NIH Public Access

Author Manuscript
Exp Gerontol. Author manuscript; available in PMC 2010 August 1.
Published in final edited form as:
NIH-PA Author Manuscript

Exp Gerontol. 2009 August ; 44(8): 541–545. doi:10.1016/j.exger.2009.05.006.

Leg Impairments Elicit Graded and Sex-specific Demographic

Responses in the Tephritid Fruit Fly Anastrepha ludens

James R. Carey1,2,*, Pablo Liedo3, Han-Georg Müller4, Jane-Ling Wang4, Wenjing Yang4,
and Freerk Molleman1
1 Department of Entomology, University of California, Davis, CA 95616

2 Center for the Economics and Demography of Aging, University of California, Berkeley 94720 USA

3 El Colegio de la Frontera Sur (ECOSUR), Tapachula, Chiapas, MEXICO

4 Department of Statistics, University of California, Davis, CA 95616

Abstract
NIH-PA Author Manuscript

This study was concerned with the impact of different levels of artificial impairment (leg
amputations) on male and female survival and female reproduction in the Mexican fruit fly, A
ludens. We monitored the demographic responses in a total of 100 flies of each sex that were
maintained individually in 4 × 4 × 10 cm and subject to 1-of-11 different leg amputations (plus intact
control) including cohorts in which either one front, one middle or one rear leg was severed (3 cohorts
total), in which two legs were severed in different front-middle-rear combinations (6 cohorts total),
or in which the two middle and one additional leg were severed (2 cohorts total). The two main
findings were that: i) although the effects on mortality of impairments were sex-specific, no universal
patterns emerged that applied to either sex; and ii) reproduction occurred in all cohorts of impaired
females. Moderately-impaired flies (e.g. amputation of a single middle leg) laid nearly as many eggs
in their lifetime as did intact controls. However, severely impaired flies (i.e. 3 legs amputated) laid
significantly fewer eggs.

Introduction
Although the concept of impairment is usually associated with humans in the context of aging,
disability, and failing health, it is also relevant to the study of fitness, longevity, and survival
NIH-PA Author Manuscript

in non-human species including insects, arachnids and other arthropods. Despite the prevalence
of impairments in nature, we are aware of only a small number of impairment-related studies
that elucidate the effects of injuries on the demographic (survival; reproduction) characteristics
of insects or on changes in insect behavior as individuals age. These include studies on supine
behavior in Mediterranean fruit flies (Carey et al., 2006; Papadopoulos et al., 2002) and the
mortality consequences of injury, damage or autotomy (self-amputation) such as the effects of
wing removal in the housefly, Musca domestica (Sohal and Buchan, 1981), leg removal (or
injury) in (Carey et al., 2006; Papadopoulos et al., 2002)Drosophila melanogaster (Carey et
al., 2007; Sepulveda et al., 2008), wing clipping in moths (Javois and Tammaru, 2004), leg
impairment in juvenile wolf (Wrinn and Uetz 2008), wing-wear in fruit-feeding butterflies

*Correspondence to: James R. Carey, Department of Entomology, One Shields Ave., University of California, Davis, CA 95616 USA,
530-752-6217, 530-752-1537 (FAX), [email protected].
Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting
proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could
affect the content, and all legal disclaimers that apply to the journal pertain.
 Carey et al. Page 2

(Molleman et al., 2008; Molleman et al., 2009), and leg autotomy in spiders (Eisner and
Camazine, 1983; Guffey, 1998; Johnson and Jakob, 1999; Moore and Tabashnik, 1989).
NIH-PA Author Manuscript

In light of the paucity of data on the influence of impairment on fitness in non-human species
and the importance of understanding the dynamics of impairment more generally, the broad
purpose of this study was to use the tephritid fruit fly, Anastrepha ludens, commonly know as
the Mexican fruit fly or Mexfly, to examine the demographic response of artificial impairment
resulting from leg amputation. We asked three specific questions: How do different levels or
configurations of leg amputations influence mortality in each sex? How do these different
impairments affect survival of males relative to females (i.e. affect the gender gap)? How do
the impairments in females affect their reproductive patterns and lifetime output? This study
builds on an earlier investigation {Carey, 2006 #194} concerned with the mortality
consequence of impairment in D. melanogaster. In this study we not only examine the effects
of impairment on mortality as was done with the D. melanogaster investigation, but also
document the effects of leg removal on age-specific reproduction.

Methods
The basic conceptual framework for impairments for humans are contained in the International
Classification of Functioning, Disability and Health (WHO, 2001) where impairments are
considered a condition that causes a loss of function. Thus the amputation of one or more legs
NIH-PA Author Manuscript

in the treatment flies in our study is considered the cause of the impairment but the impairment
itself is the expression of the (health) condition i.e. ambulatory and/or reproductive capabilities
are reduced.

Background information on both the biology of A. ludens and details of its mass production
are given in the paper by Carey and co-workers (Carey et al., 2005). We used virgin females
because the lifetime reproduction of unmated females is nearly identical to mated females in
this species (Rogina et al., 2007) and replacing dead males changes the within-cage dynamics
because of differences between the ages and/or mating histories of the original male and its
replacement. The experimental design consisted of 12 treatments including an intact control
cohort and 11 different impairment grouped into three categories: (1) Single-leg amputations:
Front (F), Middle leg (M), Rear leg (R); (2) Two-leg amputations (opposite sides): FF, FR,
FM, MM, MR, RR; and (3) Three-leg amputations: MMF, MMR. Legs were removed using
pointed foreceps.

A 1.5 μl droplet of the full diet of yeast and sugar (Carey et al., 2005) and a 6 μl droplet of
water were supplied to individual flies each day on glass slides using separate Eppendorf®
needles. Newly-emerged (virgin) individual flies were housed in 4 × 4 × 10 cm plexiglass
NIH-PA Author Manuscript

cages, each of which was part of a 24-unit cage unit. Treatments distribution was made in a
randomized block design, using different color codes to identify both the treatment and the
food slides. Females and males were placed in alternate cages to eliminate the possibility of
eggs from two females overlapping on the egg collection surface. Females laid eggs through
organdy mesh fastened to the front of the cage and were counted daily. A total of 100 individuals
of each sex were used per treatment. Daily mortality and female reproduction were monitored
daily throughout the life of each fly. Environmental conditions were 12:12 LD cycle, 24.0° C
(±2°) and 65% RH (±9%).

Results
Survival
To investigate gender-specific impairment influences on medfly lifespan, we applied non-
parametric hazard estimation to the data grouped by treatment and gender. We observed no

Exp Gerontol. Author manuscript; available in PMC 2010 August 1.

 Carey et al. Page 3

difference in hazard between males and females in the control treatment group (intact), the
least impaired groups (single legs amputated; i.e. F, M, R), and the most impaired groups (three
legs amputated; MMR, MMF). Among the intermediately impaired groups (two legs
NIH-PA Author Manuscript

amputated), males had larger hazard functions at FF, FR, and MM, while females have larger
hazard functions at FM, MR, and RR.

The life expectancies observed for all treatments with both sexes combined are presented in
Table 1 with the results of the statistical analyses presented in Table 2. Several aspects of the
results contained in these tables merit comment. First, any level of impairment caused by leg
amputation(s) reduces life expectancy. While this result was not surprising, it was conceivable
a priori that a moderate level of impairment could have resulted in an increase in longevity
through a reduction in activity as was found by Sohol and Buchan (1981) when they removed
the wings of the housefly, Musca domestica. Second, the degree to which life expectancy was
reduced was directly related to the severity of the impairment (i.e. effects on longevity of
removal of 3 legs>2 legs>1 leg). For example the average life expectancies shown in Table 1
for single-, two- and three-leg amputations were 29.5, 16.9 and 11.1 days, respectively.
Third, there were two impairment treatments that were by themselves significantly different
than other impairment treatments with respect to their impact on longevity (Table 2): i) the
amputation of a single middle leg which had the least impact on longevity relative to all other
impairment treatments; and ii) the amputation of both middle and one front leg which had the
greatest impact on longevity. That there was no statistical overlap in their effects on longevity
NIH-PA Author Manuscript

suggests that these represent impairment thresholds for minimal (i.e. with single middle leg
removed) and maximal (i.e. with both middle and one front leg removed) impact. Fourth, the
impact on life expectancy for each amputation category (i.e. number of legs removed) was
conditional on which legs or combination of legs were severed. For example, the average of
the life expectancies for the cohorts in which the front, middle legs and rear were removed
singly or in combination with other legs was 16.8, 21.7 and 18.4 days, respectively. In other
words, removal of one or both front legs had a greater impact on mortality than the removal
of one or both rear legs and removal of one or both middle legs reduced longevity less than
removal of one or two other legs.

Reproduction
The normality statistical check failed for the number of eggs laid since more than half of the
female medflies laid no eggs in their lifetimes. Therefore, we applied Wilcoxon rank sum test
(also known as Mann-Whitney U test), to check the differences of the total number of eggs
each female laid in the treatment groups. The test achieved a large χ2 statistic at 244.3 with 11
degrees of freedom and a small p-value of less than 0.0001. Thus, leg impairments have a large
negative impact on lifetime reproduction in medflies.
NIH-PA Author Manuscript

Although lifetime egg production by females was reduced in all treatments cohorts, the degree
to which reproduction was reduced depended upon the details of the impairment treatment.
For example, single leg amputations reduced lifetime reproduction by an average of only about
one third relative to reproduction in intact flies (Table 3). In contrast, the amputation of two
legs reduced egg laying by 90% relative to control flies if both front legs were amputated and
by 50% relative to controls if both rear legs were amputated. Lifetime reproduction in the two
treatments involving extreme impairments where both middle and either one of the front or
one of the rear legs was amputated averaged only around 5% of the control reproduction. The
data on both gross reproductive rates and the percent egg laying also contained in Table 3 sheds
light on these differences in net rates between impairment treatments. For example, both the
gross reproductive rates and the percent of females laying eggs for the single-leg amputations
were similar (and in some cases non-significantly higher) to the corresponding rates for the
intact control females. This suggests that the lower net reproductive rates for these treatment

Exp Gerontol. Author manuscript; available in PMC 2010 August 1.

 Carey et al. Page 4

flies were due to an increase in mortality resulting from the impairment and not to a reduction
in individual fly egg-laying capabilities. In general amputation of one or both of the front legs
had the greatest impact on lifetime reproduction whereas amputation of one or both of the
NIH-PA Author Manuscript

middle legs had the least impact on lifetime reproduction. It is remarkable that, despite the
severity of their impairment and the effects on their mobility, several of the females missing
half of their legs (i.e. 3 legs amputated) still laid a small number of eggs.

Discussion
This study yielded three main results in response to the specific questions that we addressed.
The answer to the first question was not surprising in that different levels and configurations
of leg amputations did reduce longevity in both male and female cohorts in relation to the
severity of the impairment—i.e. the greater the severity the greater the reduction in longevity.
The answer to the second question that pertained to sex-specific effects of impairment also was
not surprising in that we did identify treatments where the impact differed between the sexes.
However, the mortality response for neither sex was consistently greater across treatments.
This finding differed from the results of studies on Drosophila melanogaster where it was
reported that injuries or impairments had a greater impact on male mortality than on female
mortality (Carey et al., 2007; Sepulveda et al., 2008). The third finding was that impairment
reduced the lifetime reproduction of females due primarily to a reduction in lifespan and not
to any apparent disruptive effect on either the propensity or the ability of individuals to lay
NIH-PA Author Manuscript

eggs. Although the study on D. melanogaster by Sepulveda et al (2008) also reported a

reduction in egg laying in flies with leg injuries, they argued that the reduction was due mostly
to the disruptive effect of the injury and not to the reduced lifespan of injured females. However,
the differences may be because reproduction was measured for only 10 days in their study
whereas it was measured throughout the life of individual females in the current study. We
found that egg laying rates in moderately-impaired female flies (e.g. middle leg amputated)
was only slightly reduced relative to the reproduction in intact females. We also discovered
that even some of the most severely impaired females (i.e. loss of 3 legs) were still capable of
laying a small number of eggs despite their impairment rendering them nearly immobile.

Our data do not support the conventional argument, known as the risk-prone behavior
hypothesis (Owens, 2002), that because males typically engage in costly activities including
male-male competition (Gerhardt, 2002), sexual advertisement (Zuk et al., 1990), and mate
searching (Bell, 1990) male mortality is intrinsically higher than female mortality. All of these
activities either increase the likelihood of injury or impairment. However, the life expectancy
of intact male A. ludens in the current study as well as of medflies (Ceratitis capitata) in earlier
studies that were maintained under a range of different conditions such as dietary restriction
(Carey et al., 2008), high density (Carey et al., 1995), and starvation (Carey et al., 1999)
NIH-PA Author Manuscript

exceeded the life expectancies of females maintained under identical laboratory conditions.
Instead of a general rule on sex differentials in longevity and responses to impairments, they
may be more idiosyncratic and depend on the field biology and anatomy of the species. For
example, in the case of Mexflies females may suffer more from amputation of hindlegs because
their center of mass is probably located nearer their caudal side given that they have a large
ovipositor.

Conclusions
We have shown in a second and much larger species that higher levels of leg loss cause higher
mortality and that configuration matters. These effects were sex specific, but the mortality
response for neither sex was consistently greater across treatments, indicating species specific
idiosyncratic effects of leg impairments. Impairments impact reproduction mainly through
reducing life span in this species and even severely impaired individuals are still able to lay

Exp Gerontol. Author manuscript; available in PMC 2010 August 1.

 Carey et al. Page 5

some eggs. The results of this study not only strengthen the empirical foundation laid down
by several previous studies concerned with the effects of injury and impairment on the life
history traits in insects, but also illustrate the importance of arthropod model systems for both
NIH-PA Author Manuscript

identifying and validating general principles concerned with the dynamics of morbidity and
mortality. Consequently, we believe that disability and impairment research using arthropod
models has the potential to shed light on important unresolved questions pertaining to chronic
disability in the elderly (including humans) ranging from the mortality dynamics of morbidity
compression to age-and sex-specific patterns of mortality resulting from conditions of
comorbidity (Manton, 2008).

Acknowledgments
Research supported by NIA/NIH grants PO1 AG022500-01 and PO1 AG08761-10. We thank A. Oropeza, R.
Bustamente, E. de Leon, S. Salgado, S. Rodriguez, R. Rincon and G. Rodas for technical assistance and the Moscamed-
Moscafrut program in Mexico for their laboratory facilities at Metapa, Chiapas. We are also grateful to Kaare
Christensen for comments on the manuscript and Leslie Sandberg for editorial assistance.

Literature Cited
Bell WJ. Searching behavior patterns in insects. Ann Review of Entom 1990;35:447–467.
Carey JR, Harshman L, Liedo P, Müller HG, Wang JL, Zhang Z. Longevity-fertility trade-offs in the
tephritid fruit fly, Anastrepha ludens, across dietary-restriction gradients. Aging Cell 2008;7:470–477.
NIH-PA Author Manuscript

[PubMed: 18346215]
Carey JR, Liedo P, Müller HG, Wang JL, Chiou JM. Mortality oscillations induced by periodic starvation
alter sex-mortality differentials in Mediterranean fruit flies. J of Geront: Biol Sci 1999;54A:B424–
B431.
Carey JR, Liedo P, Müller HG, Wang JL, Vaupel JW. A simple graphical technique for displaying
individual fertility data and cohort survival: case study of 1000 Mediterranean fruit fly females. Func
Ecol 1998;12:359–363.
Carey JR, Liedo P, Muller HG, Wang JL, Senturk D, Harshman L. Biodemography of a long-lived
tephritid: Reproduction and longevity in a large cohort of female Mexican fruit flies, Anastrepha
ludens. Exper Geront 2005;40:793–800.
Carey JR, Liedo P, Vaupel JW. Mortality dynamics of density in the Mediterranean fruit fly. Exper Geront
1995;30:605–629.
Carey JR, Papadopoulos N, Kouloussis N, Katsoyannos B, Müller HG, Wang JL, Tseng YK. Age-specific
and lifetime behavior patterns in Drosophila melanogaster and the Mediterranean fruit fly, Ceratitis
capitata. Exper Geront 2006;41:91–97.
Carey JR, Pinter-Wollman N, Wyman M, Müller HG, Molleman F, Zhang N. A search for principles of
disability using experimental impairment of Drosophila melanogaster. Exper Geront 2007;42:166–
172.
NIH-PA Author Manuscript

Eisner T, Camazine S. Spider leg autotomy induced by prey venom injection: An adaptive response to
‘pain’? Proc of the National Acad of Science USA 1983;80:3382–3385.
Gerhardt, HC. Sexual dimorphism. In: Pagel, M., editor. Ency of Evol. Oxford University Press; Oxford;
2002. p. 1045-1047.
Guffey C. Leg autotomy and its potential fitness costs for two species of harvestmen (Arachnida,
Opiliones). J of Arachnology 1998;26:296–302.
Javois J, Tammaru T. Reproductive decisions are sensitive to cues of life expectancy: the case of a moth.
Anim Behav 2004;68:249–255.
Johnson SA, Jakob EM. Leg autotomy in a spider has minimal costs in competitive ability and
development. Anim Behav 1999;57:957–965. [PubMed: 10202103]
Manton KG. Recent declines in chronic disability in the elderly U.S. population: Risk factors and future
dynamics. Ann Review of Public Health 2008;29:91–113.

Exp Gerontol. Author manuscript; available in PMC 2010 August 1.

 Carey et al. Page 6

Molleman F, Ding J, Wang JL, Brakefield PM, Carey JR, Zwaan BJ. Adult diet affects life span and
reproduction of the fruit-feeding butterfly Charaxes fulvescens Entom. Experimentalis et applicata
2008;129:54–65.
NIH-PA Author Manuscript

Molleman F, Ding J, Wang JL, Carey JR. Nutrients in fruit increase fertility in wild-caught females of
long-lived Euphaedra species (Lepidoptera, Nymphalidae). The J of Insect Physiol 2009;55:375–
383.
Moore A, Tabashnik BE. Leg autotomy of adult diamondback moth (Lepidoptera: Plutellidae) in response
to tarsal contact with insecticide residues. J o Econ Entomol 1989;82:381–384.
Owens IPF. Sex differences in mortality rate. Science 2002;297:2008–2009. [PubMed: 12242430]
Papadopoulos NT, Carey JR, Katsoyannos BI, Kouloussis NA, Müller HG, Liu X. Supine behaviour
predicts time-to-death in male Mediterranean fruit flies. Proc of the Royal Soc of London: Biol Sci
2002;269:1633–1637.
Rogina B, Wolverton T, Bross TG, Chen K, Müller HG, Carey JR. Distinct biological epochs in the
reproductive life of female Drosophila melanogaster. Mech of Aging and Develop 2007;128:477–
485.
Sepulveda S, Shojaeian P, Rauser CL, Jafari M, Mueller LD, Rose MR. Interactions between injury,
stress resistance, reproduction, and aging in Drosophila melanogaster. Exper Geront 2008;43:136–
145.
Sohal RS, Buchan PB. Relationship between physical activity and life span in the adult housefly, Musca
domestica. Exper Geront 1981;16:157–162.
WHO. International Classification of Functioning, Disability and Health. World Health Organization;
NIH-PA Author Manuscript

Geneva: 2000.
Zuk M, Thornhill R, Ligon JD, Johnson K, Austad S, Ligon SH, Thornhill NW, Costin C. The role of
male ornaments and courtship behavior in female mate choice of red jungle fowl. The Amer Naturalist
1990;136:459–473.
NIH-PA Author Manuscript

Exp Gerontol. Author manuscript; available in PMC 2010 August 1.

 Carey et al. Page 7
NIH-PA Author Manuscript
NIH-PA Author Manuscript

Fig. 1.
Event history charts (Carey et al., 1998) for the intact control female A. ludens and the 11
impairment treatment cohorts. Each horizontal line corresponds to an individual female with
color-coded age segments corresponding to green=0 eggs/day, yellow = 1-20 eggs/day, and
NIH-PA Author Manuscript

red =>20 eggs/day.

Exp Gerontol. Author manuscript; available in PMC 2010 August 1.

 NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
Table 1
Life expectancies in days (both sexes combined) for different impairments (leg amputations) of the fruit fly, A. ludens. F, M, and R
denoted front, middle and rear legs, respectively. Life expectancy for intact control flies was 45.5 days. Because of the redundancy of
treatments created by the table row-by-column configuration, some table entries are also redundant.

Leg Amputation of Left Side

Carey et al.

Amputation of both Middle

Leg Amputation Right Side None F M R Average Legs

F 27.8 17.0 15.3 14.1 18.6 9.6

M 32.4 15.3 20.9 18.0 21.7
R 28.2 14.1 18.0 19.2 19.8 12.5
Average 29.5 15.5 18.1 17.1 20.0

Exp Gerontol. Author manuscript; available in PMC 2010 August 1.

Page 8
 Carey et al. Page 9

Table 2
Grouping of treatments generated by Student-Newman-Keuls Test. Means with the same letter are not significantly
different. The letters F, M and R in the treatment column denote front, middle and rear legs amputated.
NIH-PA Author Manuscript

Treatment Student-Newman-Keuls Grouping Lifespan Means

Intact A 45.469
M B 32.360
R C 28.195
F C 27.833
MM D 20.930
RR E D 19.226
MR E D F 17.995
FF E G F 16.960
FM H G F 15.335
FR H G 14.101
MMR H 12.455
MMF I 9.635
NIH-PA Author Manuscript
NIH-PA Author Manuscript

Exp Gerontol. Author manuscript; available in PMC 2010 August 1.

 NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript
Table 3
Grossa and net reproduction in the fruit fly, A ludens, for 14 different impairment (leg amputation) treatments. Gross and net reproduction
for intact control females were 695.2 and 312.7 eggs/female, the percent of the intact control females that laid any eggs was 70%, and
the percentage of life-days in which control females laid was 24.6%. Because of the redundancy of treatments created by the table row-
by-column configuration, some table entries are also redundant. (e.g. the front-mid treatment is identical to the mid-front treatment in
which females laid 255.6 eggs in their lifetime).
Carey et al.

Leg Amputation Opposite Side

Amputation Both
Leg Amputation One Side None Front Mid Rear Average Middle Legsb

GROSS Front 723.6 80.1 255.6 151.6 302.7 38.6

Mid 578.2 255.6 455.1 495.2 446.0
Rear 866.9 151.6 495.2 578.1 523.0 39.0
Average 722.9 162.4 355.4 408.3 412.3 38.8

NET Front 213.8 35.0 35.9 82.6 91.8 8.0

Mid 234.8 35.9 146.5 58.0 118.8
Rear 236.1 82.6 58.0 163.8 135.1 20.4
Average 228.2 51.2 80.1 101.58 115.3 14.2

% Laying Front 67 30 39 32 42.0 9.0

Mid 62 39 63 38 50.5 --
Rear 71 32 38 45 46.5 21.0
Average 66.7 33.7 46.7 38.3 46.4 15.0

Exp Gerontol. Author manuscript; available in PMC 2010 August 1.

% Laying days Front 24.5 19.4 16.9 15.1 19.0 11.6
Mid 29.7 16.9 20.0 19.4 21.5 --
Rear 26.4 15.1 19.4 21.4 20.6 15.5
Average 26.9 17.1 18.8 18.6 20.4 13.6

a
gross reproduction is total eggs laid by a hypothetical female that lived to the oldest age; net reproduction is eggs produced by the average female.
b
numbers in this column represent values for the two treatments in which three legs were amputated— the two middle legs plus the leg indicated for each row.
Page 10