Education Connection
Using a Haplodiploid Insect
to Teach Inheritance: Eye Color Genetics
of the Parasitoid Habrobracon hebetor
Evan Lampert and Bob Taylor
L
ive insects are often used in high
school science classes as models for
inheritance. Few, if any, studies take
advantage of the diverse genetic systems,
such as haplodiploidy, that are present in
insects. In the winter of 2006, we completed
a 1-month project, the goal of which was to
teach high school students genetics by having
them record expression of a trait in a haplodiploid insect. Also, because one of us (EL)
is an entomologist, we wanted to determine
how rearing live insects would affect students
with negative insect stereotypes. We used a
parasitic wasp, an insect group none of the
students were familiar with.
Haplodiploidy: a Unique Genetic
System
The order Hymenoptera contains an
estimated 125,000 described species, all
of which are believed to be haplodiploid
(Quicke 2003). Sex determination in Hymenoptera typically occurs through arrhenotoky, in which the haploid individuals
(unfertilized eggs) become male, and diploid
individuals (fertilized eggs) become female
(Heimpel and De Boer 2008). Exceptions
do exist for male (Whiting 1945) and female
(Beukeboom et al. 2007) Hymenoptera.
Haplodiploid insects provide novel opportunities for teaching genetics: males
typically do not have a male parent and
receive their entire genome from the female
parent. Moreover, because males only have
one allele (maternal) of a given gene, they
cannot carry an unexpressed recessive trait.
Therefore, a male used in an experimental
cross expresses either the dominant or recessive allele for a given trait. On average,
half of the male offspring of a female that
is a heterozygous “carrier” of a recessive
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trait will express the trait, regardless of the
female’s mating status.
In this project, we used the braconid parasitoid Habrobracon hebetor to demonstrate
inheritance. We chose H. hebetor because of
its recessive Oi allele that produces ivory-colored eyes when expressed (Whiting 1932).
This species was readily available because EL
completed graduate school in a research lab
that continuously reared Oi and wild-type
strains. Wild-type H. hebetor, including
those sold commercially (sources listed in
Hunter 1997), have black eyes. Our Oi mutants have been backcrossed with wild-type
individuals and differ genetically only in eye
color (Ode et al. 1995).
Biology of Habrobracon
Habrobracon hebetor is an idiobiont
ectoparasitoid of grain-feeding lepidopteran
larvae, such as the Indian meal moth (Plodia
interpunctella). Females must host feed to
mature eggs and engage in ovicide of previously laid eggs before laying 1–15 of their
own on each host (Benson 1973, Strand and
Godfray 1989). Larvae hatch in ~2 d, insert
their mouthparts into the hemocoel of the
host larva, and feed on its hemolymph until
its death (Fig. 1). After 5–7 d of feeding, H.
hebetor larvae move off of the host, produce
silken cocoons, and pupate. Adult wasps
emerge from pupae ~16 d after oviposition.
Arrhenotoky vs. CSD
We must caution here that Hymenopterans have different genetic systems and care
must be taken in choosing a species to use in
classroom activities. The primary system is
arrhenotokous sex determination, in which
any individual that is hemizygous (one allele
present rather than two or more) at sex
Fig. 1. Habrobracon hebetor larvae feeding on
a paralyzed Plodia interpunctella larva. Larvae
are four days old.
determining loci will develop into a male.
Complementary sex determination (CSD)
is another sex-determining system in some
Hymenoptera, including H. hebetor and
honeybees (Whiting 1943, 1945). In species
with CSD, individuals that are homozygous
at sex-determining loci become male, and
heterozygous individuals become female.
Homozygosity at these loci is more likely
under inbreeding; thus diploid males are
typically produced when siblings mate.
Diploid males are typically infertile or
sterile (e.g., H. hebetor) or even killed by
nestmates (e.g., honeybees), although diploid
male Cotesia vestalis can mate and produce
triploid offspring on rare occasions (de Boer
et al. 2007). Female H. hebetor avoid mating with siblings to reduce the production
of diploid males (Ode et al. 1995, Antolin
et al. 2003). In this project, we considered
the possibility of diploid males but agreed
not to teach CSD to the students.
American Entomologist • Summer 2008
The Setting
Kindred Public School, in rural Kindred,
ND, educates ~600 students in grades
2–12 (school Web site: http://www.kindred.k12.nd.us/education/school/school.
php?sectionid=3). Students who attend the
school live in seven communities (populations ranging from 180 to 580) within a
radius of ~15 mi around Kindred, as well
as on surrounding farms; a few students
also commute from West Fargo (population
20,300). The class is offered primarily to
sophomores. Juniors and seniors may also
take the course after failing previously or if they moved from another school.
Biology is split into three periods with
15–25 students in each. In the 2006-2007
school year, 58 biology students (49 sophomores, 6 juniors, and 3 seniors) participated
in this project. The activity took place 1 or
2 d a week for 5 wk through November
and December 2006. We timed the activity
in conjunction with two chapters covering
genetics in the course textbook (Johnson
1998).
Experimental Design
At North Dakota State University
(NDSU), we obtained two strains of H. hebetor, one a white-eyed strain expressing the
Oi allele and another with wild-type black
eyes. Newly emerged females were isolated
in groups of 20–25 into 16-oz. plastic cups.
Ten males were added to each of these cups
to produce the following four crosses:
Cross 1. Oi males and Oi females
Cross 2. Oi males and wild-type females
Cross 3. Wild-type males and Oi females
Cross 4. Wild-type males and wild-type
males.
Ten females from each of these four
crosses were removed from the cup after 24
h and placed singly into 100-mm Petri dishes,
where they were presented with five fifthinstar P. interpunctella larvae. Each female
was allowed 24 h to paralyze, host feed, and
oviposit onto the larvae, after which she was
removed and returned to the lab’s culture.
The parasitized P. interpunctella were placed
in a 27 °C growth chamber under a 16h:8h
light:dark cycle to allow H. hebetor larvae to
develop. These larvae, after development to
adulthood, were to be used by the students
at Kindred Public School.
Two class periods were devoted to instructing the students how to complete the
activity. These were given as the H. hebetor
were developing at NDSU, and the students
were at the beginning of the genetics unit.
First, we gave a 45-min PowerPoint presentation that provided background on meiosis,
fertilization, inheritance, haplodiploidy, and
other topics necessary to the activity. A onepage graded worksheet that was handed out
American Entomologist • Volume 54, Number 2
before the lecture was to be filled in during
the lecture to highlight key points. Students
from each of the three periods were randomly split into six groups per period (18
groups of 2–4 students each) at this time.
A quiz (scored for our purposes, but not
graded) was given the following day so we
could determine areas that needed attention during the activity. Such areas included
meiosis and haplodiploidy. The students
answered an average of 7.6 ± 0.313 correct
of 12 questions correctly (63%).
A second class period was devoted to
learning about the biology of H. hebetor. We
gave the students another one-page handout
that described the life history of the insect
and gave instructions on telling the sexes
and strains apart (Fig. 2). We brought a
variety of H. hebetor life stages, including
adults, eggs, and larvae, to the classroom.
After adults of both strains were anesthetized
with CO2, they were placed under dissecting
microscopes to allow students to differentiate between the two sexes as well as the
two eye colors. We also provided aspirators
so that students could practice transferring
adults between dishes without injuring or
losing them.
For the experiment, each group of students was randomly assigned a 16-oz. plastic
cup containing adult male and female H.
hebetor from 1 of the 4 crosses. We recorded
which cross each student group received; to
prevent bias, we did not tell the students
which cross they received. Each group was
also given a jar of 5th instar P. interpunctella
to parasitize. Caterpillar forceps were used
to transfer five larvae into each of 10 clean
60-mm Petri dishes. One adult male and one
adult female H. hebetor were then carefully
aspirated into each dish (Fig. 3). All dishes
were numbered and labeled with a group
name, then put into a single growth chamber
set at 27°C. Wasps were allowed to host feed
and oviposit overnight, and then they were
removed from the dishes.
H. hebetor eggs and larvae were allowed
Fig. 3. Two students using an aspirator to
transfer adult H. hebetor into a Petri dish of P.
interpunctella larvae.
to develop into adults (F1) over the course
of 13–15 d. Because wild-type individuals
typically develop faster than Oi mutants (EL,
personal observation), first emergence dates
were recorded for each dish. Upon emergence, one female was removed from each
dish and added to another dish containing
five 5th instar P. interpunctella larvae. The
students recorded the female’s eye color and
carefully aspirated one Oi male into each
dish to provide a mate. These insects were
placed in the incubator and allowed to mate
and oviposit. All other F1 H. hebetor were
frozen and stored outside (~0–20 °F). One
day later, the H. hebetor pair were removed
from the dishes and killed. The eye color of
the female was recorded. The F2 generation
was allowed to develop until emergence,
whereupon these adults were also frozen.
All frozen wasps were returned to the
classroom and examined together in one
class period. The students recorded several
data, including the development time of both
generations, the number of Oi females and
males, and the number of wild-type females
and males. Each group turned in their results
to us, and we summarized the total data for
the class before returning the spreadsheets
the next day.
Expected Results
We determined the proportions of black-
Fig. 2. A
comparison of adult
male H. hebetor
expressing the Oi
eye color allele (left)
and the wild-type
eye color allele
(right).
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Table 1. Expected proportions of adult H. hebetor with white eyes (expressing the Oi allele)
or black eyes (expressing the wild type allele) of each cross and generation, assuming all
Habrobracon are arrhenotokous (no CSD) or H. hebetor males are diploid (CSD).
No CSD
Cross
Prop. WT
Prop. Oi
CSD
Generation
Prop. Oi
1
F1­
—
1­
—
1
1
F2­
—
1­
—
1
2
F1
0.25
0.75
0.25
0.75
2
F2
0.25
0.75
0.5
0.5
3
F1
0.5
0.5
0.25
0.75
3
F2
0.75
0.25
0.5
0.5
4
F1
1­
—
1­
—
4
F2
1­
—
1­
—
and white-eyed adults that would result from
each of the four crosses (Table 1). These
crosses were determined using simple probability and Mendelian inheritance. Although
Hymenopteran parasitoids are known to
bias sex ratios based on environmental and
host quality cues (see Charnov 1982), for
simplicity we assumed that equal numbers
of males and females would be produced in
this activity. Two sets of predictions were
generated: one set in which all males are
haploid (arrhenotokous sex determination)
and another in which all males are diploid
(CSD plus inbreeding). To avoid biasing
their results, we did not share our predictions with the students until all of the data
were collected. Table 1 can be used for any
hymenopteran species.
Observed Results
Student participants reared, identified
by sex, and counted 5,822 adult H. hebetor. Almost identical numbers of purported
haploid (2,874 males) and diploid (2,948
females) individuals were reared. During
the second generation, the dishes that contained developing larvae from the second
and third crosses (both Oi*wild type) were
inadvertently combined. To account for this
error, we averaged the predicted ratios of
those two crosses (Table 2) and shared those
with the students. To determine whether
the predicted and collected data (Table 3)
were significantly different, we performed a
χ2 goodness-of-fit test (SAS Institute 2006).
Our predictions of the first generation were
Table 3. Recorded proportions of adult H.
hebetor with white eyes (expressing the Oi
allele) or black eyes (expressing the wild
type allele).
Cross
Prop. WT
Cross
not significantly different from the results;
however, compared with our predictions,
the results of the second generation were
significantly wild-type-biased.
Our data did not indicate in any way
that large numbers of diploid males were
produced by inbreeding under CSD. We
thus decided it was not necessary to discuss
the possibility of diploid males or the consequences of inbreeding with the students. In
the future, it may be worthwhile to consider
diploid males (which can carry the Oi allele
without expressing it) only if trait ratios appear typical of diploid species.
Reflective Writing
We devoted one class period to sharing
the data with the students, starting with the
expected ratios of white-eyed and black-eyed
adults (Tables 1 and 2 with the CSD predictions excluded). Next, we shared Table 3
with the class and highlighted the differences
in the expected data and collected data. We
did not discuss the reasons for these differences with the students; instead, we gave
them handouts and told them to write down
any reasons they could think of. They were
allowed to complete these handouts with
their groups. Students were expected to consider the data of their individual group and
the pooled data for the class. We expected
the students to reason for themselves, and
outside of explaining the predictions, we did
not give them any assistance.
These handouts were designed mainly
to assess whether students could use the
No CSD Generation
Prop. Oi
Prop. WT
2/3
F1
0.625
0.375
2/3
F2
0.5
0.5
CSD
Prop. Oi
—
0.5
Prop. WT
1
0.5
1 F1­
—
1
1 F2­
—
1
2/3
F1
0.66
0.34
2/3
F2
0.73**
0.27**
4 F1
1
—
4 F2
1
—
knowledge they gained during this activity
to answer a question. Students were penalized mainly for ignoring either the difference
between haplodiploid and diploid genetics
or the chance of experimental error. Partial
or full credit was given on any answer that
clearly demonstrated independent student
reasoning. The class average of the reflection
paper was 23.91 ± 0.441 out of 30 (79.7%)
possible points.
Did This Activity Affect Students’ Views
of Insects?
At the conclusion of this experiment,
students were given a brief elective survey
about how they viewed insects, and wasps
in particular, before and after the activity.
We also included a question about their
parents’ or guardians’ views of insects. Out
of 58 participants, 41 (70%) responded to
the survey. Sixteen of those students reported
that their parents or guardians had negative
views of insects (40%), and 10 of those students themselves feared or disliked insects.
Twenty-one students (51%) either feared or
disliked insects before beginning the activity.
Eight of the 21 students who had negative
views of insects before the activity positively
changed their views after working with H.
hebetor. In all, 27% of the respondents appreciated insects after completing the activity
(Fig. 4).
Twenty of the respondents (49%) reported a change in their opinions about wasps.
We attributed this to the fact that almost
none of the students were aware of the parasitic lifestyle of most Hymenoptera; most
were aware only of the aculeate (bees, social
wasps, ants) species. To paraphrase, many
students responded that they “didn’t realize
all wasps didn’t sting and hurt people.” One
student claimed during the activity that H.
hebetor were not “real wasps” because they
do not sting humans.
Our Reflections
This course was a mandatory general
Proportions were obtained by averaging the numbers from Table 1.
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Prop. Oi Prop. WT
** Proportion is significantly different (P < 0.05) from
the predicted proportion (Table 2).
Table 2. Expected proportion of white-eyed or black-eyed H. hebetor adults after crosses 2
and 3 were combined.
Generation
American Entomologist • Summer 2008
pae. Although no mutants are yet known for
M. digitata, they can be used to demonstrate
genetics of sex determination. M. digitata are
haplodiploid and produce female-biased sex
ratios (<10% male).
This activity received overwhelmingly
consistently positive feedback from the students. Most indicated in their surveys that
the activity was educational and fun. The
students looked forward to days that would
be dedicated to the activity. We conclude
that this activity kept the students’ attention,
reinforced the genetics unit, gave the students
experience in collecting data, and provided
us with fascinating results.
Fig. 4. Results of the insect survey taken after
this activity. Forty-one students completed the
survey.
biology course taken by students ranging
in age from 15 to 17. Genetics is often a
difficult topic for high school students, and
these students had no experience in genetics
other than an introduction in junior high.
Moreover, haplodiploid genetics were an
additional challenge. Diploid males are a
consequence of inbreeding in species such as
H. hebetor with CSD (Heimpel and De Boer
2008); however, we agreed that this concept
was too conceptually difficult for the target
age group, and therefore, we did not include
CSD in the project. We recommend that
this project should be repeated with an arrhenotokous species. This project should be
targeted to advanced placement high school
biology or genetics students, or introductory
college biology students.
The Oi mutant of H. hebetor is not
commercially available; however, other
parasitoid species can be substituted in this
project. One candidate is Nasonia vitripennis, a pteromalid parasitoid of filth fly
pupae. N. vitripennis can be purchased (as
the “jewel wasp”) from at least 10 sources
(e.g., Carolina Biological, other examples
listed in Hunter 1997). There are at least
20 visible mutations for eye or frons color
of N. vitripennis (information is available
at: http://www.rochester.edu/College/BIO/
labs/WerrenLab/nasonia/). Nasonia does not
have CSD and will inbreed.
A second species that is ideal for classroom use is the eulophid Melittobia digitata (available as WOWBugs: http://www.
wowbugs.com/index.html), a gregarious
parasitoid of vespid wasps and filth fly puAmerican Entomologist • Volume 54, Number 2
Acknowledgments
Evan Lampert and Bob Taylor were
supported by the NDSU GraSUS-II project.
GraSUS-II is supported by the GK-12 program of the National Science Foundation
(DGE-0338128) and by North Dakota State
University and the Center for Science and
Math Education. The 2006–2007 general
biology class of Kindred Public School, ND,
reared, counted, and determined the sex of
~6,000 wasps to provide us with data. Paul
Ode of the NDSU Department of Entomology
provided Habrobracon.
References Cited
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Evan Lampert received a Ph.D. in entomology from North Dakota State in 2007, and
completed this project as an NDSU GraSUSII graduate fellow. He is currently a postdoctoral research fellow in the Department
of Ecology and Evolutionary Biology at the
University of Colorado. Bob Taylor teaches
several courses at Kindred High School, including earth science, biology, and AP human
anatomy and physiology.
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