Tribolium castanaeum is a widespread insect in temperate regions. Because of its short generation time and easy handling, it serves as a model organism for various scientific questions. However, T. castanaeum, or its larger-sized relative Tenebrio molitor, can also be used at a school or college level to foster students' experimentation skills. We present a set of inquiry tasks that build stepwise on one another, using T. castanaeum or T. molitor as the model. The students will learn not only about insect ecology, but also about the influence of nutrition on several fitness parameters, such as development time and offspring number.

Scientific inquiry encompasses a wide range of activities, such as observing biological phenomena, planning experiments, and collecting and interpreting data. An experiment, as opposed to an observation, comprises variables and conditions that can be systematically altered by the experimenter. The ultimate aim of an experiment is to confirm or disprove a hypothesis or a question, to elucidate cause–effect relationships so that a causal conclusion can be drawn from the data set to explain a phenomenon. However, the terms "experiment" and "observation" are often used inconsistently, with the result that students have trouble differentiating the two inquiry methods. In particular, students misuse the term "control group" and do not understand the necessity of using replicates for every treatment.

Here, we propose a straightforward inquiry activity designed to improve students' experimentation and inquiry skills and to elucidate the ecology and life history of the red flour beetle (Tribolium castaneum). The activity also provides a foundation for exploring new questions about maintenance and life-history tradeoffs in T. castaneum, by displaying factors (such as nutrition) that influence its fitness. The students will start by observing the organism's life cycle and by discussing the possibilities of measuring life-history parameters. The subsequent experiment can be adapted for varying levels of education, from high school to university. The activity addresses various categories of knowledge in the National Science Education Standards, including life cycle, regulation, and behavior of organisms, as well as skills associated with science as inquiry (Table 1).

Table 1.

Overview of possible inquiry activities with flour beetles, depending on the students' grade level.

GradeInquiry MethodParameters To Be InvestigatedEvaluationActivityContent Standards
K–4 Observation Developmental time (T. molitor) Careful observation and documentation Observe the complete life cycle of a holometabolous insect Life science: Life cycles of organisms, metamorphosis and generation time of organisms with special focus on an holometabolous insect.
Science as inquiry:Abilities needed to do scientific inquiry: systematic observations (life cycle).
Understandings about scientific inquiry: Science can focus on different aspects of investigation, depending on the question of interest (here, observation vs. experiment). 
5–8 One-factorial experiment Number of offspring dependent on nutrition
Developmental time dependent on nutrition (T. molitor
Create an Excel graph (mean) Plan an experiment to answer the question: "Does developmental time depend on temperature?"
"Does number of offspring/developmental time depend on food quality?" (cf. Figure 3, –Y–Y and +Y+Y bars) 
Life science: Regulation and behavior: By feeding, animals gain a certain amount of resources that can be invested in life-history traits like growth, reproduction, and immune defense. If food is of decreased quality, animals gain fewer resources, and thus run into a resource-allocation tradeoff. Not enough resources are available to maintain all life-history traits. This leads to a drop in fitness (here, reduced food quality leads to a decreased number of offspring).
Science as inquiry:Abilities needed to do scientific inquiry: Students should develop research skills, such as systematic observation, making accurate measurement and identifying and controlling variables (here, –Y or +Y). In addition, students should use appropriate tools to analyze and to interpret data (here, the use of computers for the collection, summary, and display of data), drawing the relationship between evidence and explanations (here, cause-and-effect relationships: offspring rate depends on food quality). Understandings about scientific inquiry: Research may result in new ideas and phenomena for future studies. (Does food quality influence development in different developmental stages?) 
9–12 Two-factorial experiment Number of offspring dependent on nutrition
Developmental time dependent on nutrition
Nutrition in the respective developmental stage influences number of offspring differently (Figure 1)
Relationship between egg size and number of offspring (T. castanaeum
Create an Excel graph (mean and standard deviation) interpreting Figure 5  Plan an experiment to answer the question: "Do rate of offspring and developmental time depend on nutrition in different life stages?" (cf. Figures 3 and 4Life science: Resource depletion in an early life stage may be carried over to adulthood, even if the quality of resources is then sufficient.
Science as inquiry:Abilities needed to do scientific inquiry: Students should learn how to clarify a scientific question. They should further be able to conduct a scientific experiment, including controls and several variables, and display their data.
Understandings about scientific inquiry: Scientists often investigate several factors within one experiment for a variety of reasons (testing conclusions of prior investigations). 
GradeInquiry MethodParameters To Be InvestigatedEvaluationActivityContent Standards
K–4 Observation Developmental time (T. molitor) Careful observation and documentation Observe the complete life cycle of a holometabolous insect Life science: Life cycles of organisms, metamorphosis and generation time of organisms with special focus on an holometabolous insect.
Science as inquiry:Abilities needed to do scientific inquiry: systematic observations (life cycle).
Understandings about scientific inquiry: Science can focus on different aspects of investigation, depending on the question of interest (here, observation vs. experiment). 
5–8 One-factorial experiment Number of offspring dependent on nutrition
Developmental time dependent on nutrition (T. molitor
Create an Excel graph (mean) Plan an experiment to answer the question: "Does developmental time depend on temperature?"
"Does number of offspring/developmental time depend on food quality?" (cf. Figure 3, –Y–Y and +Y+Y bars) 
Life science: Regulation and behavior: By feeding, animals gain a certain amount of resources that can be invested in life-history traits like growth, reproduction, and immune defense. If food is of decreased quality, animals gain fewer resources, and thus run into a resource-allocation tradeoff. Not enough resources are available to maintain all life-history traits. This leads to a drop in fitness (here, reduced food quality leads to a decreased number of offspring).
Science as inquiry:Abilities needed to do scientific inquiry: Students should develop research skills, such as systematic observation, making accurate measurement and identifying and controlling variables (here, –Y or +Y). In addition, students should use appropriate tools to analyze and to interpret data (here, the use of computers for the collection, summary, and display of data), drawing the relationship between evidence and explanations (here, cause-and-effect relationships: offspring rate depends on food quality). Understandings about scientific inquiry: Research may result in new ideas and phenomena for future studies. (Does food quality influence development in different developmental stages?) 
9–12 Two-factorial experiment Number of offspring dependent on nutrition
Developmental time dependent on nutrition
Nutrition in the respective developmental stage influences number of offspring differently (Figure 1)
Relationship between egg size and number of offspring (T. castanaeum
Create an Excel graph (mean and standard deviation) interpreting Figure 5  Plan an experiment to answer the question: "Do rate of offspring and developmental time depend on nutrition in different life stages?" (cf. Figures 3 and 4Life science: Resource depletion in an early life stage may be carried over to adulthood, even if the quality of resources is then sufficient.
Science as inquiry:Abilities needed to do scientific inquiry: Students should learn how to clarify a scientific question. They should further be able to conduct a scientific experiment, including controls and several variables, and display their data.
Understandings about scientific inquiry: Scientists often investigate several factors within one experiment for a variety of reasons (testing conclusions of prior investigations). 

Study Organism

Tribolium castaneum was once endemic to warmer climates (originally in Indonesia and Australia) but is nowadays a widespread insect in temperate regions, where it can act as a pest on stored grain (Sokoloff, 1974). It is a prominent model organism for research in developmental biology and ecology (Park, 1948; Shippy & Brown, 2005; Roth & Kurtz, 2008; Berenos et al., 2009). Tribolium castaneum is the first beetle with a fully sequenced genome (Richards et al., 2008). Because it has a short generation time and is easy to handle, T. castaneum is a sound model system for experimental work ranging from simple to sophisticated, including experimental evolution and artificial-selection experiments (Fischer & Schmid-Hempel, 2005; Berenos et al., 2009; Roth et al., 2009). Moreover, no elaborate equipment is required for its maintenance in the laboratory. At a temperature of 34°C it takes about 2 days from egg deposition to hatching; the larvae need about 15 days to reach pupation. After 3 days, the adult beetles emerge from metamorphosis; 4 days after that, reproductive maturation is complete. Hence, it takes less than 1 month to fulfill one generation cycle. The reproductive lifetime is 3–4 months for females and 4–6 months for males. At lower temperatures, development is slower (Sokoloff, 1974).

The hallmarks of the biology of T. castaneum are only marginally different from those of its close relative T. confusum. Also, the large mealworm Tenebrio molitor can easily be bred in the laboratory without complicated requirements. Teachers might choose T. molitor for younger students because of its larger size (T. castaneum: 0.5–1.0 cm; T. confusum: 0.5–1.0 cm; T. molitor: 3.0–5.0 cm) and, thus, better handling and slower developmental time (Table 2).

Table 2.

Developmental rate (in days) of Tribolium castanaeum (30°C) and Tenebrio molitor (22°C).

EggLarvaPupaReproductive MaturationTotal
T. castanaeum 20 About 32 
T. molitor 4–7 60 6–15 60 About 140 
EggLarvaPupaReproductive MaturationTotal
T. castanaeum 20 About 32 
T. molitor 4–7 60 6–15 60 About 140 

The Experiment

The age at a life-stage transition is known to be of key importance for an individual's fitness (Moeur & Istock, 1980; Blakley, 1981). A transition at a young age will increase the chance of surviving to reproductive age, and over generations the frequency of organisms with a net shorter generation time will be higher in a population (Lewontin, 1965; Stearns, 1992). However, earlier maturation may occur at the cost of reduced size (Roff, 1992), which may affect survival (e.g., lower fat reserves) and fecundity (e.g., fewer ovarioles) (Leips & Travis, 1994). Hence, organisms face a tradeoff among body size, developmental time, and survival (Rudolf & Rödel, 2007). Thus, in developing this experiment, we recorded the developmental stages of beetles regularly to investigate whether possible delays or accelerations during might influence later reproduction. We also measured the size of the offspring and used size classes (very small, small, medium, and large individuals) to calculate correlations among offspring size, parental fecundity, and developmental time.

The biological aim of the experiment presented here is to investigate fitness differences in T. castaneum raised on different culture media, taking developmental time, fecundity, and size as readouts. It is commonly recognized that poor nutritional quality decreases offspring rates in insects (Sokoloff, 1974). In our experiment we test wthether individual fecundity depends on early imprinting by food condition during the larval phase or whether only nutritional status after metamorphosis determines fecundity.

To begin the activity, raise the red flour beetles individually from hatching on either flour or flour supplemented with yeast. After pupation, place each individual either on the food it developed in or on the other food source, thus creating four feeding treatments: (1) flour plus yeast from hatching to egg deposition (+Y+Y), (2) flour without yeast from hatching to egg deposition (-Y-Y), (3) flour plus yeast from hatching to pupation and thereafter flour only (+Y-Y), and (4) flour without yeast from hatching to pupation and thereafter supplemented with yeast (–Y+Y) (Figure 1). After the beetles reach maturity, put together breeding pairs (one female with one male, replicated 10 times) within each treatment. Reproductive success will be measured over the following 2 weeks. Be cautious with the flour; in rare cases, allergic reactions may occur.

Figure 1.

Implementation procedures and experimental setup of the Tribolium experiment.

Figure 1.

Implementation procedures and experimental setup of the Tribolium experiment.

Materials needed are 96-well plates (for T. molitor, either small plastic vials or 24-well plates are needed), flour, beetles (can be bought in most zoo stores and angling shops, or from certain universities; see, e.g., http://beetlebase.org/), yeast, sieves (normal tea sieves can be used), adhesive tape, and forceps (see Figure 1 for exact description of the experimental design and materials needed). The time from setup until final analysis is 6 weeks for T. castaneum or T. confusum and about 20 weeks for T. molitor (cf. Table 1). The experiment can be run alongside normal classroom activities or as a project conducted by certain students. Two lessons are needed for the explanation of the objectives of the study (e.g., beetle development as holometabolous insects, life-history trade-offs, and importance of nutritional quality; use Sokoloff, 1974). One additional lesson is needed for a short introduction to experimentation in general and to the design of this experiment in particular (e.g., the difference between an experiment and an observation, and why to use replicates).

In the class in which we developed this experiment, the students assumed that developmental time would be shorter (depending on sex) and the number of offspring higher if yeast were added. This is obvious when taking into account students' previous knowledge and experience: better nutrition results in higher fitness. The teacher asked the students to plan an experiment to test this assumption. They realized that two different media are needed: flour only (-Y) and flour with an additional component (+Y). We used yeast because of its high amounts of vitamins and proteins. Moreover, we introduced the factor "developmental stage" in our experiment. This means that we switched the nutritional supply (+Y or -Y) in the respective developmental stage, resulting in four combinations (Figure 1). Variables to measure are number of offspring, size of the individual offspring, and developmental time. For a basic experiment in a high school course, investigating the difference between control flour and flour with yeast may be sufficient. However, because we conducted this experiment in the context of a bachelor's thesis, we further switched the media in different developmental stages to increase the intellectual appeal. The students were asked to address the following research question (cf. Figure 2): Which parameters will influence the flour beetle's development (time and adult size) and its fitness (average contribution of each individual to the next generation – number of offspring)?

Figure 2.

Possible parameters influenced by nutritional quality and standards being addressed. The focus is on the investigation of growth, reproduction (number of offspring), and development time (Tribolium pictures © Alex Wild).

Figure 2.

Possible parameters influenced by nutritional quality and standards being addressed. The focus is on the investigation of growth, reproduction (number of offspring), and development time (Tribolium pictures © Alex Wild).

The learning objectives of this activity are as follows:

  • • The student will gain an understanding of how to design an experiment and how different variables are used.

  • • The student will experience aspects of scientific research, including testing hypotheses, evaluating a data set, and using the right forms of representation (tables and graphs).

  • • The student will understand how poor nutritional quality affects different beetle life-history traits (development, number of offspring).

There are various possibilities for adapting the experiment to the different grade levels. Table 1 presents inquiry tasks that we recommend for each and the skills that are needed for the evaluation. In K-4 or K-5, students could exclusively concentrate on the observation of the life cycle of the mealworms (e.g., document the time from larval to pupal stage and to the adult stage). More advanced students could investigate whether the developmental time of beetles depends on sex or on nutrition or whether the number of offspring depends on nutrition. However, for this, students would need to develop questions based on the observation of the life cycle that can only be answered by planning and conducting an experiment. This would give the students an opportunity to formulate their own research questions and experimentally test them. Further, the inquiry tasks proposed here could also be performed over several years in the same class. For example, students could start with the observation phase in grade 4 or 5, and continue with the experiments 2 years later.

The teacher can use Figure 2 to introduce the organism and explain that animals can invest in various life-history parameters (e.g., growth or fat reserves), then ask the students what happens when the beetle is faced with poor nutrition. The students can then hypothesize which parameters will be negatively or positively affected, and then plan an experiment to test the hypothesis (Figure 2). For this, we suggest using the POE (predict, observe, explain) process (White & Gunstone, 1992), as follows:

  1. 1. Predict: Ask the students to independently predict the effect of poor nutrition on beetles. e.g., As a result of prolonged developmenal time, number of offspring is reduced. e.g., Number of offspring will be the same in beetles feeding on high-quality nutrition and poor-quality nutition, but low-quality nutrition is compensated by a longer development time.

  2. 2. Observe, plan and carry out the experiment.

  3. 3. Explain: Results of the experiment will be discussed in the classroom. Determine whether the predictions can be confirmed or not.

Our Results

Overall support for our students' hypotheses was found: yeast was an important factor affecting developmental time and reproductive success. Most of the students argued that offspring rates are exclusively determined by the nutritional state after adult emergence. However, the results imply that the overall picture is more elaborate than the students expected: animals that spent all their life without yeast supplement produced the fewest offspring. If animals were put in food without yeast only after pupation, offspring production was still greatly reduced. But even exposure to decreased food quality (without yeast) during only the juvenile phase produced carry-over effects in adulthood. Decreased offspring production was found, however, to a smaller extent (Figure 3). This suggests that offspring production depends on conditions during the adult phase, but also that food condition before and during metamorphosis may influence reproductive success. The results are so evident that a descriptive statistic is sufficient for classroom purposes. However, in an analysis of variance with nutrition depending on developmental stage (egg to larva or larva to adult: +Y or -Y) as fixed factors, a highly significant influence of both factors was found. The nutrition during pupation and after adult enclosure affects offspring rates to a greater extent than the nutrition during the larval stage (partial eta squares: 0.59 compared with 0.35). When students were confronted with this result, they realized that nutritional quality has important effects on development and fitness during the whole lifetime and that a detrimental effect due to insufficient food quality during premature development cannot be compensated by good food quality during adulthood. Students proposed that this conclusion can also be transferred from our model system to pets and farm animals, and even to human development.

Figure 3.

Number of offspring according to treatment (mean ± SE) (Tribolium picture © Alex Wild).

Figure 3.

Number of offspring according to treatment (mean ± SE) (Tribolium picture © Alex Wild).

In our experiment, the poor food quality further slowed the development of T. castaneum, which suggests that animals need more time to compensate for the detrimental effects of the food (Figure 4). Surprisingly, size measurements demonstrated that even though metamorphosis is reached later in animals feeding on flour without yeast, animals are not able to catch up with their counterparts feeding on flour with yeast. This implies not only that reproductive success is limited but that the offspring also hatch at a smaller size if food quality is decreased. The size of offspring is positively correlated with parental reproductive success (Figure 5). This experiment suggests that food is an important factor influencing the ad hoc condition of an animal and that animals exposed to suboptimal food conditions early in life suffer detrimental carry-over effects for their whole lifetime and produce a lower number of offspring, of smaller size. Food is an important factor determining inclusive fitness of an animal.

Figure 4.

Development time according to treatment. Development of larvae on the –Y medium was, on average, 4 days longer in duration.

Figure 4.

Development time according to treatment. Development of larvae on the –Y medium was, on average, 4 days longer in duration.

Figure 5.

Number of offspring plotted versus the size category (0 5 very small, 4 5 big). In this graph, it becomes obvious that because of high variability, the use of several replicates within every treatment is necessary to fulfill the standards of an experiment. Beetles feeding on +Y+Y showed the highest number of offspring (90–190 individuals), beetles feeding on -Y-Y showed the smallest number of offspring (15–50 individuals), and beetles in the +Y-Y and -Y+Y treatments are in between. Correlation coefficient was calculated using Spearman's rho.

Figure 5.

Number of offspring plotted versus the size category (0 5 very small, 4 5 big). In this graph, it becomes obvious that because of high variability, the use of several replicates within every treatment is necessary to fulfill the standards of an experiment. Beetles feeding on +Y+Y showed the highest number of offspring (90–190 individuals), beetles feeding on -Y-Y showed the smallest number of offspring (15–50 individuals), and beetles in the +Y-Y and -Y+Y treatments are in between. Correlation coefficient was calculated using Spearman's rho.

The proposed experiment can improve students' abilities to plan experiments, analyze data sets, and interpret the results using prior biological knowledge. At the end of the activity, the difference between an experiment and an observation was discussed again with the students. They understood the observation that nutritional quality may play a general role in development to be testable in an experiment by varying and measuring single variables under controlled conditions.

Acknowledgments

We thank Christina Latzel for conducting the experiment under our guidance. Joachim Kurtz provided laboratory facilities. Alex Wild provided the Tribolium pictures used in Figures 2 and 3.

References

References
Berenos
C.
,
Schmid-Hempel
P.
&
Wegner
K.M.
(
2009
).
Evolution of host resistance and trade-offs between virulence and transmission potential in an obligately killing parasite
.
Journal of Evolutionary Biology
,
22
,
2049
2056
.
Blakley
N.
(
1981
).
Life-history significance of size-triggered metamorphosis in milkweed bugs (Oncopeltus)
.
Ecology
,
62
,
57
64
.
Fischer
O.
&
Schmid-Hempel
P.
(
2005
).
Selection by parasites may icrease host recombination frequency
.
Biology Letters
,
1
,
193
195
.
Leips
J.
&
Travis
J.
(
1994
).
Metamorphic responses to changing food levels in two species of hylid frogs
.
Ecology
,
73
,
1345
1356
.
Lewontin
R.C.
(
1965
).
Selection for colonizing ability
.
In
Baker
H.G.
&
Stebbins
G.I.
(
eds.
),
The Genetics of Colonizing Species
.
New York, NY
:
Academic Press
.
Moeur
J.E.
&
Istock
C.A.
(
1980
).
Ecology and evolution of the pitcher-plant mosquito
.
Journal of Animal Ecology
,
49
,
775
792
.
Park
T.
(
1948
).
Experimental studies of interspecies competition. 1. Competition between populations of the flour beetles, Tribolium confusum Duval and Tribolium castaneum Herbst
.
Ecological Monographs
,
19
,
265
307
.
Richards
S.
,
Gibbs
R.A.
,
Weinstock
G.M.
,
Brown
S.J.
,
Denell
R.
,
Beeman
R.W.
&
Gibbs
R.
(
2008
).
The genome of the model beetle and pest Tribolium castaneum
.
Nature
,
452
,
959
955
.
Roff
D.A.
(
1992
).
The Evolution of Life Histories: Theory and Analysis
.
New York, NY
:
Chapman & Hall
.
Roth
O.
&
Kurtz
J.
(
2008
).
The stimulation of immune defence accelerates development in the red flour beetle (Tribolium castaneum)
.
Journal of Evolutionary Ecology
,
21
,
1703
1710
.
Roth
O.
,
Sadd
B.M.
,
Schmid-Hempel
P.
&
Kurtz
J.
(
2009
).
Strain-specific priming of resistance in the red flour beetle, Tribolium castaneum
.
Proceedings of the Royal Society of London, Series B
,
276
,
145
151
.
Rudolf
V.H.W.
&
Rödel
M.-O
. (
2007
).
Phenotypic plasticity and optimal timing of metamorphosis under uncertain time constraints
.
Evolutionary Ecology
,
21
,
121
142
.
Shippy
T.D.
&
Brown
S.J.
(
2005
).
Closing the gap: comparative approaches to studying insect development in the red flour beetle Tribolium castaneum and other short and intermediate germ insects
.
Current Genomics
,
6
,
571
578
.
Sokoloff
A.
(
1974
).
The Biology of Tribolium with Special Emphasis on Genetic Aspects
.
Oxford, UK
:
Clarendon Press
.
Stearns
S.C.
(
1992
).
The Evolution of Life Histories
.
Oxford, UK
:
Oxford University Press
.
White
R.T.
&
Gunstone
R.F.
(
1992
).
Probing Understanding
.
London, UK
:
Falmer Press
.