of the 40. The apparatus of claim 31 wherein the arthropod is an control layer is used to monitor electrical excitation generated insect, crustacean or arachnid. by the probe. 41. The apparatus of claim 40 wherein the insect is a 65. The apparatus of claim 31 wherein the arthropod is an holometabolous insect. immature arthropod. 42. The apparatus of claim 40 wherein the insect is a 66. The apparatus of claim 31 wherein the incorporated hemimetabolous insect. microSystem is integrated into the body of the arthropod. 43. The apparatus of claim 65 wherein the immature arthropod is a larva. c c c c c