Boxelder bug females emerged from overwintering sites in the spring and rapidly provisioned eggs with yolk materials. Five discrete egg stages were identified based on egg size, protein content, and degree of chorion sclerotization. Females did not accumulate yolk materials into the egg until after melanization was completed, as unmelanized animals rarely possessed even stage 2 eggs. All adult females entering overwintering sites possessed only immature stage eggs (stage 1 and 2). The rate of egg vitellogenesis in the spring was rapid; a major change in numbers of more mature stage eggs (stage 3 and above) in the ovary occurred within approximately 6 days. Most mating pairs recovered in the field (92%, 12/13) possessed ovaries full of eggs in stages 3, 4 or 5. The remaining female contained only immature eggs of stage 1 and 2. This finding indicates that fully provisioned ovaries are not an absolute requirement for mating to occur. The signals that initiate vitellogenesis and control the movement of materials from fat body into eggs are unknown for the boxelder bug

Grimnes, Karin A

Miller, Deborah

Wyman, Aaron J

English

ValpoScholar

The Great Lakes Entomologist 
Volume 36 
Numbers 3 & 4 - Fall/Winter 2003 Numbers 3 & 
4 - Fall/Winter 2003 
Article 8 
October 2003 
Egg Production in the Boxelder Bug Boisea Trivittata (Hemiptera: 
Rhopalidae) 
Karin A. Grimnes 
Alma College 
Deborah Miller 
Alma College 
Aaron J. Wyman 
Alma College 
Follow this and additional works at: https://scholar.valpo.edu/tgle 
 Part of the Entomology Commons 
Recommended Citation 
Grimnes, Karin A.; Miller, Deborah; and Wyman, Aaron J. 2003. "Egg Production in the Boxelder Bug Boisea 
Trivittata (Hemiptera: Rhopalidae)," The Great Lakes Entomologist, vol 36 (2) 
Available at: https://scholar.valpo.edu/tgle/vol36/iss2/8 
This Peer-Review Article is brought to you for free and open access by the Department of Biology at ValpoScholar. 
It has been accepted for inclusion in The Great Lakes Entomologist by an authorized administrator of ValpoScholar. 
For more information, please contact a ValpoScholar staff member at scholar@valpo.edu. 
160 THE GREAT LAKES ENTOMOLOGIST Vol. 36, Nos. 3 & 4
EGG PRODUCTION IN THE BOXELDER BUG BOISEA TRIVITTATA
(HEMIPTERA: RHOPALIDAE)
Karin A. Grimnes1, Deborah Miller1 and Aaron J. Wyman1
ABSTRACT
Boxelder bug females emerged from overwintering sites in the spring and
rapidly provisioned eggs with yolk materials. Five discrete egg stages were
identified based on egg size, protein content, and degree of chorion sclerotiza-
tion. Females did not accumulate yolk materials into the egg until after mela-
nization was completed, as unmelanized animals rarely possessed even stage 2
eggs. All adult females entering overwintering sites possessed only immature
stage eggs (stage 1 and 2). The rate of egg vitellogenesis in the spring was rapid;
a major change in numbers of more mature stage eggs (stage 3 and above) in the
ovary occurred within approximately 6 days. Most mating pairs recovered in the
field (92%, 12/13) possessed ovaries full of eggs in stages 3, 4 or 5. The remain-
ing female contained only immature eggs of stage 1 and 2. This finding indi-
cates that fully provisioned ovaries are not an absolute requirement for mating
to occur. The signals that initiate vitellogenesis and control the movement of
materials from fat body into eggs are unknown for the boxelder bug.
____________________
INTRODUCTION
Boisea trivittata (Say) is considered a pest species whose primary impact
is one of nuisance to the homeowner when the aggregating bug swarms reach
huge proportions. These insects usually do not cause economic damage, al-
though they occasionally damage fruit such as strawberries (see Wheeler 1982).
The boxelder bug can be univotine or bivoltine, depending on prevailing
weather patterns. Even bivoltine populations can fail to mature a fall genera-
tion if the year is not warm enough for insects to enter overwintering sites as
adults (Yoder and Robinson 1990). Midwestern populations appear to have
predictable seasonal behavior (as summarized below from Smith and Shepherd
1937 and Tinker 1952). Boxelder bugs emerge in spring (March and April) and
feed on available foliage as well as Acer negundo L. buds and stems. Mating
occurs on warm spring days in April and May. Eggs are laid in clusters on
substrates such as young leaves, bark and man-made structures in April and
May and again in June and July. Nymphs occur between approximately May
and Oct, with adults present in June and July and again in September until the
hard frost dates in October that kill unprotected insects.
Although seasonal occurrence of eggs has been tracked for various popula-
tions, reports have been limited to observations of eggs laid on substrates (Smith
and Shephard 1937, Tinker 1952, Yoder and Robinson 1990). Descriptions of
ovarian anatomy (Payne 1936, Woolley 1949) mention the presence of eggs, but
do not quantify them in any way. Identification of discrete stages in the egg
provisioning process is needed to determine seasonal development, as is the
relationship between melanization and egg provisioning during movement of
resources from the fat body to the ovary.
1Department of Biology, Alma College, Alma, MI 48801.
1
Grimnes et al.: Egg Production in the Boxelder Bug Boisea Trivittata (Hemi
Published by ValpoScholar, 2003
2003 THE GREAT LAKES ENTOMOLOGIST 161
In this paper, we identify discrete stages of the egg provisioning process
and examine the relationship between egg provisioning and melanization. A
thorough understanding of these processes are necessary to characterize sea-
sonal egg development in the boxelder bug.
MATERIALS AND METHODS
The study site was adjacent to Lang’s Veterinary Clinic (T12N, R3W, Sec.
29, Pine River Township) in Gratiot County, Michigan where a large population
of boxelder bugs was established. This site contains numerous pistillate boxelder
trees (Acer negundo). The initial collections were made during the active sea-
sons of 1990 through 1992 and supplemented during 1994 and 1999. Insects
were collected and preserved in 70% ethanol. Live boxelder bugs also were
collected and transported to the laboratory. These bugs were maintained at
room temperature (27 ± 2°C) in 150 mm Petri dishes with boxelder tree leaves
inserted into tubing filled with 10% maple syrup (V/V) diluted with water
(Bouldrey and Grimnes 1995).
Mature preserved field-collected females were dissected in 70% ethanol,
and the eggs were separated from ovarian tissue. Egg length and width were
measured with an ocular micrometer and notes on coloration, opacity, and chorion
formation were made. We knew that dealing with tissue in alcohol would give us
slightly smaller values than fresh material, but we felt it was necessary to allow
eventual analysis of previously preserved insect samples.
The protein content of eggs was determined using the Lowry Assay (Lowry
et al. 1951). Individual eggs (N = 25 per stage) were homogenized in 0.5 ml
water using a glass homogenizer and the Lowry Assay was performed immedi-
ately. Bovine serum albumen (BSA) was used as the protein standard.
For the melanization study, newly eclosed adults (completely bright or-
ange) were collected daily from the field or from laboratory populations of fifth
instar animals. They were maintained at 27 ± 2°C in the lab and observed
hourly. Pictures were drawn noting the changes that occurred as the animals
darkened and hardened. Approximate duration of the process was estimated by
combining data from identically staged animals.
Analysis of variance was performed on the data using the JMP statistical
program (SAS Institute 1998). Comparison of means was accomplished with
the Tukey-Kramer’s HSD statistic.
RESULTS AND DISCUSSION
Egg stage characterization. We were able to visually differentiate five
stages of eggs from mature females on the basis of color, shape and the presence
and degree of chorion sclerotization (Table 1). Eggs laid on substrates were
labeled as stage 6 eggs. Eggs changed shape from round to oval, changed color
from translucent white to milky white indicating accumulation of yolk materi-
als, and began/completed chorion formation and tanning. Although the chorion
appeared brown in the alcohol-preserved specimens, our observations of freshly
laid eggs indicated the true color at laying was a light tan color that darkened
progressively to red as noted in other studies (Smith and Shepherd 1937, Yoder
and Robinson 1990).
An ANOVA was performed on egg length and width for all stages (Table
1). Egg length varied significantly with egg stage; however, all pair-wise com-
parisons with Tukey-Kramer HSD revealed that stage 3, 4 and 5 egg lengths
were not significantly different from each other. A similar (but not identical)
pattern was noted for width which was significant across stages overall; al-
though stage 3 and 4 eggs overlapped in size, stage 5 eggs were significantly
narrower than either stage 3 or 4 eggs. The correlation between egg length and
2
The Great Lakes Entomologist, Vol. 36, No. 2 [2003], Art. 8
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162 THE GREAT LAKES ENTOMOLOGIST Vol. 36, Nos. 3 & 4
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3
Grimnes et al.: Egg Production in the Boxelder Bug Boisea Trivittata (Hemi
Published by ValpoScholar, 2003
2003 THE GREAT LAKES ENTOMOLOGIST 163
width was substantial in linear regression analysis (Fig. 1, regression formula of
egg width = 0.14 + 0.51 × (egg length), r2 of 0.89). When all egg stages were
graphed together, stage 2 eggs formed a discrete collection of data points and
there were no intermediates between stage 2 and stage 3 eggs. This suggests that
the process of vitellogenesis and egg material uptake occurs very rapidly. In
addition, stage 3 and 4 eggs were somewhat variable and overlapped in size.
However, stage 5 eggs consistently were located in the center of the general “cloud”
of stage 3 and 4 eggs. As the chorion hardened and darkened in stage 5, the eggs
became compressed, possibly by the elimination of water because protein levels
still were increasing between stage 4 and 5 (data given below). Eggs laid on
substrates (stage 6) were not significantly different in size from stage 5 eggs. No
attempt was made to determine fertilization rates in this study.
Protein content analysis. Analysis of protein levels for eggs at all stages
was performed with the Lowry Assay. This assay detects free amino acids after
digestion, but does not hydrolyze proteins that have been cross-linked into ei-
ther chorion or the exoskeleton of the developing embryo. Preliminary data
indicated that levels of protein found in stage 1 eggs (under 1 µg/egg) were too
low to measure reliably so this stage was not analyzed fully. An ANOVA re-
vealed that protein content increased from stage 2 eggs to stage 5 eggs (Table 1).
Stage 6 (laid eggs) had a slightly higher protein content but this value was not
significantly different from stage 5 protein levels. Stage 3 and 4 eggs over-
lapped in detectable protein in the Lowry Assay.
Protein levels increased as egg sizes (and provisioning rates) declined
between stage 3 and stage 5 eggs. Thus, it is possible that non-protein nitrogen
sources already present in stage 3 eggs were being incorporated into amino
acids/proteins detectable by the Lowry Assay as the eggs progressed to stage 5.
Figure 1. Boisea trivittata egg sizes identified by stage (N = 25 for each stage)
4
The Great Lakes Entomologist, Vol. 36, No. 2 [2003], Art. 8
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164 THE GREAT LAKES ENTOMOLOGIST Vol. 36, Nos. 3 & 4
In addition, general increase in protein synthesis during these stages must
outpace proteins lost into chorion formation. Hatchling protein content dropped
below stage 6 by 30% (data not shown), possibly due to protein digestion or to
cross-linking of proteins during exoskeleton formation.
Melanization study. Newly molted adults progress through a series of
identifiable stages before becoming completely melanized and indistinguish-
able from older adult animals. The scale began with non-melanized newly eclosed
adults that possessed orange bodies and incompletely expanded wings. The
next steps (in order) were the full expansion of wings, the appearance of a ring
around the ocellar spot, body coloration darkening from orange to brown to
black, ventral head area fully darkened, and the sub-buccal area becoming fully
darkened. Smith and Shephard (1937) noted which areas of the bugs became
melanized, but no order for the process was given.
Newly molted boxelder bugs (N = 6) progressed to full melanization in
approximately 5-6 hours (at 27 ± 2°C). Thus, any field-collected insect that
lacked fully darkened coloration on the underside of the head was considered
incompletely melanized (molted within the past 24 hours). Animals collected in
the last stage prior to full melanization were dissected, and their eggs were
classified as to stage and size. Most eggs were in stage 1 of development. Of the
66 insects analyzed, only 3% (2/66) contained any stage 2 eggs, which consti-
tuted about 1% (7/676) of the total egg number. These data indicate that mela-
nization occurred prior to egg provisioning.
Egg kinetics. Preliminary seasonal analysis of egg stages indicated that
boxelder bugs approaching overwintering (September and October collection
dates, N = 10 for each date) contained only stage 1 eggs (100%) and had abun-
dant fat body stores when dissected. Animals emerging during early spring had
depleted fat body stores (N = 10) and no eggs in advanced egg stages of develop-
ment (stage 3 or beyond). Numbers of more mature eggs (stages 3, 4 and 5)
substantially increased within females collected 5-7 days later (N = 10), ap-
proximately three weeks after the first spring emergence dates. Females at this
time contained abundant fat body material, presumably due to spring feeding.
The mobilization of resources from the fat body to the ovary is by inference; the
vitellogenesis process has not yet been characterized.
Mating pairs collected in the field were analyzed for presence of vitellogenic
eggs at stage 3 or greater. Of twelve mating pairs thus analyzed, 11 females
(92%) had advanced eggs; the remaining female possessed only stage 1 and 2
eggs. Although these data suggest that fully provisioned eggs are not an abso-
lute requirement for the mating process, the precise relationship between mat-
ing and onset of vitellogenesis has yet to be determined.
ACKNOWLEDGEMENTS
The authors acknowledge the additional contributions of former students
Jennifer Jarrard and Matt O’Dell as well as collaborator Ken Kobylarz for vari-
ous versions of the melanization scale. We are grateful to colleagues Mark
Oemke (Alma College) and Christina Krupp (University of Vermont), as well as
the journal reviewers for helpful editorial comments. This work was partially
supported by a grant from the Kellogg Foundation entitled: The Science Teacher
Preparation Project.
LITERATURE CITED
Bouldrey, S. M. and K. A. Grimnes. 1995. An allometric study of the boxelder bug,
Boisea trivittata (Heteroptera:Rhopalidae). Great Lakes Entomol. 28: 207-212.
Lowry, O. H., N. J. Rosebrough, A. L. Farr and R. J. Randall. 1951. Protein measure-
ment with the folin phenol reagent. J. Biol. Chem. 193: 265-275.
5
Grimnes et al.: Egg Production in the Boxelder Bug Boisea Trivittata (Hemi
Published by ValpoScholar, 2003
2003 THE GREAT LAKES ENTOMOLOGIST 165
Payne, M. A. 1936. Intravitam studies on the Hemipteron (sic), Leptocoris trivittatus.
B. Description of the female reproductive organs. Zool. Jahrb. Abt. Anat. Ontog.
61: 45-50.
SAS Institute. 1998. JMP User’s manual, version 3.1. SAS Institute, Cary, NC, USA.
Smith, R. C. and B. L. Shepherd. 1937. The life history and control of the boxelder bug
in Kansas. Trans. Kansas Acad. Sci. 40: 143-159.
Tinker, M. E. 1952. The seasonal behavior and ecology of the boxelder bug Leptocoris
trivittatus in Minnesota. Ecology 33: 407-414.
Wheeler, A. G. 1982. Bed bugs and other bugs. pp. 313-345, In A. Mallis, (ed.) Hand-
book of Pest Control. Franzak and Foster Co., Cleveland, Ohio.
Woolley, T. A. 1949. Studies on the internal anatomy of the box elder bug, Leptocoris
trivittatus (Say). Ann. Entomol. Soc. Am. 42: 203-226.
Yoder, K. M. and W. H. Robinson. 1990. Seasonal abundance and habits of the boxelder
bug, Boisea trivittata (Say), in an urban environment. Proc. Entomol. Soc. Wash-
ington. 92: 802-807.
6
The Great Lakes Entomologist, Vol. 36, No. 2 [2003], Art. 8
https://scholar.valpo.edu/tgle/vol36/iss2/8

Egg Production in the Boxelder Bug \u3ci\u3eBoisea Trivittata\u3c/i\u3e (Hemiptera: Rhopalidae)

Valparaiso University

The Great Lakes Entomologist Volume 36 Numbers 3 & 4 - Fall/Winter 2003 Numbers 3 & 4 - Fall/Winter 2003 Article 8 October 2003 Egg Production in the Boxelder Bug Boisea Trivittata (Hemiptera: Rhopalidae) Karin A. Grimnes Alma College Deborah Miller Alma College Aaron J. Wyman Alma College Follow this and additional works at: https://scholar.valpo.edu/tgle Part of the Entomology Commons Recommended Citation Grimnes, Karin A.; Miller, Deborah; and Wyman, Aaron J. 2003. "Egg Production in the Boxelder Bug Boisea Trivittata (Hemiptera: Rhopalidae)," The Great Lakes Entomologist, vol 36 (2) Available at: https://scholar.valpo.edu/tgle/vol36/iss2/8 This Peer-Review Article is brought to you for free and open access by the Department of Biology at ValpoScholar. It has been accepted for inclusion in The Great Lakes Entomologist by an authorized administrator of ValpoScholar. For more information, please contact a ValpoScholar staff member at scholar@valpo.edu. 160 THE GREAT LAKES ENTOMOLOGIST Vol. 36, Nos. 3 & 4EGG PRODUCTION IN THE BOXELDER BUG BOISEA TRIVITTATA(HEMIPTERA: RHOPALIDAE)Karin A. Grimnes1, Deborah Miller1 and Aaron J. Wyman1ABSTRACTBoxelder bug females emerged from overwintering sites in the spring andrapidly provisioned eggs with yolk materials. Five discrete egg stages wereidentified based on egg size, protein content, and degree of chorion sclerotiza-tion. Females did not accumulate yolk materials into the egg until after mela-nization was completed, as unmelanized animals rarely possessed even stage 2eggs. All adult females entering overwintering sites possessed only immaturestage eggs (stage 1 and 2). The rate of egg vitellogenesis in the spring was rapid;a major change in numbers of more mature stage eggs (stage 3 and above) in theovary occurred within approximately 6 days. Most mating pairs recovered in thefield (92%, 12/13) possessed ovaries full of eggs in stages 3, 4 or 5. The remain-ing female contained only immature eggs of stage 1 and 2. This finding indi-cates that fully provisioned ovaries are not an absolute requirement for matingto occur. The signals that initiate vitellogenesis and control the movement ofmaterials from fat body into eggs are unknown for the boxelder bug.____________________INTRODUCTIONBoisea trivittata (Say) is considered a pest species whose primary impactis one of nuisance to the homeowner when the aggregating bug swarms reachhuge proportions. These insects usually do not cause economic damage, al-though they occasionally damage fruit such as strawberries (see Wheeler 1982).The boxelder bug can be univotine or bivoltine, depending on prevailingweather patterns. Even bivoltine populations can fail to mature a fall genera-tion if the year is not warm enough for insects to enter overwintering sites asadults (Yoder and Robinson 1990). Midwestern populations appear to havepredictable seasonal behavior (as summarized below from Smith and Shepherd1937 and Tinker 1952). Boxelder bugs emerge in spring (March and April) andfeed on available foliage as well as Acer negundo L. buds and stems. Matingoccurs on warm spring days in April and May. Eggs are laid in clusters onsubstrates such as young leaves, bark and man-made structures in April andMay and again in June and July. Nymphs occur between approximately Mayand Oct, with adults present in June and July and again in September until thehard frost dates in October that kill unprotected insects.Although seasonal occurrence of eggs has been tracked for various popula-tions, reports have been limited to observations of eggs laid on substrates (Smithand Shephard 1937, Tinker 1952, Yoder and Robinson 1990). Descriptions ofovarian anatomy (Payne 1936, Woolley 1949) mention the presence of eggs, butdo not quantify them in any way. Identification of discrete stages in the eggprovisioning process is needed to determine seasonal development, as is therelationship between melanization and egg provisioning during movement ofresources from the fat body to the ovary.1Department of Biology, Alma College, Alma, MI 48801.1Grimnes et al.: Egg Production in the Boxelder Bug Boisea Trivittata (HemiPublished by ValpoScholar, 20032003 THE GREAT LAKES ENTOMOLOGIST 161In this paper, we identify discrete stages of the egg provisioning processand examine the relationship between egg provisioning and melanization. Athorough understanding of these processes are necessary to characterize sea-sonal egg development in the boxelder bug.MATERIALS AND METHODSThe study site was adjacent to Lang’s Veterinary Clinic (T12N, R3W, Sec.29, Pine River Township) in Gratiot County, Michigan where a large populationof boxelder bugs was established. This site contains numerous pistillate boxeldertrees (Acer negundo). The initial collections were made during the active sea-sons of 1990 through 1992 and supplemented during 1994 and 1999. Insectswere collected and preserved in 70% ethanol. Live boxelder bugs also werecollected and transported to the laboratory. These bugs were maintained atroom temperature (27 ± 2°C) in 150 mm Petri dishes with boxelder tree leavesinserted into tubing filled with 10% maple syrup (V/V) diluted with water(Bouldrey and Grimnes 1995).Mature preserved field-collected females were dissected in 70% ethanol,and the eggs were separated from ovarian tissue. Egg length and width weremeasured with an ocular micrometer and notes on coloration, opacity, and chorionformation were made. We knew that dealing with tissue in alcohol would give usslightly smaller values than fresh material, but we felt it was necessary to alloweventual analysis of previously preserved insect samples.The protein content of eggs was determined using the Lowry Assay (Lowryet al. 1951). Individual eggs (N = 25 per stage) were homogenized in 0.5 mlwater using a glass homogenizer and the Lowry Assay was performed immedi-ately. Bovine serum albumen (BSA) was used as the protein standard.For the melanization study, newly eclosed adults (completely bright or-ange) were collected daily from the field or from laboratory populations of fifthinstar animals. They were maintained at 27 ± 2°C in the lab and observedhourly. Pictures were drawn noting the changes that occurred as the animalsdarkened and hardened. Approximate duration of the process was estimated bycombining data from identically staged animals.Analysis of variance was performed on the data using the JMP statisticalprogram (SAS Institute 1998). Comparison of means was accomplished withthe Tukey-Kramer’s HSD statistic.RESULTS AND DISCUSSIONEgg stage characterization. We were able to visually differentiate fivestages of eggs from mature females on the basis of color, shape and the presenceand degree of chorion sclerotization (Table 1). Eggs laid on substrates werelabeled as stage 6 eggs. Eggs changed shape from round to oval, changed colorfrom translucent white to milky white indicating accumulation of yolk materi-als, and began/completed chorion formation and tanning. Although the chorionappeared brown in the alcohol-preserved specimens, our observations of freshlylaid eggs indicated the true color at laying was a light tan color that darkenedprogressively to red as noted in other studies (Smith and Shepherd 1937, Yoderand Robinson 1990).An ANOVA was performed on egg length and width for all stages (Table1). Egg length varied significantly with egg stage; however, all pair-wise com-parisons with Tukey-Kramer HSD revealed that stage 3, 4 and 5 egg lengthswere not significantly different from each other. A similar (but not identical)pattern was noted for width which was significant across stages overall; al-though stage 3 and 4 eggs overlapped in size, stage 5 eggs were significantlynarrower than either stage 3 or 4 eggs. The correlation between egg length and2The Great Lakes Entomologist, Vol. 36, No. 2 [2003], Art. 8https://scholar.valpo.edu/tgle/vol36/iss2/8162 THE GREAT LAKES ENTOMOLOGIST Vol. 36, Nos. 3 & 4Table 1. Characteristics of Egg Stages in Boisea trivittata using JMP analysis (Mean ± SE)Egg Stage1Egg Length (µm)Egg Width (µm)Protein content (µg/egg)Description of egg stage1308 ± 64a284 ± 62aNot Determinedsmall, round, translucent, non-vitellogenic2806 ± 179b560 ± 112b8.4 ± 1.1asquare-shaped, white, non-vitellogenic31632 ± 130c996 ± 105c21.5 ± 1.1boval, white, vitellogenic41568 ± 69c994 ± 58c28.7 ± 1.8bchorion light tan, subtle hatch line51590 ± 63c926 ± 30d68.6 ± 4.0cchorion dark brown, obvious hatch line61556 ± 8c2907 ± 13d276.8 ± 1.8claid eggs taken from Acer negundo leaves1 N = 25 eggs/stage unless otherwise noted; means sharing a letter within a column are not significantly different at P < 0.05 in theTukey-Kramer HSD test.2 N = 15 eggs/stage3Grimnes et al.: Egg Production in the Boxelder Bug Boisea Trivittata (HemiPublished by ValpoScholar, 20032003 THE GREAT LAKES ENTOMOLOGIST 163width was substantial in linear regression analysis (Fig. 1, regression formula ofegg width = 0.14 + 0.51 × (egg length), r2 of 0.89). When all egg stages weregraphed together, stage 2 eggs formed a discrete collection of data points andthere were no intermediates between stage 2 and stage 3 eggs. This suggests thatthe process of vitellogenesis and egg material uptake occurs very rapidly. Inaddition, stage 3 and 4 eggs were somewhat variable and overlapped in size.However, stage 5 eggs consistently were located in the center of the general “cloud”of stage 3 and 4 eggs. As the chorion hardened and darkened in stage 5, the eggsbecame compressed, possibly by the elimination of water because protein levelsstill were increasing between stage 4 and 5 (data given below). Eggs laid onsubstrates (stage 6) were not significantly different in size from stage 5 eggs. Noattempt was made to determine fertilization rates in this study.Protein content analysis. Analysis of protein levels for eggs at all stageswas performed with the Lowry Assay. This assay detects free amino acids afterdigestion, but does not hydrolyze proteins that have been cross-linked into ei-ther chorion or the exoskeleton of the developing embryo. Preliminary dataindicated that levels of protein found in stage 1 eggs (under 1 µg/egg) were toolow to measure reliably so this stage was not analyzed fully. An ANOVA re-vealed that protein content increased from stage 2 eggs to stage 5 eggs (Table 1).Stage 6 (laid eggs) had a slightly higher protein content but this value was notsignificantly different from stage 5 protein levels. Stage 3 and 4 eggs over-lapped in detectable protein in the Lowry Assay.Protein levels increased as egg sizes (and provisioning rates) declinedbetween stage 3 and stage 5 eggs. Thus, it is possible that non-protein nitrogensources already present in stage 3 eggs were being incorporated into aminoacids/proteins detectable by the Lowry Assay as the eggs progressed to stage 5.Figure 1. Boisea trivittata egg sizes identified by stage (N = 25 for each stage)4The Great Lakes Entomologist, Vol. 36, No. 2 [2003], Art. 8https://scholar.valpo.edu/tgle/vol36/iss2/8164 THE GREAT LAKES ENTOMOLOGIST Vol. 36, Nos. 3 & 4In addition, general increase in protein synthesis during these stages mustoutpace proteins lost into chorion formation. Hatchling protein content droppedbelow stage 6 by 30% (data not shown), possibly due to protein digestion or tocross-linking of proteins during exoskeleton formation.Melanization study. Newly molted adults progress through a series ofidentifiable stages before becoming completely melanized and indistinguish-able from older adult animals. The scale began with non-melanized newly eclosedadults that possessed orange bodies and incompletely expanded wings. Thenext steps (in order) were the full expansion of wings, the appearance of a ringaround the ocellar spot, body coloration darkening from orange to brown toblack, ventral head area fully darkened, and the sub-buccal area becoming fullydarkened. Smith and Shephard (1937) noted which areas of the bugs becamemelanized, but no order for the process was given.Newly molted boxelder bugs (N = 6) progressed to full melanization inapproximately 5-6 hours (at 27 ± 2°C). Thus, any field-collected insect thatlacked fully darkened coloration on the underside of the head was consideredincompletely melanized (molted within the past 24 hours). Animals collected inthe last stage prior to full melanization were dissected, and their eggs wereclassified as to stage and size. Most eggs were in stage 1 of development. Of the66 insects analyzed, only 3% (2/66) contained any stage 2 eggs, which consti-tuted about 1% (7/676) of the total egg number. These data indicate that mela-nization occurred prior to egg provisioning.Egg kinetics. Preliminary seasonal analysis of egg stages indicated thatboxelder bugs approaching overwintering (September and October collectiondates, N = 10 for each date) contained only stage 1 eggs (100%) and had abun-dant fat body stores when dissected. Animals emerging during early spring haddepleted fat body stores (N = 10) and no eggs in advanced egg stages of develop-ment (stage 3 or beyond). Numbers of more mature eggs (stages 3, 4 and 5)substantially increased within females collected 5-7 days later (N = 10), ap-proximately three weeks after the first spring emergence dates. Females at thistime contained abundant fat body material, presumably due to spring feeding.The mobilization of resources from the fat body to the ovary is by inference; thevitellogenesis process has not yet been characterized.Mating pairs collected in the field were analyzed for presence of vitellogeniceggs at stage 3 or greater. Of twelve mating pairs thus analyzed, 11 females(92%) had advanced eggs; the remaining female possessed only stage 1 and 2eggs. Although these data suggest that fully provisioned eggs are not an abso-lute requirement for the mating process, the precise relationship between mat-ing and onset of vitellogenesis has yet to be determined.ACKNOWLEDGEMENTSThe authors acknowledge the additional contributions of former studentsJennifer Jarrard and Matt O’Dell as well as collaborator Ken Kobylarz for vari-ous versions of the melanization scale. We are grateful to colleagues MarkOemke (Alma College) and Christina Krupp (University of Vermont), as well asthe journal reviewers for helpful editorial comments. This work was partiallysupported by a grant from the Kellogg Foundation entitled: The Science TeacherPreparation Project.LITERATURE CITEDBouldrey, S. M. and K. A. Grimnes. 1995. An allometric study of the boxelder bug,Boisea trivittata (Heteroptera:Rhopalidae). Great Lakes Entomol. 28: 207-212.Lowry, O. H., N. J. Rosebrough, A. L. Farr and R. J. Randall. 1951. Protein measure-ment with the folin phenol reagent. J. Biol. Chem. 193: 265-275.5Grimnes et al.: Egg Production in the Boxelder Bug Boisea Trivittata (HemiPublished by ValpoScholar, 20032003 THE GREAT LAKES ENTOMOLOGIST 165Payne, M. A. 1936. Intravitam studies on the Hemipteron (sic), Leptocoris trivittatus.B. Description of the female reproductive organs. Zool. Jahrb. Abt. Anat. Ontog.61: 45-50.SAS Institute. 1998. JMP User’s manual, version 3.1. SAS Institute, Cary, NC, USA.Smith, R. C. and B. L. Shepherd. 1937. The life history and control of the boxelder bugin Kansas. Trans. Kansas Acad. Sci. 40: 143-159.Tinker, M. E. 1952. The seasonal behavior and ecology of the boxelder bug Leptocoristrivittatus in Minnesota. Ecology 33: 407-414.Wheeler, A. G. 1982. Bed bugs and other bugs. pp. 313-345, In A. Mallis, (ed.) Hand-book of Pest Control. Franzak and Foster Co., Cleveland, Ohio.Woolley, T. A. 1949. Studies on the internal anatomy of the box elder bug, Leptocoristrivittatus (Say). Ann. Entomol. Soc. Am. 42: 203-226.Yoder, K. M. and W. H. Robinson. 1990. Seasonal abundance and habits of the boxelderbug, Boisea trivittata (Say), in an urban environment. Proc. Entomol. Soc. Wash-ington. 92: 802-807.6The Great Lakes Entomologist, Vol. 36, No. 2 [2003], Art. 8https://scholar.valpo.edu/tgle/vol36/iss2/8

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Egg Production in the Boxelder Bug \u3ci\u3eBoisea Trivittata\u3c/i\u3e (Hemiptera: Rhopalidae)

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