How Should We Foster the Professional Integrity of Engineers in Japan? A Pride-Based Approach

I discuss the predicament that engineering-ethics education in Japan now faces and propose a solution to this. The predicament is professional motivation, i.e., the problem of how to motivate engineering students to maintain their professional integrity. The special professional responsibilities of engineers are often explained either as an implicit social contract between the profession and society (the “social-contract” view), or as requirements for membership in the profession (the “membership-requirement” view). However, there are empirical data that suggest that such views will not do in Japan, and this is the predicament that confronts us. In this country, the profession of engineering did not exist 10 years ago and is still quite underdeveloped. Engineers in this country do not have privileges, high income, or high social status. Under such conditions, neither the social-contract view nor the membership-requirement view is convincing. As an alternative approach that might work in Japan, I propose a pride-based view. The notion of pride has been analyzed in the virtue-ethics literature, but the full potential of this notion has not been explored. Unlike other kinds of pride, professional pride can directly benefit the general public by motivating engineers to do excellent work even without social rewards, since being proud of themselves is already a reward. My proposal is to foster a particular kind of professional pride associated with the importance of professional services in society, as the motivational basis for professional integrity. There is evidence to suggest that this model works.
There is an on-going shift in emphasis in the professional-ethics literature from misconduct to integrity [1, 2]. Integrity within this context generally means maintaining high moral standards and doing superior work in a professional capacity, as opposed to merely avoiding misconduct. The profession of engineering is of course no exception. However, even though the importance of professional integrity in society is obvious, the question of motivating students to maintain this integrity is not as straightforward a matter. Engineering-ethics courses are usually expected to do the job. Ethics teachers do not have to spend a great deal of time to explain why professionals should avoid misconduct, as long as students have already acquired everyday morality. Professional integrity and other special responsibilities by professionals, on the other hand, go beyond everyday morality, and need a different basis to convince students. How then can engineers (and other professionals) be motivated to provide efficient services to society? How can we foster professional integrity in engineers? These issues, which I will henceforth refer to as “the problem of professional motivation,” have been discussed by many authors, and many different solutions have been proposed.
To clarify the issue, we need to distinguish the motivation to obey the codes of ethics and the justification of such codes. Professional codes of ethics have clear societal virtues, and often essential for the normal functioning of the society. In this sense, the existence of the code is morally desirable and justified. However, there are obligations in codes of ethics that seem to go beyond common sense obligations, such as “engineers shall be guided in all their relations by the highest standards of honesty and integrity,” and “engineers shall at all times strive to serve the public interest,” both listed as “professional obligations” in the Code of Ethics for Engineers by the NSPE (National Society for the Professional Engineers). To use a deontological term, these acts are usually regarded as supererogatory acts for ordinary people, and without special reasons, this should not be different for engineers. Thus, there is a gap between what society needs and what the society can require of engineers as ordinary obligations. The gap can be filled either by justifying the special obligations in a way engineers themselves feel happy with them, or by motivating engineers in other ways to accept these obligations. The problem of professional motivation can be solved and professional integrity will be fostered in either way, but the motivational aspect becomes more crucial when we have to take the second strategy.
Since the effectiveness of a solution to the problem is partly dependent on the circumstances engineers are placed in, the most effective solution may differ from one situation to another. One of the main conclusions I intend to draw in this paper is that Japanese ethics teachers face a special predicament that teachers in many other countries (including the U.S.) might not. The quandary, if it is real, calls for a different solution to the problem of professional motivation. I will also provide a solution to the problem based on the notion of “pride”, which I take to be at least more promising in the current Japanese circumstances than the alternatives.
Grounds for Professional Integrity of Engineers
Let us first examine some common solutions to the problem of professional motivation. This issue is often discussed within the context of the nature of codes of ethics. Although the scope of professional integrity (which is my main concern here) often goes beyond written texts of codes of ethics, for now let us ignore this difference; for if we fail to persuade students to obey the codes of ethics of relevant professional societies, how can we persuade them to have professional integrity?
Probably the most prevalent view is the so-called “social contract” view (or, more accurately, the “contract-with-society” view). This view sees an implicit contract between society and the professions and is based on particular notions of what professions are. According to this notion, professions have some characteristic features, such as autonomous professional societies, ethics codes, licenses, and privileges [3, 4]. These features are the basis of the contract, where society gives professions prestige, autonomy, educational opportunity, and other support for them to function; in exchange, professions maintain high moral standards and return to society high-quality services.
If students accept this scheme exists, it is rather easy to persuade them to participate in the contract, i.e., the contract is advantageous for them. This account of professional ethics is widely accepted (e.g., Harris et al. [5] discusses this view).1
Another model, which I call the “membership-requirement” view, has some strong supporters such as Davis [6, 7]. According to this model, engineers have special responsibilities simply because they have decided to take part in the profession, and all members are required to follow special standards (and the standards in turn are beneficial for the profession as a whole). According to this interpretation, professional responsibilities are basically derived from the principle of fairness (“do not cheat”), rather than the assumed implicit contract with society.2
Both views have some advantages and disadvantages, but I will not discuss them here. What I would like to ask is a rather different question: can these views justify special obligations of engineers and motivate engineers to have a sense of professional integrity, especially in Japan? There are some reasons for believing that the situation is apparently different in this eastern country, and this is what I will try to demonstrate in the following.
Japanese Peculiarities
The peculiarities of the Japanese situation I have in mind involve the environment Japanese engineers are placed in. Both the “social-contract” view and the “membership-requirement” view presuppose the existence of the profession of engineering as a matter of fact (rather than as an ideal), and the former view further presupposes the existence of an implicit contract between the profession and society (again, as a matter of fact). However, neither of these conditions is actually met in Japan.
Underdevelopment in Profession of Engineering
First, until quite recently, engineers in Japan had never characterized themselves as professionals in a relevant sense. As a matter of fact, the very notion of profession was foreign to them. Engineering societies in Japan are not professional organizations in the proper sense, but academic societies; their main purpose is to organize academic meetings, and they rarely play a role in defending the interests of engineers. In fact, the lack of the notion of profession in engineering is frequently mentioned in the engineering-ethics literature in Japan as the main obstacle to the development of professional engineering ethics [8, 9]. Since they were (and still largely are) academic societies, most of the societies never had an ethics code (until recently). There was no accreditation system for engineering education, and professional engineer (PE) licenses did not exist.
This was the situation in Japan 10 years ago. Now, there is an on-going change toward the professionalization of engineering in Japan. Engineering societies have started to establish codes of ethics. An organization for accreditation, the Japan Accreditation Board of Engineering Education (JABEE), was established in 1999 and some engineering departments have already been accredited. To meet the accreditation criteria, engineering schools started to offer engineering-ethics courses, one of which I am teaching myself. The government amended the licenses for Consulting Engineers into those for Professional Engineers in 2000, and introduced some new features, such as the Continual Professional Development (CPD) requirement, to the licenses.3 Since licensing and accreditation have been used by various professions to attain privilege and autonomy, these changes seem to be steps toward the professionalization of engineering in Japan.
However, the flip side of these positive developments is that those who are actively involved in these changes are still in the minority. Such changes are mainly caused by external factors irrelevant to most engineers. An effort was made in 1997 to make Japanese engineers internationally competitive.4 To achieve this purpose, the authorities had to establish an accreditation system compatible with ABET (Accreditation Board for Engineering and Technology) of the United States and other Washington Accord countries, but one conspicuously missing element in the Japanese engineering-education system was ethics education. They studied engineering-ethics education systems in other countries (especially in the U.S.), and noticed that such systems are firmly based on the notion of profession. They thus started the series of changes in professionalization.
International competitiveness and the establishment of an accreditation system are not problems of their own making for most individual engineers in Japan. Even though codes of ethics as written texts have been established, what gives life to such texts is the members of the societies who consciously observe them, and in this sense the codes do not yet have real lives. It will take a long time for Japanese engineers to become aware of the importance of codes of ethics and regard engineering as a profession in the proper sense.
Even after the recent amendments, there are still almost no privileges associated with PE licenses, and most engineers can still conduct their business without any license whatsoever.5 Many engineering schools (including most of the best engineering programs in the country) are not yet accredited, but their graduates do not seem to have any trouble finding jobs. Thus, the change toward professional privilege and autonomy is still nominal.
In summary, no autonomous professional groups existed for engineering students to take part in 10 years ago, and even now these are quite underdeveloped. Anyway, there are no advantages gained by becoming a member. Given these considerations, the “membership-requirement” view is hopeless as a clue to motivating engineering students, and therefore does not seem to solve the problem of professional motivation, at least for the time being, in Japan. This also means that an important part of the supposed community deal in the “social-contract” view is also missing. Do we have to wait until the profession is firmly established before teaching professional ethics?
Lack of Implicit Social Contract in Japan
Even if engineering is not a profession in the proper sense, if there is an implicit social contract between engineers and society as a matter of fact, the “social-contract” view will do. Privilege and autonomy are not the only things given to engineers by society. For example, if their social status is sufficiently high, that can be an implicit term in the social contract. However, there is evidence that suggests that the social status of engineers is not as high as they might wish. In the following, let us look at a couple of suggestive surveys in this regard.
There was an interesting survey conducted in 1998 by Matsushige et al. [10] that showed that Japanese engineers do not earn that much (at least not enough to make the “social-contract” view persuasive).6 Osaka University is one of seven major national (former Imperial) universities in Japan. They were classified by the degree they earned, and four of the categories were: Bachelor of Engineering, Master of Engineering, Bachelor of Social Science, and Bachelor of Humanities. They were further divided into age groups and the average salaries were calculated for all categories. The results for male graduates are summarized in Table 1.7Table 1Average salary in 1998 of Osaka University male graduates (reproduced from [10], p. 5)22–29 years old30–39 years old40–49 years old50–60 years oldBachelor of Social Sciences¥4,626,000 ($38,600)¥9,133,000 ($76,100)¥13,067,000 ($108,800)¥15,682,000 ($130,700)Bachelor of Engineering¥5,088,000 ($42,400)¥7,036,000 ($58,600)¥10,988,000 ($91,500)¥14,566,000 ($121,400)Master of Engineering and higher¥4,370,000 ($36,400)¥7,392,000 ($61,600)¥11,457,000 ($95,500)¥14,004,000 ($116,700)Bachelor of Humanities¥4,844,000 ($40,300)¥7,365,000 ($61,300)¥11,792,000 ($98,300)¥12,329,000 ($102,700)Dollar amounts are an approximation with $1 = ¥120 at the current rate
There are several things to note here. First, social-science-major graduates are consistently better paid than other majors after they reach their thirties. Up to their forties, humanities majors are paid as much as engineering majors. In their fifties, the average salary of humanities graduates becomes lower than that of engineering graduates. However, according to the analysis done by Matsushige et al., this difference is not caused by recognizing the status of engineers, but by the tendency for engineers to stay at a single company, while humanities majors leave their original company at around this age.8 Given that engineering majors spend longer in doing course work than social science or humanities majors, there is no point in majoring in engineering if one is interested in earning a good salary. There seems to be no recognition that the status of engineers is higher than that of the others in terms of salary.
Of course, the data should be compared with similar data from other countries to analyze it objectively, especially that from the U.S., but so far I have found no comparable surveys.9 We also should note that as Osaka University is by no means an average university, it is possible that these results will not apply to less prestigious universities. Still this is a very suggestive case study.
Even if they do not earn much, it is still possible for engineers to be highly regarded in society. However, another social survey in 1995 (part of a large survey called Social Stratification and Mobility) suggested that the social status of engineers is not as high as that of other professions [11]. Four-thousand ordinary people were asked to rank the social status of 56 occupations, and “automobile design engineers” was one of them. This occupation was ranked 14th in the 56 occupations (Table 2). This may not sound that serious, but other professions, such as doctors, judges, architects and accountants ranked much higher than engineers.10Table 2Occupational Prestige Scores in 1995 “Stratification and Social Mobility” Survey in Japan (reconstructed from [11]; the original result has 56 jobs listed)Doctor 90.1 (highest)Nurse 59.7CEO of large company 87.3Police officer 57.9Lawyer 86.9Municipal officer 56.8University professor 84.3Carpenter 53.1Congressman 74.9Restaurant cook 51.6Architect 71.9Mechanical assembly worker 51.1Accountant 70.8Farmer 45.6Manager of small company 68.9Insurance sales person 44.2Automotive design engineer 66.3Waitress 38.0School teacher 63.5Coal miner 36.6 (lowest)
A similar poll on occupational prestige is conducted in the U.S. every year by Harris Interactive, and engineers in the 2006 poll had higher prestige than professions such as architects, athletes, lawyers, and accountants, even though firefighters, doctors, and nurses had yet higher prestige (Table 3).11 This high status for engineers has been fairly stable since this poll began in 1977. Since the questions in the Japanese and American surveys were different, we cannot simply compare the two surveys; but the difference in the trend is remarkable.
Table 3The percentage of people who think that the occupation has “very great prestige”. Based on survey conducted by Harris Interactive in 2006Firefighter 63%Athlete 23%Doctor 58%Lawyer 21%Nurse 55%Entertainer 18%Scientist 54%Accountant 17%Teacher 52%Banker 17%Military officer 51%Journalist 16%Police officer 43%Union Leader 12%Priest/Minister/Clergyman 40%Actor 12%Farmer 36%Business executive 11%Engineer 34%Stockbroker 11%Member of Congress 28%Real estate agent/broker 6%Architect 27%
To sum up, most elements of the “social-contract” view, i.e., the engineering profession, codes of ethics, privilege, high income, and prestige, are missing or underdeveloped in Japan. How can there be a social contract under such circumstances? Thus, this view does not seem to solve the problem of professional motivation in Japan either.
Pride-based Approach
Outline of Pride-based Approach
What I have argued so far is that both the “social-contract” view and the “membership-requirement” view lack a factual basis in Japanese society. This also means that they are hard to use in engineering-ethics education in Japan. What then are the alternatives available in Japan, especially in teaching professional integrity?
An appeal to common morality may do for many items in codes of ethics. For example, the avoidance of harm to the public can be derived from the harm principle. However, as I pointed out at the beginning of this paper, there are other obligations in codes of ethics that seem to go beyond common sense obligations, and the “social contract” and “membership requirement” are invoked because common morality do not seem sufficient for engineers.
The model of ethics education I will outline in the following is based on the notion of pride. The basic idea is simple. One can have pride as a professional even where the profession as an autonomous body is absent. Such pride will help engineers to adhere to high moral standards, even without being rewarded by society. We can expect to promote moral behavior in engineers (including engineering students) by developing their professional pride in engineering-ethics education.12 In terms of justification, this model does not try to justify special obligations of engineers; rather, the model tries to motivate students to accept supererogatory acts as their own obligations.
Concept of Professional Pride
To explicate this approach, I need to say more about the very notion of pride in general, and professional pride in particular. First, let us examine the notion of pride itself. Pride is a positive feeling toward oneself and things associated with oneself. Let us call these things the “object” of pride, following David Hume, whose analysis of this notion is one of the earliest [13].13 Groundless pride might be psychologically possible, but pride usually involves some beneficial properties about the object of pride as grounds (again, following Hume, let us call this the “cause” of pride). Since Hume, several moral philosophers have focused on this notion, especially in terms of virtue ethics. Some authors have considered pride as a vice (i.e., as contrary to the virtue of modesty), and others as a virtue [14, 15].14 When virtue ethicists (including Hume) talk about pride as a virtue, their reason for the positive evaluation of pride is based on its effect on oneself; pride is, according to these authors, a virtue because it promotes self-esteem and personal happiness.
Of course, pride as a virtue is being considered within the present context. Just like other virtues in virtue ethics, such as courage, a misplaced pride and an excessive pride can be a vice rather than a virtue. Thus, just like a courage shown in a robbery does not count as a virtue, a pride as a robber does not count as a virtue. It is hard to make precise the distinction between misplaced and appropriate prides. This is a problem with the virtue ethics approach in general.15
This may be true of pride in general, but professional pride in particular can have a different other-regarding moral function. Professionals in various professions seem to have pride in their trade. What kind of pride is it? The object of professional pride may be the professional him or her as a professional, the profession as a collective body, or other professionals in the same profession. The cause of professional pride may be excellence in professional skill, the importance of the service they have done to society, or possibly high moral standards. To be justly proud of these things, professionals have to maintain these qualities in their professional work. This will of course benefit society. Here, we can see the possibility for professional pride to be an other-regarding virtue. The beautiful part of this scheme is that society does not necessarily have to reward good work; the feeling of pride itself functions as a reward. Of course, such feelings will be reinforced by the recognition of excellence by others.16
When we think of professional integrity, professional pride has another virtue. Since pride is a positive feeling, it can motivate professionals to overachieve, i.e., to do things that are not required or expected (they overachieve simply because it feels good). This is in strong contrast to negative feelings, such as fear of punishment and feelings of guilt, which do not motivate professionals to achieve more than required. Given these limitations of negative feelings, some sort of positive feelings will be necessary as motivation in the transition from minimal professional responsibility to professional integrity, and professional pride is a good candidate for this.
Let us now examine whether this will solve the problem of professional motivation in Japan. Given that the concept of the engineering profession as an autonomous body is absent in Japan, the profession itself may not be suitable as an object of pride. However, we do have individual engineers, and of course they can be the objects of their own professional pride. Some possible causes of professional pride (such as the high moral standards of the profession as a whole) are missing in Japan, but other important ones, such as their excellence as engineers and the importance of services they give to society, do exist. Rewards from society, which will reinforce this pride, can hardly be expected in Japan, but, as I said, pride is self-rewarding. Thus, the pride-based approach seems (at least) a possible solution to the problem.
Prospects for Pride-based Education
The pride-based approach is yet to be developed. There are many problems to be solved and many questions to be answered before the model outlined here can be implemented in engineering-ethics education. First, does real-world professional pride have desirable characteristics? It is possible for professionals to feel pride because of something other than their skills or services. For example, the sole cause of their pride may be their high income and prestige. If this is the case, the pride-based model does not work where the “social-contract” view does not. This is a psychological question that calls for an empirical investigation. Second, can professional pride become strong enough to support professional integrity? The “social-contract” view seems to provide a fairly strong motivational basis for professional ethics, where it works. Can pride be as strong as such motivation? This is another psychological issue, but harder to answer, given the speculative nature of the question. Third, is it possible to teach pride in the first place? Is pride not something we acquire by ourselves, rather than something taught in school? This is a pedagogical inquiry, which calls for classroom experiments.
There is an interesting survey that may illuminate some of these issues. Okamoto et al. surveyed firefighters in Japan on their occupational self-esteem [17]. The results revealed that there were two independent factors in the sense of occupational self-esteem firefighters had (they conducted factor analysis on the replies to their questionnaire). The first was the esteem based on the service they gave, which Okamoto et al. called “self-esteem from occupational tasks”, and the second was the esteem stemming from the very nature of the occupation (e.g., intense training and dangerous work), which they called “self-esteem from occupational capacity” (pp. 37–38). They further found that the self-esteem from occupational tasks was negatively correlated with delinquency, while the correlation between the self-esteem from occupational capacity and delinquency was not statistically significant (pp. 51–53).
Since there are many differences between firefighting (which is not even a profession in the strictest sense) and engineering, we need to use a great deal of caution in applying the results of Okamoto et al.’s study. However, their results constitute positive empirical evidence that occupational self-esteem (which is often associated with, or even equated with, pride) can be caused by the importance of the service being provided, and that that kind of self-esteem helps the person to maintain high moral standards. If this is true, there are practical implications for engineering-ethics education under the pride-based approach. The best way to foster the desirable kind of professional pride in engineering students in the classroom is to stress the importance of the service provided by engineers. Professional pride acquired in this way will be conducive to professional integrity. Placing emphasis on professional capacity (e.g., higher education and skill) may also foster professional pride, but we cannot expect this kind of pride to be related to professional integrity.
Even though I limited my discussion to Japan, it is plausible that many aspects of my discussion apply to other countries. Professionals play similar roles in other countries, and it is only natural that they have the similar motivational structure. Thus, in countries where the social contract model does not work, the pride-based approach may be a solution. In countries where we can assume the social contract between the society and engineering profession, we may not need the pride-based model, but still it may help in motivating engineering students.
To summarize, the notion of pride is a practical one, and engineering-ethics education based on this concept seems promising. Although we have to accumulate more empirical data and undertake more conceptual analyses before drawing conclusions on the effectiveness of such approaches, this approach is worth trying in a country where the profession of engineering and its implicit social contract with society are almost absent.

professional integrity
japan
pride
engineering ethics


Usefulness of three-dimensional computed tomography of the larynx for evaluation of unilateral vocal fold paralysis before and after treatment: technique and clinical applications

Laryngoplasty is well-known technique for unilateral vocal fold paralysis (UVFP). However, operation result are sometimes not as good as expected before surgery. Three-dimensional Computed tomography (3DCT) is useful for visualizing complicated intralaryngeal structures. Moreover, 3DCT is suited for analyzing the movement of the vocal fold and arytenoid cartilage because the technique is based on actual data from live patients. We have been used 3DCT of the Larynx for evaluation of UVFP before and after treatment. We uncovered some new findings about UVFP and reasons of unsatisfactory outcomes after operation. Technique and clinical applications of 3DCT for UVFP are outlined in this paper.
Introduction
The movement of the arytenoid cartilage is complicated [1, 2]. Three-dimensional computed tomography (3DCT) is useful for visualizing the location of the arytenoid cartilage. However, complete understanding of the laryngeal anatomy is necessarily to reconstruct the cartilages in three dimensions. In our institution, an otolaryngologist who specializes in the larynx utilizes 3DCT reconstruction. The use of Windows® XP-compatible image-processing software enables various analyses in unilateral vocal fold paralysis. This study reports the clinical application of 3DCT in the laryngeal region using the image-processing software Mimics (Materialise, Yokohama, Japan).
Production of 3DCT
Computed tomography was performed in the horizontal plane using a CT scanner (Sensation Cardiac 64; Siemens, Munich, Germany) with a 1 mm slice width, a helical pitch of 1, and an image production interval of 0.5 mm. Several other studies have used slice widths of around 1 mm [3–6].
Scans were made when the glottis was closed (with phonation) and open. Each patient was scanned for about 2 s. The speed of the scan enables scanning of patients with unilateral vocal fold paralysis during a brief phonation and minimizes motion artifact. Before scanning, doctors (the authors) were consulting the patient about the timing between phonation and the scan.
The data were transferred using the Dicom system. Mimics ver. 9.01 (Materialise) was used to create 3DCT images through its surface-rendering protocols. Using Windows® XP (Microsoft®, Redmond, WA, USA), 3DCT images can be shown in axial, coronal, sagittal, and 3D display windows (Fig. 1).
Fig. 1Actual view seen with the Mimics image-processing software. Using Windows® XP (Microsoft®), 3DCT images can be created with axial, coronal, sagittal, and 3D display windows. The use of Windows XP-compatible image-processing software enables various analyses in unilateral vocal fold paralysis
The threshold was set from 69 to 85 HU for the thyroid and cricoid cartilages and 52 to 69 HU for the arytenoid cartilage. Visualization of the vocal process is usually limited to its base because of its elastic cartilage. In addition, the laryngeal lumen was visualized. The threshold was set between −600 and −100 HU [7].
Stereoscopic location of the arytenoid cartilage in unilateral vocal fold paralysis
First, we present a case of severe unilateral vocal fold paralysis with a mean flow rate (MFR) of 1,200 ml/s (Fig. 2). The patient was an 80-year-old man suffering from paralysis following aortic aneurysm surgery.
Fig. 2The locations of the arytenoids in severe unilateral vocal fold paralysis. During inspiration, the healthy side (yellow) was located in the lateral part of the cricoarytenoid joint, while the paralyzed side (red) was located in the dorsocranial part of the cricoarytenoid joint. During phonation, the healthy side rocked and moved to the ventromediocaudal part of the cricoarytenoid joint, while the paralyzed side glided dorsocranially. This movement is a passive movement
During inspiration, the arytenoid cartilage on the healthy side was located in the lateral part of the cricoarytenoid joint. Simultaneously, the vocal process was located outside and above the joint. In comparison, the arytenoid cartilage on the paralyzed side was located in the dorsocranial part of the cricoarytenoid joint, while the paralyzed vocal process was located inferiorly and the arytenoid cartilage dropped forward. This position on the paralyzed side was the result of posterior cricoarytenoid muscle paralysis.
During phonation, the healthy side rocked and moved to the ventromediocaudal part of the cricoarytenoid joint to adduct the vocal cord, and the vocal process moved inward and downward. Conversely, the paralyzed side glided dorsocranially in comparison with its position during inspiration. Therefore, the arytenoid cartilage showed a rocking movement on the healthy side, and a gliding movement on the paralyzed side. This gliding movement on the paralyzed side was a passive movement. the paralyzed arytenoid underwent passive movement caused by contact with the mobile side during phonation. The vocal fold blown up may affect this passive movement. We call such arytenoids “flaccid arytenoids.” The position of the paralyzed arytenoid depends on the severity of paralysis.
Surgical simulation
This section describes type I thyroplasty. It is important to determine the level of the vocal folds in this procedure. Note that the arytenoid cartilage on the paralyzed side glides dorsocranially, as mentioned above. The paralyzed vocal fold becomes higher than the vocal fold on the healthy side during phonation. The type I thyroplasty window should be based on the vocal cord level on the healthy side during phonation. Therefore, the vocal fold level of the healthy side must be projected onto the thyroid cartilage of the paralyzed side.
Figure 3 compares the upper surface of the vocal fold between our 3DCT simulation and the original type I procedure reported by Isshiki et al. [8]. The figure shows that the upper surface level of the vocal fold in the original procedure is consistent with the vocal fold level on the paralyzed side detected by 3DCT. This might have arisen because the upper surface level of the vocal cord in the original procedure was based on cadaveric studies of the larynx. However, the vocal process on the healthy side during phonation is lower than that on the paralyzed side. Therefore, the design should be made consistent with the healthy side. Individualized simulation for each case is needed because of the shape difference between left and right laminas of the thyroid cartilage (especially the laryngeal deviation) [9, 10]. The program Mimics allows distances to be measured on a personal computer; for example, the distances from the inferior notch or inferior nodule to the surface of the vocal fold may be measured. The 3DCT could contribute to a better oriented and designed thyroplasty window.
Fig. 3A comparison of the upper surface of the vocal fold between the 3DCT simulation (red dotted line) and the original type I procedure reported by Isshiki (black dotted line). The figure shows a 3D image processed to make the thyroid cartilage more radiolucent, allowing the healthy vocal fold to be visualized. The upper surface level of the vocal fold with the original procedure is consistent with the vocal fold level of the paralyzed side detected by 3DCT. However, the vocal process of the healthy side during phonation is located below than that of the paralyzed side
Postoperative evaluation
Case 1
A 69-year-old woman underwent arytenoid adduction for right recurrent nerve paralysis after thyroid carcinoma surgery. According to the postoperative 3DCT images (Fig. 4), the vocal process adducted to the appropriate position. However, a large glottal chink was observed during phonation. In this case, the cricothyroid joint was released, and a thread placed on the muscular process was fixed to the anterior part of the thyroid cartilage. Since the cricothyroid joint was released, the cricoid cartilage (arytenoid cartilage) and thyroid cartilage were close together, which shortened the anteroposterior diameter of the vocal fold. The vocal fold relaxed and lost its tension.
Fig. 4Case 1: A 69-year-old woman underwent arytenoid adduction for right recurrent nerve paralysis after thyroid carcinoma surgery. a In the postoperative 3DCT images, the vocal process appears to adduct to the appropriate position. b A large glottal chink is observed during phonation. c Since the cricothyroid joint is released, the cricoid (arytenoid cartilage) and thyroid cartilages are close together. d The anteroposterior diameter of the vocal fold (double-headed arrow) is shortened. The vocal fold relaxes and loses its tension
Case 2
A 64-year-old man underwent type I thyroplasty for left recurrent nerve paralysis after esophageal cancer surgery (Fig. 5). This patient had relatively severe paralysis, but still underwent a type I thyroplasty only. The postoperative 3DCT image shows that the Gore-Tex® is inserted at the proper level of the vocal fold. However, the posterior parts of the vocal fold were at different levels because the flaccid paralyzed arytenoid cartilage was blown toward the dorsocranial part of the cricoarytenoid joint, as mentioned above. The MFR was improved from 770 ml/s preoperatively to 290 ml/s postoperatively. If the posterior support of the vocal fold had been fixed, the vocal fold would have developed tension against the flow of expired air during phonation, further improving the voice.
Fig. 5Case 2: A 64-year-old man underwent type I thyroplasty for left recurrent nerve paralysis after esophageal cancer surgery. a The postoperative 3DCT image shows that the Gore-Tex® was inserted at the proper level of the vocal fold. b A level difference is seen in the posterior part of the vocal fold because the flaccid paralyzed arytenoid cartilage was blown toward the dorsocranial part of the cricoarytenoid joint during phonation
Case 3
A 70-year-old man was given a silicon injection 20 years earlier to treat left recurrent nerve paralysis after thoracic aortic aneurysm surgery (Fig. 6). The MFR was 650 ml/s, which improved insufficiently. The 3DCT showed that the silicon was injected widely from the anterior to posterior parts of the vocal fold. Silicon is also injected near the arytenoid cartilage. It was thought that the original surgeon had intended to adduct the arytenoid cartilage. However, the arytenoid cartilage glides dorsocranially, increasing the vertical difference in the level of the posterior part of the vocal fold. The vocal process was not adducted and the upper part of the arytenoid cartilage moved medially in an unnatural manner. As described earlier, during phonation, the arytenoid cartilage on the healthy side moves by rocking to the ventromediocaudal part of the cricoarytenoid joint, and the vocal process is moved inward. Arytenoid adduction [10, 11] is a procedure that resolves the level difference between the two vocal fold by reproducing the rocking motion of the paralyzed arytenoid cartilage. In contrast, injection and type I surgery are techniques to medialize the vocal fold by increasing its volume. An injection near the arytenoid cartilage moves the arytenoids to a dorsocranial position, and they never adduct naturally. If arytenoid adduction is scheduled after these techniques, the injected materials could interfere with the second procedure. The effect of the injection on posterior vocal fold closure arises mainly from the increased volume in the posterior part of the vocal fold.
Fig. 6Case 3: A 70-year-old man underwent a silicon injection 20 years earlier to treat left recurrent nerve paralysis after thoracic aortic aneurysm surgery. a The 3DCT shows that the silicon is injected widely from the anterior to posterior parts of the vocal fold. b Silicon is also injected near the arytenoid cartilage. The arytenoid cartilage glides dorsocranially; the vocal process is not adducted and the upper part of the arytenoid cartilage is unnaturally shifted medially. The vertical difference in the level of the posterior part of the vocal fold increased. c The effect of the injection on posterior vocal fold closure is thought to be due mainly to the volume increase in the posterior part of the vocal fold
Case 4
A 77-year-old man received a fat injection for left recurrent nerve paralysis after esophageal cancer surgery. Three years after the fat injection, the maximum phonation time improved from 3 s before the injection to 7 s after the injection. The 3DCT image indicates that the vocal cord closure was sufficient (Fig. 7a). In the lateral view, remaining fat is seen 3 years after the injection and it appears to be pushing the entire vocal fold (Fig. 7b). However, the coronal section CT image revealed that the level difference of the two vocal folds had not improved. The vocal folds overlapped so that the incomplete closure was resolved (Fig. 7c). Fat, unlike silicon, may not interfere with the vocal fold vibration of the healthy side, even if the vocal cords overlap, because fat is soft.
Fig. 7Case 4: A 77-year-old man underwent fat injection for left recurrent nerve paralysis after esophageal cancer surgery. a The 3DCT (CT endoscopic image) suggests that the vocal cord closure is sufficient. b The lateral view shows fat remaining 3 years after the injection, which seems to be pushing the entire vocal fold. c The coronal view revealed that the difference in the levels of the vocal fold had not improved
Conclusions
Laryngeal 3DCT is useful for determining the stereoscopic configuration of the arytenoid cartilage. In addition, 3DCT enables observation of the laryngeal framework from every angle, and is also useful for surgical simulation and feedback after surgery.

three-dimensional computed tomography
unilateral vocal fold paralysis
thyroplasty


Storage hexamer utilization in two lepidopterans: differences correlated with the timing of egg formation

Most insects produce two or more storage hexamers whose constituents and developmental profiles are sufficiently different to suggest specialization in the ways that they support metamorphosis and reproduction. Hexamerin specializations are compared here in the Cecropia moth (Hyalophora cecropia), which produces eggs during the pupal-adult molt, and the Monarch butterfly (Danaus plexippus), which produces eggs under long-day conditions after adult eclosion. In both sexes of both species, reserves of arylphorin (ArH) were exhausted by the end of metamorphosis. In Cecropia, the same was true for the high-methionine hexamerins, V-MtH and M-MtH. But in short day Monarch females 20–30% of the pupal reserves of V-MtH and M-MtH survived metamorphosis, persisting until long-day conditions were imposed to stimulate egg formation. Differences in storage sites have been documented in other lepidopterans, with MtH reserves being found primarily in fat body protein granules and the ArH reserve being found primarily in the hemolymph. Similar differences could explain how a fraction of the MtH's, but not of ArH, escapes utilization during metamorphosis in a species with post-eclosion egg formation. No differences in utilization schedules were detected between V- and M-MtH, despite divergent compositions and antigenic reactivity.
Introduction
Insects prepare for the synthetic demands of molting, metamorphosis and reproduction by accumulating hexamerins in their hemolymph and fat body. First to be described were two soluble storage hexamerins isolated from larvae of Calliphora erythrocephala (Munn and Greville, 1969). Two or more hexamerins were later reported in the hemolymph and fat body of many other insects, with some lepidopterans having as many as four. Hexamerins occurring within a single species can differ in amino acid composition, stage of synthesis, distribution between hemolymph and fat body, timing of clearance from the hemolymph, and antigenic reactivity (reviewed by Wyatt and Pan, 1978; Riddiford and Law, 1983; Kanost et al., 1990; Telfer and Kunkle, 1991).
The first storage proteins to be described in lepidopterans were two methionine-rich hexamerins from Hyalophora cecropia (Tojo et al., 1978). They differed in electrophoretic mobility in native PAGE and also in amino acid composition, methionine contents being 7.0% for the more slowly migrating and 4.9% for the faster. (We adopt here descriptive acronyms, V-MtH and M-MtH, for very and moderately methionine-rich hexamerins.) The two appear in the hemolymph late in the last larval instar, rise to maximum concentrations early in the larval-pupal molt, and are then largely endocytosed by the cells of the fat body, which store them in crystalline form until they are utilized during adult development. MtH's have been identified in a variety of moths, either as isolated proteins or from cDNA sequencing (Tojo et al., 1978; 1980; 1985; Ryan et al., 1985; Bean and Silhacek, 1988; Jones et al., 1990; Jones et al., 1993; Memmel et al., 1994). They are identified here in a butterfly by antigenic cross-reactions. They tend to be more abundant in female pupae than in males, and this has led to a prediction that they play special roles in egg formation.
A third lepidopteran hexamerin, first described in Manduca sexta (Kramer et al., 1980), has a high aromatic amino acid content, resembling in this regard one of the prototypical hexamerins of Calliphora. The importance of aromatic amino acids in sclerotization led to the suggestion that hexamerins in this class, now known as arylphorins (ArH) (Telfer et al., 1983), are adapted to support cuticle deposition. While many lepidopterans produce just these three, some also contain a riboflavin-binding hexamerin, RbH (Telfer and Massey, 1987; Miller and Silhacek, 1992; Magee et al., 1994). Like ArH, RbH is a major component of pupal hemolymph that disappears during adult development (Pan and Telfer, 1999).
Labeling experiments in saturniid moths failed to confirm the targeting of hexamerins for special developmental processes. In Actias luna M-MtH and ArH proved to be equivalent sources of labeled amino acids for the production of eggs and cuticle, as well as other adult tissues (Pan and Telfer, 1996), and RbH and ArH were found to be similarly equivalent in H. cecropia (Pan and Telfer, 1999). These tests were performed in species that produce eggs at the same time that they form the soma of moths. They thus left open the question of which of the hexamerins remain available to support egg formation when the latter is delayed until after adult eclosion.
To investigate this we compared the timing of hexamerin depletion in two lepidopterans that form eggs at different times relative to eclosion. The Cecropia silkmoth, Hyalophora cecropia, ecloses with a full complement of eggs that are ready to be fertilized and laid. The Monarch butterfly, Danaus plexippus, delays egg formation until it experiences a long-day photoperiod after eclosion (reviewed by Ackery and Vane-Wright, 1984). The strategy we used was to time in each species the disappearance of V-MtH, M-MtH and ArH during metamorphosis and egg formation. The accumulation of vitellogenin (Vg) was simultaneously measured, since synthesis of this abundant product by the adult fat body can be correlated with hexamerin depletion (Wheeler and Buck, 1996; Wheeler et al., 1999). RbH was not included because we could not detect it with rabbit antibodies in Monarch larvae or pupae (Pan and Telfer, 1999).
Materials and Methods
1. Experimental Insects
Diapausing pupae of field-reared Hyalophora cecropia were stored at 6°C for at least five months and then allowed to initiate adult development by transfer to 25°C. At this temperature development from apolyis to eclosion requires 23 days in females and 21 days in males.
Migrating butterflies of Danaus plexippus were captured during mid-October in eastern Tennessee. Most of the catches were sufficiently fresh to suggest recent emergence in nearby regions. They were kept in cages at outdoor temperature and day length, and fed once per day with a 30% honey solution. After two to three weeks under these conditions the ovaries contained only small, translucent follicles, indicating an absence of yolk formation. Their fat bodies, relative to those of freshly caught butterflies, were greatly enlarged and full of lipid droplets.
To obtain pupae and second generation adults, some captured Monarchs were kept at 30°C under long day conditions, and fed twice daily with the honey solution. They terminated reproductive diapause and began mating in 1 to 2 weeks. Potted milkweed, Asclepias curasavica, was provided for egg laying. Hatched caterpillars were left on the plants until they reached the 3rd instar, and were then transferred to field-collected cuttings of Asclepias syriaca. Additional eggs and caterpillars were generously provided by Mr. Paul deMarrais of Afton, Tennessee.
2. Antibodies
V-MtH, M-MtH, ArH and Vg were measured in both species with rabbit antibodies against the corresponding proteins of H. cecropia. The reactions with Monarch proteins, though weaker than those with Cecropia, were still strong enough to be useful. This relationship is shown in Figure 1 for antibodies to Cecropia V-MtH.
ArH, Vg and what we identify below as M-MtH have been isolated from the hemolymph of Cecropia pupae by chromatographic methods, and the reactions of the antisera employed to measure them have already been described (Telfer et al., 1983; Telfer and Pan, 1988; Pan and Telfer, 1992). Each of the three antisera reacted with its homologous isolate to produce a single zone of heavy precipitation in immunodiffusion tests and showed no additonal reactions when combined with unfractionated hemolymph, whole-body extracts, or isolates other than the one used for immunization.
The antiserum against V-MtH has not been described before. We used native PAGE to isolate this hexamerin from pupal male hemolymph. Slab gels (0.3 × 12 × 17 cm) containing 4% acrylamide and lacking lane dividers were overlain with 40 µl of pupal male hemolymph diluted with twice its volume of native PAGE sample buffer. After electrophoresis a vertical strip from the center of the gel was stained with Coomassie Blue (Figure 2, lane 1). Horizontal strips corresponding to the positions of the two MtH's were cut from the unstained remainder of the gel and stored by freezing.
The two MtH's were identified by N-terminal amino acid microsequencing at a facility of the University of Pennsylvania School of Veterinary Medicine. For the faster of the two in native PAGE, the N-terminal sequence was RPDNDDVNFVVSM and for the slower band was SVVNDANYSF (40% of the fast component lacked the N-terminal RP but was otherwise identical to the other 60%.) These sequences matched N-terminal sequences deduced from cDNA clones of, respectively, M-MtH and V-MtH after signal polypeptide deletion (Massey, 1995; accession number AF032398 for M-MtH and AF032399 for V-MtH). The faster band corresponded to a 738 amino acid polypeptide with a methionine content of 4.76%, while the slower corresponded to a 737 amino acid polypeptide with a methionine content of 8.54%. Protein extracted from the slower band, thus identified as V-MtH, was used to immunize a rabbit (Harlow and Lane, 1988). In a variety of immunodiffusion tests with pupal hemolymph and extracts from H. cecropia, anti-V-MtH produced a single band of precipitation (Figure 1).
3. Tissue Preparation
Hemolymph was drained through a dorsal slit into a pre-chilled, graduated test tube containing 0.2 ml of a buffer solution—either, pH 7.2 phosphate-buffered saline (PBS) for Cecropia or 0.15 M Tris-citrate, pH 8.2, for the Monarch. (The Tris-citrate buffer used for the Monarch extraction was 6X that of the running buffer strength employed for Rocket Immunoelectrophoresis (Pan and Telfer, 1990). It was used in case the smaller amount of tissues collected from this species should prove to require a more sensitive technique for protein quantification, but this proved eventually not to be the case.) Both extraction media contained in addition a protease inhibitor cocktail, Control® (Boehringer Mannheim Biochemical), and 5mM phenylthiourea (PTU). The midgut and, when present, the bursa copulatrix were discarded and the rest of the soft tissues were scraped out of the abdominal cuticle and added to the collected blood. Additional buffer with the protease inhibitor and PTU was added to bring the suspended tissues to a volume that would yield antigen concentrations convenient to measure by the method described below. For Cecropia the final volume was 6 ml per pupa, and 3 or 1.5 ml per adult female and male, respectively. For Monarch pupae the final volume was 3 ml and for adults 0.75 ml. Comparisons between stages were made possible by normalizing to the pupal volume; e. g., concentrations for adult Cecropia females were divided by two and for males by four in order to compare them with the contents of pupal extracts.
The collected samples were stored frozen. For extraction, the tissue mixtures were thawed and transferred to a glass homogenizer. When chorionated eggs were present, they were crushed separately in a ceramic mortar and then combined with the rest of the tissues in the homogenizer.
4. Measurement of Antigen Concentrations
An immunodiffusion method was used to measure changes in extractable stores of the four proteins. Antigen concentrations were measured by layering the extracts over antisera that had been solidified in 3 mm (i. d.) glass tubes with 0.3% agarose, PBS and 0.05% sodium azide. The rate of penetration of an antigen-antibody precipitation front through the agarose is a function of the rate of diffusion of the antigen into the agarose layer and this in turn is a measure of antigen concentration in the upper reservoir. Procedures for setting up the tubes, for measuring rates of advance of precipitation fronts, and for constructing standard curves have already been described (Telfer et al., 1983; Telfer and Pan, 1988). The method was developed for studies on human serum proteins (Oudin, 1948), and has been used many times to measure antigen concentrations in insect hemolymph and egg extracts. It requires larger volumes and higher antigen concentrations than rocket immunoelectrophoresis (Pan and Telfer, 1990, 1992), but when these conditions are satisfied, as they proved to be for hexamerin measurements in Cecropia and Monarchs, it is convenient and reliable.
The measurements are of relative concentrations and are expressed as percentages of the concentration in a standard solution. For the hexamerins, extracts of abdomens from female pupae of each species were used as the standards; for VG, extracts of females containing chorionated eggs were used. The latter included newly eclosed adults in Cecropia and day 7 adults in long day Monarchs.
5. SDS-PAGE Electrophoresis
Insoluble residues that would escape detection by immunodiffusion were monitored by SDS-PAGE. For this purpose 0.1 ml of each of the homogenized samples from a chosen stage were vortexed and pooled. The pooled sample was centrifuged in a microfuge (Beckman®) for 4 min. The lipid cap was removed, and the supernatant decanted. Pellet and supernatant were then separately dissolved in SDS-PAGE sample buffer so that equal volumes contained equal fractions of, respectively, the total pellet and supernatant material in the uncentrifuged pool. The samples were compared in neighboring lanes of pre-cast 4–15% gradient mini gels from Bio-Rad®. Electrophoresis was conducted at constant voltage (100 v), and terminated after 1 hr and 12 min. Coomassie Brilliant Blue staining was used to reveal the banding patterns.
Results
1. Vitellogenin
Cecropia produced Vg earlier than the Monarch (Figure 3). Like many other saturniids, Cecropia synthesizes part of its Vg, in this case about 30 mg per 6 g female, during the larval-pupal molt, and stores it in the hemolymph during the months of diapause (Telfer, 1954). Beginning 10 days before eclosion, when yolk formation begins, there is a second period of Vg synthesis (Pan, 1971) (Figure 3) that draws on precursors released from the hexamerins present at that time (Pan and Telfer, 1996, 1999). In Monarchs, Vg was not detected in either pupae or short day adults. In females raised under long day conditions it was first detected 2–3 days after eclosion (Figure 3) and it continued to accumulate until day 7 when eggs began to be laid.
In SDS-PAGE, Coomassie-stained Vg subunits were identified at 180 and 40 kDa by their presence in females and absence from males (Figure 4, Vg). They were found in pupal and adult extracts of Cecropia and in extracts of long day Monarch adults, the same samples in which Vg had been detected by immunodiffusion.
Vg is stored as a solute in the hemolymph of female Cecropia pupae and accordingly centrifugal pellets of extracts at that stage did not produce detectable 180 and 40 kDa bands. In adults most Vg has been transferred to the yolk; pellets at this stage produced weak Vg bands due, presumably, to small yolk spheres that are not readily disrupted by homogenization.
2. Hexamerin utilization in females
Cecropia
All three Cecropia hexamerins were consumed during the first 13 days of somatic tissue metamorphosis, 55% of the soluble MtH's and 33% of soluble ArH disappeared before VG began its second rise (Figure 5A). The last eight days of adult development encompass late somatic tissue development as well as the second rise in Vg content, and during this period the three hexamerins fell to less than 2% of pupal levels.
Coomassie staining of SDS-PAGE gels of pupal extracts was concentrated in a cluster of circa 80 kDa bands (Figure 4A, asterisks). Mini gels resolved the hexamerin subunit bands in the clusters poorly, but for present purposes that was not a problem. Low 80 kDa staining in centrifugal pellets of adult Cecropia extracts (Figure 4A, lanes A1 and A2) showed that PBS-insoluble reserves, like the soluble reserves assayed by immunodiffusion, were essentially negligible after eclosion.
Monarch
Nearly all of the soluble ArH stored in Monarch pupae was consumed during metamorphosis, but substantial MtH reserves remained. This was first seen in individuals that had been collected in the field in October and then fed as captives for four weeks under short day conditions (Figure 6A). At the end of the four weeks, short-day females still contained about 20% as much M-MtH and 30% as much V-MtH as female pupae. When captive females were held for two weeks under short day conditions and then for two additional weeks in the long days that promote egg formation, these figures were much lower—1.5% for M-MtH (p < 0.01) and 0.7% for V-MtH (p < 0.02) (Figure 6A). These results were confirmed by SDS-PAGE, which detected a strong 80 kDa band in short day adults (Figure 4B, lanes Ast) and very little staining in long-day adults (lanes Alg). The time-course of the decrease was examined in a set of females that had been raised from eggs in the laboratory under long day conditions (Figure 7). In this case 32% of extractable pupal M-MtH and 47% of V-MtH remained at the time of eclosion. During one week of feeding as adults under continuing long day conditions three quarters of the reserves present at eclosion were consumed.
3. Sexual Differences.
While males contained roughly as much soluble ArH as females in Cecropia pupae, their contents of V-MtH and M-MtH were significantly lower (Figure 5B). A sexual difference was also seen during adult development: from days 13 to 21, when vitellogenesis and chorion deposition occur, utilization of V-MtH and M-MtH was more rapid in females than in males (Figure 5A and B). In males, as described above for females, immunodiffusion failed to detect more than 1–2% of the pupal levels of any of the three hexamerins at the time of eclosion (Figure 5B, Adult). In confirmation of this, SDS-PAGE revealed only traces of 80 kDa material in centrifugal pellets (Figure 4A, stages A1 and A2, lanes P).
Monarchs differed from the lepidopteran norm in showing no sexual differences in the amounts of soluble V- and M-MtH stored in pupae (Figure 6B, green bars). In four week old adults raised under short day conditions, however, females retained larger reserves than males (Figure 6A and B, red bars), but had consumed more of these two proteins under conditions permitting egg formation (Figure 6A and B. blue bars).
Discussion
1. Egg formation versus metamorphosis
The results are clear in showing that methionine-rich hexamerins in the absence of ArH support egg formation in Monarchs. Short-day adults, whose ovaries do not produce eggs, retained 20–30% of their pupal reserves of the MtH's, but only 0.3% of ArH (Figure 6A). Reduction of V-and M-MtH to only 1–2% of pupal levels in long-day females that had begun to lay eggs confirms that adult MtH's are in fact consumed during egg formation. The Monarch results also show that V- and M-MtH support somatic tissue development, as 50% of pupal V-MtH and 70% of M-MtH disappeared during adult development (Figure 7). The depletion of pupal V- and M-MtH in males (Figure 6B) also indicates functions other than the support of egg formation.
ArH, too, is multi-functional. It is known to support larval molting in the absence of the MtH's in Manduca sexta (Kramer et al., 1980; Riddiford and Hice, 1985; Webb and Riddiford, 1988) and Cecropia (Telfer et al., 1983). This situation differs in the dictyopteran, Blatta orientalis, whose two hexamerins are both present in larval hemolymph and cycle together during molting (Duhamel and Kunkel, 1978). ArH can also support reproductive functions, for it was as effective as M-MtH in providing precursors for the synthesis of Vg and chorions in pharate adult Luna moths (Pan and Telfer, 1996).
Selectivity in the consumption of the multiple hexamerins within a lepidopteran species is shown by these results to be related to availability: the MtH's are not used in the larval molts because they are not synthesized until late in the last instar (Tojo et al., 1978; Riddiford and Hice, 1985; Jones et al, 1993; Memmel et al, 1994); and pupal reserves of ArH do not support egg formation in adult Monarchs because they are fully consumed during metamorphosis. When both are at hand, however, both contribute to whatever metamorphic and reproductive functions are under way.
Two kinds of processes are apparent that affect availability. Most obvious is the early synthesis of ArH that makes it uniquely available for the support of larval molting. The second involves differences in mode of storage. In insects generally, storage hexamers are secreted into larval hemolymph, where they can attain extraordinary concentrations. When metamorphosis approaches they are reclaimed to varying degrees by the fat body, and packaged in cytoplasmic storage granules. In an earlier study over 99% of injected M-MtH was cleared from the hemolymph of pharate pupae, compared with only 35% of ArH (Pan and Telfer, 1992). A consequence of this in both Luna and Cecropia pupae is that the MtH's are the most prominent proteins of fat body extracts while ArH is the most prominent protein of hemolymph (Figure 2 in Pan and Telfer, 1996, and Figure 17 in Tojo et al, 1978). There can also be differences in the disposition of proteins within the fat body, for in Cecropia the hexamerin storage granules contain protein crystals embedded in an amorphous material (Tojo et al., 1978). Differences in accessibility provide a plausible speculation on how V- and M-MtH, but not ArH, might survive metamorphosis in sufficient amounts to support post-eclosion egg formation.
2. How hexamerins support egg formation
Hexamerins presumably support egg formation primarily by providing precursors for protein synthesis, for the synthesis of yolk proteins by fat body and of chorion proteins by the follicle cells both entail transcription and the incorporation of free amino acids (Wyatt, 1991; Paul and Kafatos, 1975; Nadel and Kafatos, 1980). Cases of intact hexamerin incorporation by ovaries have been described, but these are idiosyncratic rather than general. Antigenic epitopes of all four lepidopteran hexamerins are present among yolk proteins of saturniid moths, but they are only 0.2% as concentrated as those of Vg (Telfer and Pan, 1988). In Hyphantria cunea antigenic activity of an M-MtH-like protein was detected in intercellular spaces and yolk during vitellogenesis, but was absent from mature eggs (Seo et al., 1998). The authors speculated that this protein provides an internal source of amino acids for protein synthesis in the maturing egg. A more striking exception occurs in Riptortus clavatus, a hemipteran whose yolk contains approximately equal amounts of Vg and a 500 kDa, biliverdin-binding hexamerin (Chinzei et al., 1990; Miura et al., 1998). The yolk form of this otherwise conventional storage protein is secreted by the fat body of adult females in synchrony with Vg and selectively endocytosed by the vitellogenic oocyte. In another unusual case, the principal hemolymph storage proteins of last instar larvae and pupae of Bombyx mori are not hexamerins but a family of 30 kDa proteins that persist into the adult stage and are accumulated during vitellogenesis as major constituents of yolk (Zhu et al., 1986; Chen and Yamashita, 1990). But immunodiffusion and SDS-PAGE revealed nothing comparable to either the Riptortus or Bombyx storage proteins in egg-filled abdomens of long-day Monarchs.
3. Hexamerin synthesis in adults?
There are other cases of the resumption of hexamerin synthesis in eclosed adults (e.g., Kunkel and Pan, 1976; Wyatt et al., 1992), but these occur in males as well as females and are thus not necessarily related to egg formation. Post-eclosion synthesis is not ruled out as a source of V- and M-MtH in adult Monarchs, but if it occurs it is not sufficient to prevent the depletion that accompanies egg formation. It would not, in any case, affect the argument that the consumption of residual pupal V- and M-MtH, rather than ArH, is correlated with post-ecdysial egg formation.
4. V-MtH versus M-MtH
The two methionine-rich hexamerins are electrophoretically distinct and show no detectable similarities in precipitation by polyclonal rabbit antibodies. Yet they behaved here identically in the timing of their disappearance during adult development and in the fraction of their pupal reserves that survived metamorphosis. Cladistic analyses of amino acid sequences deduced from cDNA clones suggest that they are descended from products of a gene duplication occurring early in lepidopteran evolution (Burmester et al., 1998). But we are as yet left with no clue as to why natural selection has favored a pair of diverging MtH's over a single protein that might have averaged their amino acid compositions.


The autonomic nervous system at high altitude

The effects of hypobaric hypoxia in visitors depend not only on the actual elevation but also on the rate of ascent. Sympathetic activity increases and there are increases in blood pressure and heart rate. Pulmonary vasoconstriction leads to pulmonary hypertension, particularly during exercise. The sympathetic excitation results from hypoxia, partly through chemoreceptor reflexes and partly through altered baroreceptor function. High pulmonary arterial pressures may also cause reflex systemic vasoconstriction. Most permanent high altitude dwellers show excellent adaptation although there are differences between populations in the extent of the ventilatory drive and the erythropoiesis. Some altitude dwellers, particularly Andeans, may develop chronic mountain sickness, the most prominent characteristic of which being excessive polycythaemia. Excessive hypoxia due to peripheral chemoreceptor dysfunction has been suggested as a cause. The hyperviscous blood leads to pulmonary hypertension, symptoms of cerebral hypoperfusion, and eventually right heart failure and death.
Introduction
High altitude places are amongst the most inhospitable on earth. According to WHO [70] in 1966 there were approximately 140 million people living at altitudes over 2,500 m and there are several areas of permanent habitation at over 4,000 m. These are in three main regions of the world: the Andes of South America, the highlands of Eastern Africa, and the Himalayas of South-Central Asia. This review is concerned with the effects of the altitude on visitors and the ways by which the permanent high altitude dwellers have adapted to their environment.
The two main challenges to life at high altitude come from hypobaric hypoxia and the low ambient temperatures. Temperature decreases about 1°C for each 150 m elevation, so that at 4,500 m temperature is roughly 30°C lower than at sea level. Barometric pressure falls progressively with increasing altitude. Up to about 2,500 m there are few if any effects of hypoxia. Above 3,000 m some effects of hypoxia are likely to be experienced and above 4,000 m adverse effects would be experienced by most unacclimatized visitors. However, many people live and work at altitude with no apparent adverse effects. One such example is Cerro de Pasco a busy mining town of 72,000 population at 4,300 m in the Peruvian Andes, where much high altitude research has been undertaken. At this altitude barometric pressure is 450 mmHg and without hyperventilation alveolar oxygen tension would be only 34 mmHg.
Autonomic control in visitors
Most adaptive changes occur in the first days and weeks following arrival at altitude, and this is the period when acute mountain sickness with cerebral and/or pulmonary oedema may occur. Recent studies in animals and man have highlighted the role of the autonomic nervous system in adaptation and in particular the importance of sympathetic activation following high altitude exposure.
Cardiovascular effects
Acute hypobaric hypoxia results in an increase in resting heart rate and blood pressure and this is seen both during altitude exposure [5] and during simulated exposure using a hypobaric chamber [68]. Vogel et al. [68, 69] demonstrated that the rate of ascent influenced the magnitude of the tachycardia. Gradual increases in altitude over two weeks resulted in larger heart rate changes compared with an abrupt ascent. Later, as subjects acclimatise at altitudes up to about 4,500 m, much of the increase in heart rate is lost and resting heart rates return towards their sea level values.
Acute hypoxia also causes an increase in cardiac output both at rest and for given levels of exercise. This was seen both when breathing hypoxic gas at sea level [34, 68, 69] and on acute exposure to high altitude [21]. As subjects acclimatise to the altitude cardiac output decreases although the heart rate can remain high with a low stroke volume. This may be due to a loss of plasma volume [23, 54].
The effect of hypoxia on the pulmonary circulation is rapid, resulting in an increase in pulmonary vascular resistance and pulmonary hypertension [49]. The maximum response occurs within 5 min [65]. Breathing 11% oxygen for 30 min increases pulmonary artery pressure from 16 to 25 mmHg [72]. The effect of hypoxia on the pulmonary circulation is even more pronounced during exercise, as demonstrated in the Operation Everest II studies [24] where pulmonary artery pressure increased during near-maximal exercise at 8840 m to 54 mmHg.
The mechanism of pulmonary artery vasoconstriction initially involves inhibition of O2 sensitive K+ channels leading to depolarization of pulmonary artery smooth muscle cells and activation of voltage gated Ca2+ channels causing Ca2+ influx and vasoconstriction [48]. This process is immediately reversed by breathing oxygen. However, lowlanders exposed to high altitude for 2–3 weeks develop pulmonary hypertension that is not completely reversed by oxygen breathing [24] suggesting remodelling of pulmonary arterioles. Remodelling involves proliferation of smooth muscle cells and thickening of the artery wall [57].
Sympathetic activity
Acute hypoxia is a potent activator of sympathetic activity [39, 56]. Studies in several species, including dogs, rats and rabbits, showed that hypoxia stimulated the sympathoadrenal system. Acute hypoxia in spontaneously breathing anaesthetized animals causes increases in sympathetic nerve activity, increased release of catecholamines, increases in heart rate and regional vasoconstriction [28, 60]. However the effects of hypoxia on the human sympathetic nervous system are more difficult to determine and often indirect methods of assessment have been employed.
One method of assessment of sympathetic activity in humans is from blood or urine levels of catecholamines. However, catecholamine levels are the net resultant of secretion, spill-over, reuptake and excretion [59, 18] and results must be interpreted with caution. Mazzeo et al. [40] measured arterial noradrenaline and adrenaline concentrations in subjects at sea level, then after 4 h then 21 days at altitude (4,300 m). They reported an initial decrease in noradrenaline but by day 21 it had increased to 52% above sea level values. Arterial adrenaline values doubled following acute altitude exposure then declined to only 26% above sea-level by day 21. In a later study, however, the same authors [41] measured 24 h urinary noradrenaline and adrenaline excretion and venous plasma catecholames in women at sea level and during 12 days of exposure to 4,300 m and reported increases in both urinary noradrenaline and adrenaline excretion after only one day at altitude and increases in plasma catecholamines on day 4 at altitude. During the 12 day period noradrenaline continued to increase as assessed both from urinary excretion and in the plasma samples. Adrenaline values however fell back to those recorded at sea level. Rostrup [59] reported initial decreases in both plasma noradrenaline and adrenaline which subsequently recovered. Results from simulated hypoxia are also conflicting with an increase in urinary adrenaline but no change in noradrenaline [29] or no change in either [71]. These results illustrate the difficulty in assessing autonomic activity from blood or urine catecholamines, but they do suggest that in the early stages of exposure to altitude there is an increase mainly in adrenaline, but that later it is noradrenaline that predominates.
The changes in catecholamines are more consistent during exposure to chronic hypoxia. Calbet [10] measured systemic and skeletal muscle noradrenaline and adrenaline spillover in lowlanders after exposure to 5,260 m. After 9 weeks plasma noradrenaline and adrenaline concentrations were approximately 4 and 2 fold higher than the sea level values. These values were similar to those in patients with compensated chronic heart failure [2].
The heart rate at maximal exercise is reduced at altitude. In Operation Everest II maximal heart rates decreased from 160 at sea level to 118 at 8,848 m [54]. Given the evidence for elevated catecholamines at altitude, at least during chronic exposure, this suggests a down regulation of the cardiac β -adrenergic receptors. Studies in rats following prolonged exposure to hypobaric hypoxia have shown a decreased β-adrenergic receptor density [30, 67]. Short exposures to hypoxia (1–15 days) did not affect β-adrenergic receptor density. However by 21 days there was a 24% reduction. Leon-Velarde et al [35] exposed rats to a simulated altitude of 5,500 m for 21 days and also found a 24% reduction in β-adrenergic receptor density in both left and right ventricles. They also reported an increase in α1-adrenergic receptor density in the left ventricle, although Morel et al [48], who exposed rats to 5,500 m for 15 days, found no change in α1-adrenergic receptor density in either left or right ventricle. Density of adenosine receptors has also been shown to be decreased by 46% following 30 day exposure to 5,500 m simulated altitude in the rat while muscarinic receptor density increased by 49% [31].
Changes in receptor density have also been estimated indirectly in man by determining the rate of isoprenaline infusion required to increase heart rate by 25 beats/min. The required rate increased with increasing exposure to altitude and this was attributed to a down regulation of the β-adrenergic receptors [56]. Platelet α2-adrenergic receptor density decreased after 4 weeks exposure to 5,050 m [19]. Changes in α2-adrenergic receptor density on platelets may indicate a similar change in the central nervous system [53]. In the central nervous system α2-adrenergic receptors are known to play an important role in cardiovascular regulation [22]. Stimulation of these receptors in the ventrolateral medulla has been shown to reduce sympathetic and increase parasympathetic outflow [55]. If a change in the density of these receptors occurred it may explain many of the effects of altitude on the autonomic system.
The effect on the parasympathetic system has been assessed in humans from the responses to muscarinic blockade. Following short exposures to hypoxia the changes in heart rate following muscarinic blockade became smaller [11]. However, Boushel et al. [8] exposed subjects to an altitude of 5,260 m for 9 weeks and then studied the effects of muscarinic blockade both at rest and during exercise, and suggested that there was an enhanced parasympathetic activity and that this was responsible for the reduction in heart rate seen during chronic adaptation to altitude.
Muscle sympathetic nerve activity
Several studies have examined the effects of acute hypoxia on muscle sympathetic nerve activity and reported increases in discharge [17, 27]. Chronic hypoxia also enhances sympathetic activity and Hanson and Sander [27] reported that, following exposure to an altitude of 5,260 m for 4 weeks, activity remained elevated 3 days following descent.
Heart rate variability
Cardiac autonomic nerve activity is often assessed noninvasively from spontaneous changes in heart rate. This is influenced by respiration [9] and this complicates many studies of hypoxia due to the concomitant respiratory stimulation. High frequency R–R interval power is considered to be associated with cardiac parasympathetic activity whereas the low frequency components are associated with both parasympathetic and sympathetic activity. The ratio of low to high frequency power is said to be an index of the “sympathovagal balance”. Power in both high and low frequency bands were found to decrease at altitude although the ratio of low to high frequency power increased [32, 16, 17]. The increase in the ratio is believed to imply that the sympathetic activity is dominant compared to parasympathetic. These findings imply that acute exposure to hypoxia causes decreased parasympathetic and increased sympathetic tone and during acclimatization there is a progressive shift toward still higher sympathetic tone.
Arterial baroreflex
Studies in humans of baroreflex control at altitude or simulated altitude have yielded contradictory results. Sagawa et al. [61], using the neck chamber method, reported that acute exposure to simulated altitude of 4,300 m had no effect on carotid baroreflex set point for heart rate control but there was a 50% reduction in the gain of the reflex. Halliwill and Minson [25, 26], on the other hand, using nitroprusside and phenylephrine, found that hypoxic breathing increased set point but did not change gain. Studies of “spontaneous” baroreflex gain have indicated that it decreases at altitude or simulated altitude [5, 6, 7, 63]. Interpretation of these findings is complicated by the differences in baseline blood pressures and heart rate. Recently, Cooper et al. [15] reported that although acute hypoxia did not change baroreceptor control of heart rate it did decrease the gain of the vascular resistance response without changing “set point”.
Mechanisms for sympathetic activation at high altitude
The increase in sympathetic activity at altitude is caused by both the direct and indirect effects of hypoxia. The role of chemoreceptors is reviewed by Marshall [39]. Hypoxia acts directly on vascular smooth muscle in the systemic circulation causing relaxation and therefore hypotension. This, in turn, would lead to baroreceptor-mediated sympathetic excitation. Alterations in baroreflex function: an increase in “set point” and possibly a decrease in gain, are likely to contribute. An additional mechanism for exciting sympathetic activity may also arise through stimulation of pulmonary arterial baroreceptors. Hypoxia induces pulmonary hypertension and we have recently reported that, in the anaesthetized dog, increases in pulmonary artery pressure increase systemic vascular resistance [38, 46].
Autonomic function in high altitude residents
Healthy highlanders
Healthy high altitude dwellers show excellent adaptation to their environment. These adaptations are likely to be associated with altered gene expression as the expression of genes associated with vascular control and reactions to hypoxia have been found to be high in altitude dwellers [1]. Different communities, however, seem to adopt different adaptation strategies [4]. For example Andeans hyperventilate to decrease end-tidal and arterial CO2 levels to as low as 25 mmHg and have haemoglobin levels well above those at sea-level. Ethiopian highlanders, on the other hand, have CO2 and haemoglobins levels similar to sea-level dwellers [13, 33].
Despite the successful adaptation of permanent residents, some differences do occur. The carotid body shows age-related hyperplasia but blunted responses to hypoxia [62]. High altitude dwellers have been reported to show earlier cardiovascular degenerative changes with aging. Arterial wall stiffness is greater and blood pressures are higher [51, 52]. High altitude residents, both healthy subjects and patients with chronic mountain sickness (see below) have higher catecholamine levels when measured at altitude than following descent to sea level [20]. Baroreceptors, however, appear to function normally at altitude [5]. We [45] examined cardiac and vascular responses to neck chamber pressures and found that responses were unaffected by descent to sea-level. Moore et al. [47] also studied baroreceptor function in anaesthetized dogs, which allows better control of relevant variables. There was no difference in the gain of the baroreflex control of vascular resistance between lowland animals and animals reared at altitude (4,330 m). Set point, however, was lower in high altitude animals which seems surprising in view of the known hypertensive effect of hypoxia.
Blood volumes are larger in high altitude dwellers. In Andeans this is due to a large packed cell volume whereas in Ethiopians plasma volume was large [12, 13]. Probably as the result of the large blood volumes, tolerance to orthostatic stress was greater than that in sea-level residents [12, 14].
Patients with chronic mountain sickness
Chronic mountain sickness (CMS, Monge’s disease; [43]) is a condition frequently found in long-term residents of high altitudes particularly in the Andes where it is a major public health problem. It also occurs in residents on the Tibetan plateau, although not in Ethiopians [13].
Patients with CMS develop excessive polycythemia and various clinical features including dyspnoea, palpitations, insomnia, dizziness, headache, confusion, loss of appetite, lack of mental concentration and memory alterations. Patients may also complain of decreased exercise tolerance, bone pain, acral paraesthesia and occasionally haemoptysis. The impairment of mental function may be reversed by phlebotomy [42]. Physical examination reveals cyanosis, due to the combination of polycythemia and low oxygen saturation, and a marked pigmentation of the skin exposed to the sun. Hyperaemia of conjunctivae is characteristic and the retinal vessels are also dilated and engorged. The second cardiac sound is frequently accentuated and there is an increased cardiac size, mainly due to right ventricular hypertrophy. As the condition progresses, overt congestive heart failure becomes evident, characterized by dyspnoea at rest and during mild effort, peripheral oedema, distension of superficial veins, and progressive cardiac dilation. A clinical score was devised in the attempt to assess the severity of the syndrome and to compare CMS cases within and between different countries in the world [37].
CMS is most likely the result of several influences acting on subjects living at high altitudes. Hypoventilation associated with aging has been proposed as one of the main underlying mechanisms [64]. Many of the clinical features may be attributed to the excessive polycythemia which leads to hyperviscosity of the blood and consequently impaired blood flow and impaired oxygen delivery to several organs including the brain [44].
Sime et al. [64] reported a blunted ventilatory response to hypoxia (VRH) in subjects with CMS and suggested this as the basic underlying cause. However, the same effect may also occur in some subjects without CMS [3, 62]. The function of the peripheral chemoreceptors has been shown to be abnormal [36].
Studies have been carried out to examine the plasticity of chemoreflexes to both long and short term changes in blood gas tensions of chronically hypoxic high-altitude natives with blunted respiratory responses to hypoxia. It was found that natives who had migrated to live at sea level had ventilatory responses to acute hypoxia (few minutes) which had become similar to those of sea-level controls [21]. However, responses to sustained hypoxia (20 min) remained markedly blunted. These results may explain the apparent discrepancy in previous studies.
Recent studies have examined cardiovascular and cerebrovascular control in subjects with CMS. Bernardi et al. [6] assessed baroreflex function and the chemo- and baroreflex interactions in Andean subjects with and without CMS. They found that subjects with CMS showed a reduction in the responses to stimulation of peripheral chemoreflexes. In addition, these subjects also showed a reduction in the baroreflex control of heart rate and blood pressure. The reduction in the arterial baroreflex correlated with an increase in CMS score, and with an increase of haemoglobin levels. They interpreted these findings as suggestive of a functional, reversible central depression rather than of the presence of an organic dysautonomia in CMS. They further suggested that the observed baroreflex alteration might be involved in the causation of some of the symptoms of CMS. In another study on subjects with CMS, performed at high altitude, the same authors showed that increasing oxygen saturation for an hour by a slow breathing pattern or by oxygen administration was associated with increased arterial baroreflex sensitivity [33].
The major mechanism for the control of blood pressure is through regulation of peripheral vascular resistance, but most studies have examined only the control of heart rate. We have recently studied the responses of forearm vascular resistance to carotid baroreceptor stimulation in high altitude residents with and without CMS, both at their resident altitude and shortly after descent to sea level. Results showed that baroreflex “set point” was higher in CMS, but only at altitude. At sea level, values were similar [45].
Cerebrovascular control Cerebral blood flow is known to be less in CMS patients than in healthy controls and this is attributed to the high viscosity of the blood. Villafuerte et al. [66] estimated that the ideal haemoglobin level for maximal oxygen delivery is 14.7 g/dl, a value similar to that of sea-level dwellers. The cerebral circulation normally shows an efficient autoregulation whereby changes in cerebral perfusion pressure have minimal effect on flow. Claydon et al. [14] assessed autoregulation from the correlation between flow and pressure during orthostatic stress, where a significant correlation with a high coefficient indicates poor autoregulation. Results of this study showed that cerebrovascular autoregulation was impaired in CMS patients. These findings are compatible with those of Roach et al. [58] who reported reduced cerebrovascular sensitivity to carbon dioxide in the presence of hypobaric hypoxia in subjects with CMS.
Norcliffe et al. [50] determined the cerebrovascular responses to hypoxia and hypercapnia, separately and together, in CMS patients and normal high altitude dwellers. CMS patients did not respond differently from the normals, but in both groups at altitude the sensitivity of the cerebral circulation to hypoxia was less than that in sea level residents. Shortly after descent to sea level, however, sensitivity increased. Sensitivity to hypercapnia during hypoxia decreased after descent.

autonomic nervous system
altitude
hypoxia
chemoreceptors
baroreceptors
mountain sickness
respiration
polycythemia