Order 2303456: the effect of bean type amount of egg laid by female bean bettles

BIOL1108LTLAS04-2019-01 xexperiment_1_data_sheet.xlsxFirstLabReportRubric2020-01 xhandbook totalclassdataversion1.xlsb.xlsx
 

• Type of paperLab Report
• SubjectBiology
• Number of pages3
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my lab report about bean type and beans color affect to the number of egg laid. ( Navy beans vs Black beans) Hypothesis format : X happen … because .. Prediction format : If ( I do on experiment) then ( I′ll get a result) The format of lab report i will send in file attachment

BIOL1108L – Thinking Like a Scientist – Part 4

Before coming to lab, you should read through this entire handout and the appropriate pages in chapter 4 in McMillan (2017).

Copyright Stephen Burnett, 2009-2019, Clayton State University, Morrow, GA. Do not copy or use this document without permission.

Objectives

Upon completion of this exercise, you will be able to:

1. Properly read or write a discussion section for a scientific paper.

Introduction

The final section of a scientific paper is the Discussion (or Conclusion) section. In this section, you summarize past research, your results, and the implications for your hypothesis. This is the section where you interpret the information that you have accumulated and suggest future research that could/should be done. This is the section in the paper where you have the most freedom to make speculation, but it is important that you keep your speculations within reason!

Because you are addressing both your specific results and the wider implications of those results, you always want to start this section with the specific information from your experiments, then move on to the general ideas, where you discuss what your results mean in the big picture. You need to address every variable you measured in your experiment. If you address it in the methods section, you need to address it here as well, even if it’s to say that the particular variable wasn’t important/significant. Your results should be the foundation for your argument so it is vital that you interpret them correctly or everything else will be inaccurate. However, it’s also important that you limit your conclusions to things that your data can support. This means it’s generally best to be conservative when you interpret your data so that you don’t overextend your results!

The first thing you do in this section is refer back to your original hypothesis and predictions for your experiment and state why your results did or did no support them. Remember, supporting or not are the only possible options. We never prove or disprove our ideas and it is also not possible partially support them.

Part of your job in this section is to try and convince others who might doubt your results that your conclusions are a sound way to interpret those results. As such you need to provide a convincing argument, however, you must avoid the temptation to simply point out data that agreed with your ideas. Data that contradicts your ideas is something that others will notice and if you fail to address it, they will discount your conclusions as a result. Also avoid making “excuses” for why the results do not support your hypothesis. Students often feel the need to make the data match their hypothesis even when this is clearly not the case. In science, it is more common to be wrong than to be right, so there is no problem with having a hypothesis that isn’t supported, and there are no points off on your grade in this class! This extends to the desire that students feel to provide the sources of “error” that caused problems in their experiment. While errors do occur, you should only bring them up if you have evidence they occurred – suggesting that an error might have occurred is unscientific and not helpful. If you mention problems/errors in earlier parts of the report, you should definitely address them here.

In this section, it is important to think towards the future. In this section you would need to provide suggestions for future questions or areas of study that were brought up by your results. When you make this suggestion, you do not need to have all the details for the experiment you would do, but you have to provide enough to make it clear what you would do and why it is relevant. The experiment you provide also needs to make sense in the context of the topics you addressed. If you have a report that addresses multiple experiments, then each section of the report needs to have suggestions for future research. These suggestions must be for experiments you haven’t done already. This means that if you write up a report that includes two experiments in it, your follow-up suggestion for experiment 1 cannot be to suggest doing what you did in experiment 2.

Next, you need to use this section to discuss larger issues than you addressed directly. It is important that you do this in a focused way – there are many topics that you could include that aren’t important/relevant to what you did, so you need to avoid going overboard. One important thing that you should do is tie your results to what other researchers who did similar/related experiments found. This will often include the references you used to provide background in your introduction, but it might refer to other studies as well. Do your results agree with those references or do they differ and why do you think that happened? When referring to other research it is important to put the information in your own words – scientific writing does not use quotations. Plagiarism is also a concern if you don’t fully understand what you are citing, so you want to be careful to avoid things that will cost you points or have worse repercussions. You also need to make sure you Also it is common for a lab report to address multiple experiments. In such a case you would use this section to tie together all the experiments, explain what you learned by looking at all of them, what you better understand overall as a result, etc.

Lastly, you need to tie your results into the “big picture” that you were addressing. This would include how your results fit into the larger understanding of the organisms you were studying, why those are important, and how our understanding is changed by them.

One last thing you can consider as you finish your lab report is how well the ideas flow and follow the standard format. Overall a well-written lab report should have a symmetrical, hourglass shape, where the width of the particular portion specifies how broad the information should be. Narrow sections represent items that are specific to your project, while wider parts represent areas where you should be talking about material that fits into a broader context (Figure 1).

Figure 1: the “hourglass” model of a lab report. Width of the red section corresponds to how broad that material is in that section. Each section of the report is marked with yellow brackets. For sections that vary in width, some indication has been given to indicate where a particular portion fits in.

Thinking Like a Scientist – Part 4 – Page 1

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>Sheet

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Bean # # eggs Bean # # eggs Bean # # eggs

1

1 1 1 0 1 0

2 1 2 1 2 0 2 0

0 3 0 3 0 3 0

0 4 1 4 0 4 0

0 5 0 5 0 5 0

0 6 0 6 0 6 0

0 7 1 7 0 7 0

1 8 0 8 0 8 0

1 9 1 9 0 9 0

1 10 0 10 0 10 0

1 11 0 11 0 11 0

1 12 0 12 0 12 0

1 13 0 13 0 13 0

1 14 0 14 0 14 0

1 15 0 15 0 15 0

1 16 0 16 0 16 0

0 17 0 17 0 17 0

1 18 0 18 0 18 0

0 19 0 19 0 19 0

1 20 0 20 0 20 0

1 21 0 21 0 21 0

2 22 0 22 0 22 0

0 23 0 23 0 23 0

0 24 0 24 0 24 0

1 25 0 25 0 25 0

1 26 1 26 0 26 0

1 27 0 27 0 27 0

1 28 0 28 0 28 0

1 29 0 29 0 29 0

1 30 0 30 0 30 0

																																																																																																			Navy bean – dish 1	Black bean – dish 1	Navy bean – dish 2	Black bean – dish 2
			Bean #				# eggs
																																																																																													0
			3
			4
			5
			6
			7
			8
			9
			10
			11
			12
			13
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First Biology Lab Report Rubric

Name: Score:

2

2

2

2

2

3

Excellent (+0.5)	Good (+0. 2 5)	Acceptable (0)	Poor (-0.5)	Unacceptable (-1)
					2	Abstract
Abstract is an accurate summary of the lab report
Abstract covers all portions of the report (the purpose, design, findings, and conclusion)
Introduction
Provides relevant background information on experiment
Hypotheses and predictions are clearly stated, in proper format, and logically connected
Methods/Treatments/Controls
Methods give enough details to allow for replication of procedure from all experiments.
Methods are described accurately
Figure/tables (these may be used in multiple sections of the report, so this applies to any figures or tables in the report
Format of tables and figures is clear and correct (including required parts such as captions, axis labels with proper unit measurements, etc.)
Sufficient number of figures/tables were provided to summarize information effectively
Results
Quantifies results if possible and uses statistics.
Highlights important results in the text of the report
Discussion
Uses data obtained in the experiment(s) to logically explain why results support or refute student’s hypothesis.
Suggests and describes follow-up experiment or question (this must go beyond just repeating or making slight modifications to the current experiment).
	3	References
Information in the text that is not from the student’s direct knowledge is cited in text and the source is listed at the end.
Citations and references adhere to proper format both at end and in text.
Suitable number and type of references were used
Presentation
Report is written in scientific style and is clear and to the point.
Grammar and spelling are correct.
Report shows evidence of work that went into it (research, writing, avoiding last-minute corrections, etc.)

A Handbook on Bean Beetles,

Callosobruchus maculatus

Christopher W. Beck
Department of Biology, Emory University

christopher.beck@emory.edu

and

Lawrence S. Blumer
Department of Biology, Morehouse College

lawrence.blumer@morehouse.edu

201

9

This handbook was developed as part of a project titled: Developing Bean
Beetles as a Model System for Undergraduate Laboratories. The most current

version of this handbook may be downloaded from www.beanbeetles.org

This project was supported by the National Science Foundation, DUE-0535903,
DUE-0815135, and DUE-0814373.

National Science Foundation

Copyright © by Christopher W. Beck and Lawrence S. Blumer, 2019. All rights
reserved. The content of this document may be freely used for non-profit
educational purposes, with proper acknowledgement of the source. All other
uses are prohibited without prior written permission from the copyright holders.

Disclaimer: Any opinions, findings, and conclusions or recommendations
expressed in this material are those of the authors and do not necessary reflect
the views of the National Science Foundation, Emory University, or Morehouse
College.

Acknowledgments:

Figure 1. A molecular phylogeny for Callosobruchus, was reprinted with
permission from Tuda, M., J. Rönn, S. Buranapanichpan, N. Wasano, and G.
Arnqvist. 2006, Evolutionary diversification of the bean beetle genus
Callosobruchus (Coleoptera: Bruchidae): traits associated with stored-product
pest status. Molecular Ecology (2006) 15, 3541–3551.

Figure 3, Karyotypes of bean beetles, Callosobruchus maculatus, was reprinted
with permission from Yadav, J.S. 1971. Karyological studies on the three
species of Bruchidae (Coleoptera). Caryologia 24(2);157-166.

Figure 4, Size frequency distribution of larval head capsules, was reprinted from
Journal of Stored Products Research, Volume 39, Devereau, A.D., I. Gudrups,
J.H. Appleby, and P.F. Credland, Automatic, rapid screening of seed resistance
in cowpea, Vigna unguiculata (L.) Walpers, to the seed beetle Callosobruchus
maculatus (F.) (Coleoptera: Bruchidae) using acoustic monitoring., Pages 117-
129, Copyright (2003), with permission from Elsevier.
http://www.sciencedirect.com/science/journal/0022474X

Figure 6, Dorsal view of male and female Callosobruchus maculatus, was
reprinted with permission from, Brown, L. and J.F. Downhower. 1988. Analyses
in Behavioral Ecology: A Manual for Lab and Field. Sinauer Associates, 19

4

pages.

Photographs were taken by L. Blumer.

Table of Contents

Introduction

1

Background Information 1

Natural History 1

Life Cycle

3

Genetics

5

Development 5

Culture and Handling Methods

6

Culture Techniques 6

Generation Time

7

Identifying the Sexes

8

Handling Techniques 9

Measuring Beetles 9

Mating Beetles

10

Isolating Virgins 10

Disposal of Cultures

11

Literature Cited 1

2

1

Introduction

This handbook provides basic information about raising, handling, and
maintaining cultures of bean beetles, Callosobruchus maculatus. Our intended
audience is the faculty and staff who teach or coordinate undergraduate
laboratory courses. The information provided here is based on our own
experiences working with this insect in undergraduate laboratory courses, and
information available in the research literature. Our references to specific
commercial vendors for supplies are intended to assist you in finding the types of
supplies we have used, but these references are not an endorsement of these
particular vendors. Comments to the authors with corrections or suggestions for
additional information to include in future versions of this handbook would be
much appreciated.

Background Information

Natural History

Bean beetles, Callosobruchus maculatus (Coleoptera: Chrysomelidae:
Bruchinae), are agricultural pest insects of Africa and Asia that presently range
throughout the tropical and subtropical world. This species also is known as the
southern cowpea weevil. The larvae of this species feed and develop exclusively
on the seed of legumes (Fabaceae) hence the name bean beetle. The adults do
not require food or water and spend their limited lifespan (one – two weeks)
mating and laying eggs on beans. The systematic placement of bean beetles is
as follows: Callosobruchus is one of the genera in the subfamily Bruchinae
(seed beetles) that is in the family Chrysomeloidae (Kergoat et al. 2007). This
group is part of the order of beetles, Coleoptera (from Greek “sheath-winged”
referring the stiff outer, first pair of wings (elytra) that protect the membranous
second pair of flight wings). The Coleoptera is largest of the orders that
comprise the class Insecta. Insects are the largest and most diverse (750,000
described species) of all the animal classes that are found in all but marine
environments. Insects are protostomous animals and are thus more closely
related to mollusks and crustacea than to the deuterstomous vertebrate classes.
See the Integrated Taxonomic Information System on the subfamily Bruchinae
for more information on the systematic placement of Callosobruchus (ITIS 2011
) The systematic placement of seed beetles in the family
Chrysomelidae (rather than in their own family) is relatively recent so some
websites on animal taxonomy may not yet reflect this change. Additional
information about the taxonomy of C. maculatus, ecological associations, and
information about other species of Callosobruchus may be found at the
Encyclopedia of Life website (EOL 2011 ).
Callosobruchus maculatus forms a monophyletic group with C. analis, C.

2

rhodesianus, and C. subinnotatus, all species that use dry beans in the genus
Vigna as their natural hosts (Figure 1, Tuda et al. 2006).

Figure 1. A molecular phylogeny for Callosobruchus. The circle on the left
indicates the ability to use dry mature seeds and the circle on the right indicates
the host species subtribe. (Reprinted with permission from Fig. 1 in Tuda, M., J.
Rönn, S. Buranapanichpan, N. Wasano, and G. Arnqvist, 2006, Evolutionary
diversification of the bean beetle genus Callosobruchus (Coleoptera: Bruchidae):
traits associated with stored-product pest status. Molecular Ecology (2006)
15:3541–3551).

Bean beetles exhibit two adult forms (morphs), a sedentary (flightless) form and
a dispersal (flying) form. The dispersal morph is induced by high larval density in
stored beans or laboratory cultures, and is caused by density dependent micro-
habitat temperature increases (Utida 1956, 1972). Induction of the dispersal
morph allows individuals to move to new, higher quality habitats. These two
morphs have very different life history characteristics such as longer adult
lifespan in the dispersal morph and significantly reduced fecundity compared to

3

the sedentary morph (Utida 1956, 1972). In the sedentary form, the sexes are
highly dimorphic and readily distinguished but sex differences are very subtle in
the dispersal form. Thus, it is essential to maintain laboratory cultures at low
density (one or two larvae per bean) and temperatures no greater than 30°C, if
individual beetles need to be unambiguously identified by sex (see Identifying
the Sexes).

We are frequently asked, by students and even fellow academics, what is the
purpose of bean beetles? A very short adult life span and a larval stage in which
most or all life-time feeding occurs is not unusual in insects (for example the
Order Ephemeroptera, mayflies), but this life cycle seems strange compared to
the dominance of adult stages in familiar birds and mammals. Ultimately, in
evolutionary terms, the purpose of bean beetles is the same as in all other living
things, reproduce and leave descendants. Ecologically, bean beetles are
herbivores that have specialized on seed consumption. They are a part of food
webs in that eggs and larvae are prey for parasitoid wasp species (Boeke et al.
2003), and adults may be prey for birds, reptiles and amphibians, so they do
have the purpose of providing food for other organisms.

Life Cycle

Once inseminated, adult females will lay (oviposit) single fertilized eggs on the
external surface of a bean. Individual eggs (0.75mm long) are oval or spindle
shaped, clear, shiny and firmly glued to the bean surface (Figure 2a). The larva
that hatches from the egg burrows from the egg through the seed coat and into
the bean endosperm without moving outside the protection of the egg. Once the
larva burrows into the bean, the remaining egg (shell) becomes opaque white
(Figure 2a) or mottled as it fills with frass (feces) from the larva. The larva
(Figure 2b) burrows and feeds on the bean endosperm and embryo, undergoes a
series of molts, and burrows to a position just underneath the seed coat prior to
pupation. Although the seed coat of the bean is still intact, a round 1-2mm
window is apparent at the location where the beetle is pupating (Figure 2c).
Pupation (Figures 2d) is the complete metamorphosis of the larval maggot to a
winged adult. The adult that results from pupation chews through the seed coat
and emerges from the bean (Figure 2e and 2f). The adults are fully mature 24 to
36 hours after emergence. Males seek females to inseminate (see Mating
Beetles) and females store viable sperm in their spermatheca (a structure in the
female reproductive tract for storing sperm). Neither male nor female adults
require food or water during their short adult lifetime (10-14 days).

4

Figure 2a. Single newly laid egg Figure 2b. Larval bean beetle. The dark area at the
(upper right arrow) and an old egg upper right of the isolated larva is the mouth. A
(center) on mung bean. The graph larva in a mung bean is at the arrow. The graph paper
paper squares are 1mm. squares are 1mm.

Figure 2c. “Window” in Figure 2d. Pupa of bean beetle. A young pupa (left)
seed coat of cowpea at the and an older pupa (right) in a head down position.
location of a pupating beetle. The graph paper squares are 1mm.

Figure 2e. Adult bean beetle. Figure 2f. Adult emergence holes. These holes are the result of
An adult female, sedentary an adult bean beetle emerging after pupation. The graph paper
morph, on a mung bean. squares are 1mm.

5

Genetics

Callosobruchus maculatus has a karyotype of ten chromosome pairs (2N=20)
(Yadav 1971). Chromosome 10 is a sex chromosome and males are the
hetergametic sex (Figure 3). One Mendelian trait has been described for bean
beetles, body color which is autosomal and has alleles with incomplete
dominance (Eady 1991). Heritable variation in body size is well described (Fox
et al. 2004) as are life-history and behavioral traits such as longevity and
copulation duration (Brown et al 2009). Some genes have been isolated and
characterized, for example, genes with environmentally relevant transcription
regulation (Chi et al. 2011). The nucleotide database in the NCBI GenBank has
more than 80 entries for C. maculatus
. When a whole genome sequence becomes
available, it may be accessed by entering Callosobruchus in the NCBI Entrez
search engine .

Figure 3. Karyotypes of bean beetles, Callosobruchus maculatus. The upper
image shows the ten chromosome pairs of a male and the lower image is that of
a female. The chromosomes pairs on the right side are the sex chromosomes.
(Figure reprinted from Figures 31 and 32 of Yadav 1971 Caryologia 24:157-166
with permission).

Development

Fertilization occurs as females lay eggs and glue them to the surface of a bean
seed (Fabaceae). Early embryonic development occurs inside the transparent
egg until the first instar larva (maggot) burrows through the seed coat into the
seed endosperm directly from the egg. Bean beetles development is long-germ
(Patel et al. 1994) similar to Drosophila in which body segment determination
occurs by the end of the blastoderm formation. The empty egg shell is typically
filled with white frass (fecal waste) as the larva feeds (Figure 2a). There are four
larval instars (Figure 4, Devereau et al. 2003) all feeding inside the endosperm of
the seed on which the egg was laid. Pupation occurs inside the seed and an
adult emerges by chewing and removing a circular piece of the seed coat to form
a round exit hole (Figure 2f). Temperature, the species of bean chosen for egg
laying, and relative humidity all influence development time and success (see
Generation Time).

6

Figure 4. Size frequency distribution of larval head capsules. The head capsule
size frequency distribution of 100 randomly selected C. maculatus larvae shows
four relatively distinct size classes corresponding to the four larval instars. (This
figure was reprinted from Journal of Stored Products Research, Volume 39,
Devereau, A.D., I. Gudrups, J.H. Appleby, and P.F. Credland, Automatic, rapid
screening of seed resistance in cowpea, Vigna unguiculata (L.) Walpers, to the
seed beetle Callosobruchus maculatus (F.) (Coleoptera: Bruchidae) using
acoustic monitoring., Pages 117-129, Copyright (2003), with permission from
Elsevier. ).

Culture and Handling Methods

Culture Techniques

Starting new cultures requires no more than containers to contain beans and
beetles. Virtually any closable containers will work successfully: lidded plastic
Petri dishes, screen covered glass jars, snap lid vials, and cotton plugged shell
vials are all suitable containers. We prefer to use disposable plastic containers in
our teaching laboratories to minimize breakage and to keep cultures relatively
small but replicated. Although bean beetles are the easiest of insects to
successfully culture, sometimes a culture will fail if adults were very old when
introduced to the new culture container or too few adults were introduced for
adequate numbers of eggs to be laid. Therefore, we always start stock cultures
in pairs (or more if needed) on the same date and we never dispose of old
cultures until we see that a new culture has successfully yielded adults. It is
always a good practice to check a new culture a few days after it is started to see
if numerous eggs have been laid. Plastic Petri dishes 150 x 25 mm (Falcon
353025, Fisher Scientific 08-772-6, VWR 25379-048, Carolina Biological 199279)
are ideal containers from which students can easily view cultures and remove
selected adults. Although the lids fit loosely on the plates, Petri dishes will
confine adults and permit adequate ventilation without any modification.
Covering the bottom of a Petri dish with a single layer of beans (approximately 50

7

ml volume) and introducing 10 adults males and 10 adult females is sufficient to
produce a dense culture. Cultures established in this manner will typically
sustain two or three sequential generations without adding additional beans and
without inducing the production of dispersal morph adults. Cultures older than
three generations on the same set of beans should be discarded (see Disposal
of Cultures). We also have had good results using
plastic snap-lid containers (300 ml Corning Snap-
Seal Sample Containers, Fisher Scientific 02-540-
23) with pin-holes punched in the lid for ventilation.
As with the Petri dishes, we use only 50 ml of beans
in each 300 ml snap-lid container. The ideal seeds
(beans) to use are mung (Vigna radiata = Phaseolus
aureus), blackeye peas or cowpeas (Vigna
unguiculata) and adzuki (Vigna angularis) (Figure 5).
Although bean beetles can be reared successfully on
these three species, they differ in nutrient quality
(USDA Agricultural Research Service) and
secondary compounds (Bisby et al. 1994). We find
that raising beetles on blackeye peas is best done in
150 mm petri dishes, which minimizes mold growth
on the beans. The successful completion of the
bean beetle life cycle on most other bean species is
minimal (Janzen 1977), but we have successfully
cultured bean beetles on pigeon peas (Cajanus cajan), chickpeas or garbanzo
beans (Cicer arietinum) and hyacinth beans (Lablab purpureus). Dark
(completely black or brown) varieties of blackeye peas will support normal
development of bean beetles and have the advantage of making it easier to see
newly laid eggs. These varieties should not be confused with black beans
(Phaseolus vulgaris) that are toxic to bean beetles (Janzen et al. 1976). We
prefer to use organically grown beans to minimize pesticide problems in our
cultures, but it is not essential for successful culturing of beetles. Dry beans and
adult beetles in a container that keeps the beetles from escaping are all that you
need. Keep the cultures at temperatures between 22° – 30°C (not in direct
sunlight and away from radiators).

Generation Time

The elapsed time from newly laid eggs to the emergence of adult beetles varies
between bean beetle strains and environmental conditions. Previous studies
indicate that temperature and relative humidity (Howe and Currie 1964, Schoof
1941) are the most important variables influencing generation times (egg to
adult) when beetles are raised on preferred host beans. Within a limited range,
increasing temperature will decrease the generation time. In our laboratory, we
have observed generation times as short as 3-4 weeks in a 30°C incubator
(12:12 day:night light cycle) and ambient humidity (averaging 30% RH and

Figure 5. Beans for
culturing bean beetles. top
left: blackeye peas
(cowpeas) dark variety,
hyacinth beans light and
dark varieties; middle left:
pigeon peas, chickpea
(garbanzo), adzuki; bottom
left: blackeye peas light
variety and mung beans.

8

ranging from 20%-40% RH). Cultures raised in a 25°C incubator (12:

12

day:night light cycle) and ambient humidity (averaging 50% RH and ranging from
40%-60% RH) had generation times of 4-5 weeks. Cultures maintained on a
laboratory bench at room temperature (22°C) with indirect outdoor window
lighting and ambient humidity (averaging 50% RH and ranging from 40%-60%
RH) had a generation time of a full 7 weeks. Reliably obtaining newly emerged
adults for a specific date (a scheduled laboratory class meeting) requires that you
grow cultures for a few months under your laboratory conditions so you can
predict emergence times. In low humidity locations, or during the winter in North
America, when RH is typically low, the emergence rates of beetles may be
improved by increasing the RH of an incubator or culture container. Simply
placing a tray of water in a temperature controlled incubator may be all that is
necessary to bring RH to the 40%-60% range, and improve emergence success
rates.

Generation time also depends on the host species of bean you choose to use.
We have found longer generation times in adzuki beans compared to either
mung or black-eyed peas. At 30°C, it takes seven weeks for emergence from
adzuki beans compared to 3-4 weeks from mung beans. Bean beetles grown on
pigeon peas, garbanzo, and hyacinth beans have generation times similar to
those of adzuki beans.

Identifying the Sexes

Male and female bean beetles (of the sedentary morph) are easily distinguished
from one another by general appearance. The most distinguishing characteristic
is the coloration on the plate covering the end of the abdomen. In the female, the
plate is enlarged and is darkly colored on both sides (Figure 6). In the male, the
plate is smaller and lacks stripes. Generally, females are larger in size than
males, but there is much variation. In some strains, females are black in
coloration and males are brown (Figure 6), but in others both sexes are brown or
black.

Figure 6. Dorsal view of male and female Callosobruchus maculatus. The sex
specific coloration of the posterior abdominal plate (pygidium) is shown (Figure
from Brown and Downhower, 1988 reprinted with permission). Photographs of a
male and female are at the same scale. The squares are 1mm.

9

Handling Techniques

Although sedentary morph bean beetles are capable of flying, they rarely do. As
a result, they are easy to handle. Beetles can be moved either using Drosophila
sorting brushes (Carolina Biological 17-3094 or Ward’s Natural Science 15 V
3846) or soft forceps (BioquipTM featherweight forceps 4748 or 4750; Ward’s
Natural Science 14 V 0520).

When removing beetles from stock cultures, individual Petri dishes, or well
plates, tap the containers lightly on the lab bench before removing the lid to
prevent beetles from crawling out immediately. If the lid is left off for more than a
minute or so, beetles will escape from the culture dishes.

Especially when kept individually, bean beetles will often “play dead.” Don’t be
fooled! A gentle prod with forceps or brush will cause them to move.

Measuring Beetles

Body Mass – To weigh individual beetles, a 0.1 mg analytical balance, at a
minimum, is necessary (for example, Ohaus Analytical Balance Model PA64,
Carolina Biological 70-2498, Fisher Scientific S97282). Individual beetles can be
placed in the bottom of a 35mm Petri dish (for example, Falcon 351008, Fisher
Scientific 08-757-100A or 60mm dish, Carolina Biological 741246) to be weighed.

Linear Measures – Linear measures of body size, such as the length of the elytra
(the hard wing covers), may be readily collected on dead adults. Such
measurements may be facilitated by using an inexpensive microscope video
camera, such as the Moticam 352 (Carolina Biological 591282) attached to the
eyepiece of a dissection microscope. This video camera connects directly to a
computer (Mac or Windows PC) via the USB port and measurements are made
by using image analysis software (included with the camera) to evaluate the
length of a line drawn on a body part in a captured image or in a live video
image. Dead animals may be sorted by sex and glued to file cards for
measurement under a dissection microscope. A free image analysis program
(NIH Image J) may be used to make measurements on any digital image,
including those captured with a Motic camera. A very inexpensive alternative is
to provide a microscope ocular cell phone mount (such as Gosky universal cell
phone adapter mount) that will permit students to capture images with a known
size background or scale on their own cell phone camera. Students may then
use Image J on those photographs downloaded to a computer.

10

Mating Beetles

Both virgin and non-virgin beetles will mate readily. However, virgin males may
not produce fully formed spermatophores until 24 hours after emergence. In
addition, females may not mate for several hours after a previous mating.

To mate beetles, place beetles into a 35mm
Petri dish (for example, Falcon 351008,
Fisher Scientific 08-757-100A or 60mm dish,
Carolina Biological 741246). Males will
chase females until they are able to mount
and copulate with females (Figure 7).
Copulation generally begins within 10-15
minutes, but sometimes may not begin for 30
minutes to an hour.

Figure 7. Bean beetles mating.

To determine if a male transferred a spermatophore successfully during
copulation, weigh the male before and after copulation. Males may lose as much
as 5% or more of their body mass due to spermatophore transfer. However,
spermatophore size will decrease with subsequent mating by a given male.

Isolating Virgins

To isolate virgin beetles, place a mated female in a 35mm Petri dish (for
example, Falcon 351008, Fisher Scientific 08-757-100A or 60mm dish, Carolina
Biological 741246) with a single layer of beans. The vast majority of females in a
stock culture will have mated and are capable of laying
fertile eggs. After 12-24 hours, females will begin to lay
eggs on beans. With an excess of beans, females will
lay only a single egg on each bean. Finding eggs on
beans can be facilitated by using a dissection
microscope (at 10x total magnification such as Carolina
Biological 593290, VWR 15147-844 or Fisher Scientific
S94912) or a large magnifying glass (2.0x magnifier
Carolina Biological 602106 or 2.5x magnifier Fisher
Scientific 14-648-19 or VWR 62379-535). Remove the
beans with single eggs (Figure 8) and place each bean
in an individual 35mm Petri dish or the well of a 6 or 12-
well flat bottom tissue culture well plate (tissue culture
plates, Fisher Scientific or Carolina Biological 703466-703467). Replenish the
beans as they are removed. A single female can produce more than 100 eggs in
her lifetime. In general, the sex ratio is 1:1.

11

Disposal of Cultures

Bean beetles are a potential agricultural pest insect that is not distributed
throughout the United States and Canada. This species is absent in much of
North America because it is intolerant to freezing temperatures and suitable host
plant species are not among our native (non-agricultural) flora. None-the-less, it
is prudent and appropriate to dispose of living adults, and beans that have had
contact with living adults, in a manner that will prevent their release to the natural
environment. The USDA requires that live cultures and beans exposed to live
adult beetles be placing in a freezer (0°C) for a minimum of 72 hours (3 days)
prior to disposal to ensure that beetles at every life cycle stage are dead. Then,
double bag and dispose the frozen culture in the same manner as food waste.

12

Literature Cited

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Dictionary of the Leguminosae. Volume 1. Plants and their Constitutents.
Chapman and Hall, London. 1180 pages.

Boeke, S.J., A.A.C. Sinzogan, R.P. de Almeida, P.W.M. de Boer, G. Jeong, D.K.

Kossou, and J.J.A. van Loon. 2003. Side-effects of cowpea treatment with
botanical insecticides on two parasitoids of Callosobruchus maculatus.

Entomologia Experimentalis et Applicata 108:43–51.

Brown, E.A, L. Gay, R. Vasudev, T. Tregenza, P.E. Eady, and D.J. Hosken.

2009. Negative phenotypic and genetic associations between copulation
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345

Brown, L. and J.F. Downhower. 1988. Analyses in Behavioral Ecology: A Manual

for Lab and Field. Sinauer Associates, 194 pages.

Chi, Y.H., J.-E. Ahn, D.-J. Yun, S.Y. Lee, T.-X. Liu, and K. Zhu-Salzman. 2011.

Changes in oxygen and carbon dioxide environment alter gene expression
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Devereau, A.D., I. Gudrups, J.H. Appleby, and P.F. Credland. 2003. Automatic,

rapid screening of seed resistance in cowpea, Vigna unguiculata (L.)
Walpers, to the seed beetle Callosobruchus maculatus (F.) (Coleoptera:
Bruchidae) using acoustic monitoring. Journal of Stored Products Research
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Eady, P.E. 1991. Sperm competition in Callosobruchus maculatus (Coleoptera:

Bruchedae): a comparison of two methods used to estimate paternity.
Ecological Entomology 16:45-53.

EOL. 2011. Callosobruchus maculatus (Fabricius, 1775)”. Encyclopedia of Life,

available from . Accessed 20 Mar 2011.

Fox, C.W., M.L. Bush, D.A. Roff, and W.G. Wallin. 2004. Evolutionary genetics of

lifespan and mortality rates in two populations of seed beetles,
Callosobruchus maculatus. Heredity 92:170-181.

Howe, R.W. and J.E. Currie. 1964. Some laboratory observations on the rates of

development, mortality and oviposition of several species of Bruchidae
breeding in stored pulses. Bulletin of Entomological Research. 55:437-477.

13

ITIS. 2011. Bruchinae Latreille, 1802 Integrated Taxonomic Information System.
ITIS Report accessed 20 March 2011.

Janzen, D.H. 1977. How southern cowpea weevil larvae (Bruchidae

Callosobruchus maculatus) die on non-host seeds. Ecology 58:921-927.

Janzen, D.H., H.B. Juster, and I.E. Liener. 1976. Insecticidal action of the

phytohemagglutinin in black beans on a bruchid beetle. Science 192:795-
796.

Kergoat, G.J., J.-F. Silvain , A. Delobel , M. Tuda, and K.-W. Anton. 2007.

Defining the limits of taxonomic conservatism in host–plant use for
phytophagous insects: Molecular systematics and evolution of host–plant
associations in the seed-beetle genus Bruchus Linnaeus (Coleoptera:
Chrysomelidae: Bruchinae). Molecular Phylogenetics and Evolution 43
(2007):251–269.

Patel, N.H., B.G. Condron, and K. Zinn. 1994. Pair-rule expression patterns of
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367:429-434.

Schoof, H.F. 1941. The effects of various relative humidities on the life proceses
of the southern cowpea weevil, Callosobruchus maculatus (Fabr.) at 30 C.,
+/- 0.8 degrees. Ecology 22(3):297-305.

Tuda, M., J. Rönn, S. Buranapanichpan, N. Wasano, and G. Arnqvist. 2006,

Evolutionary diversification of the bean beetle genus Callosobruchus
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at

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14

Yadav, J.S. 1971. Karyological studies on the three species of Bruchidae
(Coleoptera). Caryologia 24(2):157-166.

2

>Sheet

1 Navy beans Black beans Bean # # eggs

Bean # # eggs

1 1 1 1
2 1 2 2
3

1 3 1
4

1 4 1
5

1 5 1
6

1 6 1
7

2 7 1
8

1 8 1
9

2 9

0 10

1 10 1
11

0 11 1
12

2 12 1
13

1 13 2
14

0 14 1
15

1 15 1
16

2 16 1
17

1 17 2
18

1 18 2
19

1 19 2
20

1 20 2
21

0 21 2
22

0 22 1
23

1 23 0
24

1 24 1
25

0 25 1
26

1 26 1
27

0 27 1
28

1 28 0
29

0 29 1
30

1 30 1
31

0 31 1
32

0 32 0
33

0 33 0
34

0 34 1
35

0 35 2
36

1 36 0
37

0 37 0
38

0 38 0
39

0 39 1
40

0 40 0
41

1 41 0
42

0 42 0
43

0 43 0
44

0 44 0
45

0 45 0
46

0 46 0
47

0 47 0
48

0 48 0
49

0 49 0
50

0 50 1
51

0 51 0
52

0 52 0
53

0 53 0
54

0 54 0
55

0 55 0
56

0 56 0
57

0 57 0
58

0 58 0
59

0 59 0
60

0 60 0
61

0 61 0
62

0 62 0
63

0 63 1
64

0 64 0
65

0 65 0
66

0 66 0
67

0 67 0
68

1 68 1
69

1 69 0
70

0 70 0
71

0 71 1
72

1 72 0
73

1 73 0
74

2 74 0
75

0 75 0
76

0 76 0
77

0 77 1
78

0 78 1
79

0 79 0
80

0 80 0
81

0 81 0
82

0 82 0
83

0 83 0
84

0 84 0
85

0 85 0
86

0 86 0
87

0 87 0
88

0 88 0
89

0 89 0
90

0 90 0
91

0 91 0
92

0 92 0
93

0 93 0
94

0 94 0
95

0 95 0
96

0 96 0
97

0 97 0
98

0 98 0
99

0 99 0
100

0 100 0
101

0 101 0
102

0 102 0
103

0 103 0
104

0 104 0
105

0 105 0
106

0 106 0
107

0 107 0
108

0 108 0
109

0 109 0
110

0 110 0
111

0 111 0
112

0 112 0
113

0 113 0
114

0 114 0
115

0 115 0
116

0 116 0
117

0 117 0
118

0 118 0
119

0 119 0
120

0 120 0
121

1 121 2
122

1 122 1
123

2 123 1
124

2 124 1
125

2 125 0
126

2 126 1
127

2 127 2
128

1 128 3
129

1 129 2
130

2 130 2
131

1 131 1
132

2 132 1
133

2 133 1
134

1 134 1
135

1 135 2
136

1 136 1
137

1 137 2
138

1 138 1
139

2 139 2
140

1 140 1
141

1 141 1
142

2 142 2
143

2 143 2
144

2 144 1
145

1 145 1
146

2 146 2
147

3 147 2
148

2 148 1
149

1 149 3
150

1 150 1
151

0 151 0
152

0 152 0
153

0 153 0
154

0 154 0
155

0 155 0
156

0 156 0
157

0 157 0
158

0 158 0
159

0 159 0
160

0 160 0
161

0 161 0
162

0 162 0
163

0 163 0
164

0 164 0
165

0 165 0
166

0 166 0
167

0 167 0
168

0 168 0
169

0 169 0
170

0 170 0
171

0 171 0
172

0 172 0
173

0 173 0
174

0 174 0
175

0 175 0
176

0 176 0
177

0 177 0
178

0 178 0
179

0 179 0
180

0 180 0
181

3 181 2
182

2 182 2
183

2 183 2
184

3 184 1
185

2 185 1
186

2 186 4
187

1 187 1
188

1 188 1
189

1 189 1
190

2 190 2
191

3 191 2
192

1 192 1
193

2 193 2
194

1 194 1
195

2 195 1
196

2 196 2
197

1 197 1
198

0 198 1
199

3 199 1
200

2 200 2
201

1 201 3
202

2 202 2
203

2 203 2
204

1 204 1
205

3 205 1
206

2 206 2
207

3 207 1
208

2 208 2
209

4 209 1
210

2 210 3
211

2 211 2
212

3 212 2
213

1 213 4
214

2 214 3
215

4 215 3
216

2 216 2
217

2 217 2
218

3 218 2
219

2 219 2
220

2 220 3
221

2 221 2
222

2 222 3
223

4 223 4
224

3 224 1
225

1 225 2
226

2 226 1
227

2 227 2
228

2 228 3
229

3 229 3
230

4 230 3
231

2 231 3
232

3 232 4
233

2 233 0
234

2 234 2
235

2 235 2
236

1 236 2
237

3 237 3
238

3 238 2
239

2 239 2
240

1 240 2
241

0 241 1
242

1 242 1
243

0 243 0
244

0 244 1
245

0 245 0
246

0 246 0
247

0 247 1
248

1 248 0
249

1 249 1
250

1 250 0
251

1 251 0
252

1 252 0
253

1 253 0
254

1 254 0
255

1 255 0
256

1 256 0
257

0 257 0
258

1 258 0
259

0 259 0
260

1 260 0
261

1 261 0
262

2 262 0
263

0 263 0
264

0 264 0
265

1 265 0
266

1 266 1
267

1 267 0
268

1 268 0
269

1 269 1
270

1 270 0
271

0 271 0
272

0 272 0
273

0 273 0
274

0 274 0
275

0 275 0
276

0 276 0
277

0 277 0
278

0 278 0
279

0 279 0
280

0 280 0
281

0 281 0
282

0 282 0
283

0 283 0
284

0 284 0
285

0 285 0
286

0 286 0
287

0 287 0
288

0 288 0
289

0 289 0
290

0 290 0
291

0 291 0
292

0 292 0
293

0 293 0
294

0 294 0
295

0 295 0
296

0 296 0
297

0 297 0
298

0 298 0
299

0 299 0
300

0 300 0
Observed

229 216
Expected 222.5 Chi-square 0.1898876404

0.1898876404
0.3797752809

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 1 1 1 1 1 1 2 1 2 1 0 2 1 0 1 2 1 1 1 1 0 0 1 1 0 1 0 1 0 1 0 0 0 0 0 1 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 1 1 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 2 2 2 2 2 1 1 2 1 2 2 1 1 1 1 1 2 1 1 2 2 2 1 2 3 2 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3 2 2 3 2 2 1 1 1 2 3 1 2 1 2 2 1 0 3 2 1 2 2 1 3 2 3 2 4 2 2 3 1 2 4 2 2 3 2 2 2 2 4 3 1 2 2 2 3 4 2 3 2 2 2 1 3 3 2 1 0 1 0 0 0 0 0 1 1 1 1 1 1 1 1 1 0 1 0 1 1 2 0 0 1 1 1 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

# navybeans

#eggs

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300 1 2 1 1 1 1 1 1 0 1 1 1 2 1 1 1 2 2 2 2 2 1 0 1 1 1 1 0 1 1 1 0 0 1 2 0 0 0 1 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 1 0 0 1 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 1 1 1 0 1 2 3 2 2 1 1 1 1 2 1 2 1 2 1 1 2 2 1 1 2 2 1 3 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 2 2 1 1 4 1 1 1 2 2 1 2 1 1 2 1 1 1 2 3 2 2 1 1 2 1 2 1 3 2 2 4 3 3 2 2 2 2 3 2 3 4 1 2 1 2 3 3 3 3 4 0 2 2 2 3 2 2 2 1 1 0 1 0 0 1 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

# black beans

# eggs