text:
|UFDC Home
|myUFDC Home | Help
This item has the following downloads:
|Full Text
LIFE HISTORY OF Triaspis eugenii Wharton and Lopez-Martinez (HYMENOPTERA:
CONTROL OF PEPPER WEEVIL Anthonomus eugenii Cano (COLEOPTERA:
Copyright 2006
Esteban Rodriguez-Leyva
This work is dedicated to my parents, Benito Rodriguez and Pilar Leyva, and brothers,
Martin, Ver6nica, and Victor, who have been teaching me the importance of having
resolutions in life, social gathering and education included, within an
admirable familial strength. Without their support it would be
harder to complete this study, to my nieces and nephew.
Este trabajo lo dedico a mis padres, Benito Rodriguez y Pilar Leyva, y a mis hermanos,
Martin, Ver6nica, y Victor, quienes me han mostrado la importancia de tener
metas en la vida, incluido el convivir y la educaci6n, en el seno de una
extraordinaria fuerza familiar. Sin su apoyo completar este trabajo
hubiese sido mas dificil. A mis sobrinas y sobrino.
Many people and institutions contributed to the success of this dissertation. I am
extremely grateful to Consejo Nacional de Ciencia y Tecnologia (National Council of
Science and Technology) and Colegio de Postgraduados from Mexico which supported
tuitions, living expenses, and laborer situations through all the process for getting the
goals of this study. My gratitude and appreciation go to my advisor, Dr. Philip A. Stansly,
he found resources for conducting and assisting my dissertation and gave me
independence to express ideas and objectives. Additionally, for the many hours that he
spent reading, correcting, and re-reading drafts of this dissertation. I am grateful to Dr.
David J. Schuster who offered encouragement, support, and always friendship. I would
like to recognize the rest of my committee, Drs. Norman C. Leppla, Ru Nguyen, and
Steven A. Sargent, for being available and offering experiences to improve my academic
development. Special thanks go to Dr. Susan Webb for allowing me to use resources in
her laboratory in Gainesville, and Mike Miller for being such an amiable person.
Special recognition goes to Debbie Hall, Myrna Litchfield, and Nancy Sanders for
providing constant support, always with professionalism and amiability.
I want to thank also the personnel in the methods and quarantine facilities at the
Division of Plant Industry, Gainesville, FL, and many people of the South West Florida
Research and Education Center (SWFREC) at Immokalee, which supported me in the
implementation of a bunch of activities. I thank James M. Conner, Carmen G6mez, Jaime
Martinez, Alexandra Delgado, and Ronald D. French. At the same time, I thank those
who helped me have a nice time in the center, C. Vavrina, F. Jaber, R. Pluke, M. Ozores,
P. Roberts, R. Muchovej, S. Sukla, K. Cushman, J. Knowles, K. Jackson, C. Pandey, M.
Triana, J. Castillo, F. Roka, J. & K. Hill, K. & R. Systma, B. & B. Hyman, A. Garcia, D.
Spencer, and L. Rigby. I also thank Emily Vasquez and Steven Davis of the entomology
group at Gulf Coast Research and Education Center (GCREC) in Bradenton.
I thank Eugenio Mariscal for his help during the field trips to Nayarit, Mexico. I
thank my friends in Gainesville, I will always remember academic, cultural, and
gastronomical moments, excursions and of course "intoxicating moments of life" that I
spent with all of them.
A C K N O W L E D G M E N T S ................................................................................................. iv
LIST OF TABLES ......................................... .................. ix
LIST OF FIGURES ......... ......................... ...... ........ ............ xi
ABSTRACT ........ .............. ............. .. ...... .......... .......... xii
L literature R review ................................................................... ......... ..... ....... .. .4
Origin of Capsicum spp. and Distribution of Pepper Weevil.............................4
E conom ic Im portance................................... ......................... ............... 5
D e sc rip tio n ....................................................... ................ 6
B iology and L ife H history ............................................ .................. ...............
Host Plant Association ............................ ...... ..... .... ...............
Capsicum spp. ............................................................9
Solanum spp. .................................................. ................ 11
M anagem ent Strategies ............................................... ............................ 12
H ost plant resistance ................................. ...... ......... ......... ... ...... .....12
Cultural and chem ical control ............................. ..... ....... ............... 13
B biological control ...................................... .......... .... .............. ..14
O bjectiv es .................................................... ........... .. ..... ........ 19
Introduction............ .. ....... ................................ .......................... ..... 20
M materials an d M eth od s .................................................................... .....................22
Laboratory W eevil Colony .............. ...................................................... ... 22
Crowding Effects on Oviposition................................................24
Fruit Q quality ..............................................................25
A dult Feeding and Oviposition ........................................ ....................... 26
L ife T able ........................................27
Nitrogen Content of Fruits and Buds ................. ................ ............. 28
R results ............. ...... ..... ......... .............................................29
Crowding Effects and Oviposition ............................ ................................... 29
Fruit Q quality ...................................... .............. ..................30
Feeding and Oviposition in Peppers versus Floral Buds (7d) ...........................30
Oviposition with or without Floral Buds (7d)................... ................................31
L ife T ab le ................. ......................................................... 3 1
Nitrogen Content of Fruits and Buds.................................. .................. 32
D discussion ................ .. .... .... .............. ............... ............ 33
Crowding Effects and Oviposition ............................ ................................... 33
F ruit Q quality .......................................................................34
A dult Feeding and Oviposition ........................................ ....................... 35
L ife T ab le .........................................................................3 7
3 LIFE HISTORY OF Triaspis eugenii Wharton and Lopez-Martinez
In tro du ctio n ...................................... ................................................ 4 0
M materials and M methods ....................................................................... ..................43
S u rv e y s ................................................................4 3
P arasitoid R hearing .............................. .... ...................... .. ........ .... ............44
H ost-A ge Range ................ ... ......... .... ........ .. .. ...... ... 46
Role of the Oviposition Plug in Host Location ................................................47
Effect of Weevil: Fruit Ratio on Parasitoid Production .................................. 49
L ife C y cle .........................................................................4 9
F e cu n d ity .............................................. .. ................ ................ 5 1
A dult L longevity ............. ................. .......... ...... ......................... 52
R e su lts ................................................................................................................... 5 2
S u rv e y s ................................................................5 2
H ost-A ge R ange ................ ... ......... .... ........ .. .. ...... ... 54
Role of the Oviposition Plug in Host Location ..........................................54
Effect of Weevil: Fruit Ratio on Parasitoid Production .............................. 55
L ife C y cle .........................................................................56
F e cu n d ity ....................................................... ................ 6 1
A dult Longevity ....................................... ....... ........ .. ........ .... 62
D isc u ssio n ............................................................................................................. 6 3
S u rv e y s ................................................................6 3
H ost-A ge Range ................ ... ......... .... ........ .. .. ...... ... 64
Role of the Oviposition Plug in Host Location ................................................66
L ife C y cle .......................................... ........... ....... ...... ..... ............... 6 7
Potential of T eugenii as a Biological Agent of the Pepper Weevil .................69
4 RELEASE AND RECOVERY OF Triaspis eugenii Wharton and Lopez-Martinez
Introdu action ...................................... ................................................. 72
M materials and M methods ....................................................................... ..................74
In se c ts ......................................................................................................7 4
F field C ag e s ................................................................7 4
Effect of Honey Supplement .................................. .......................... 76
Survival ............................................................................... 78
Effect of H ost-D am aged Plants.................................... .................................... 78
Field Release and Establishm ent ......................................................................80
R esu lts ............................................................................................... 8 1
Effect of Honey Supplement .................................. .......................... 81
Survival ............................................................................... 82
Effect of H ost-D am aged Plants.................................... .................................... 82
Field Release and Establishm ent ......................................................................84
D iscu ssio n ........................... ............ ....................................................... ............... 8 6
Effect of Honey Supplement .................................. .......................... 86
Effect of H ost-D am aged Plants.................................... .................................... 87
Field Release and Establishm ent ......................................................................89
Rearing Methodology and Influence of Food Quality on Biology of Pepper
W e e v il ....................................................................................................9 2
Life H history of Triaspis eugenii ............................... ................................. 93
Field C age and Field R release ........................................ ......................... 94
Table page
1 Biological data of pepper weevil, selected references until 2005 ............................9
2 Non Capsicum Solanaceae used by pepper weevil* ..........................................12
3 Natural enemies of the pepper weevil, references until 2005 .............................17
4 Characteristics of 'Jalapefio Mitla' used for nitrogen content determinations.........28
5 Numbers of eggs per female per day for different numbers of females and males
per single 'Jalapefio' pepper fruit....................................... .......................... 30
6 Feeding and oviposition of pepper weevil adults when given a choice of
'Jalapefio' pepper fruit or floral buds (7d) .................................... ............... 30
7 Oviposition of pepper weevil females fed with 'Jalapefio' pepper fruits only or
w ith pepper fruits plus floral buds....................................... ......................... 31
8 Life table parameters for A. eugenii females (n =14) reared on 'Jalapefio'
peppers at 27 + 20C, and 14:10 (L:D) photoperiod.................... ...............32
9 Nitrogen content (Mean + SD) of anthers in floral buds and 'Jalapeio' fruits at
three m aturation stages ............... ............................................ .. .. ........ ...... 33
10 Life table parameters for the pepper weevil............. .. ........... .............. 38
11 A. eugenii and parasitoids emerged from approximately 50 kg of infested hot
peppers collected in the state of Nayarit, Mexico, April and May, 2003 ..............53
12 Parasitism of pepper weevil eggs by T. eugenii when given the choice of two
ages of eggs ........................................... ............................ 54
13 Parasitism of pepper weevil eggs by T. eugenii when given no choice of egg age .54
14 Parasitism of pepper weevil eggs by T. eugenii when given the choice of eggs
covered with an oviposition plug and eggs with no plug...................................55
15 Parasitism of pepper weevil eggs by T. eugenii when given no choice of eggs
covered with an oviposition plug and eggs with no plug...................................55
16 T eugenii produced with two pepper weevil densities ........................................ 56
17 Developmental times of T eugenii in days (Mean SD) at 27 1C .................57
18 Demographic parameters (+ 95% CL) for T eugenii reared on pepper weevil
eggs in 'Jalapefio' fruits for different exposure intervals at 27 + 2C ...................63
19 Life table parameters (+ 95% confidence limits) estimated for 10 T. eugenii and
14 pepper w eevil adults at 27 + 2 C ............................. ... ......................... 71
20 Releases of T eugenii during 2005 at the SWFREC in Immokalee, FL.................80
21 Number of T eugenii adults released in field cages and subsequently observed
on pepper fruits (n=5) hung on pepper plants at 28.2 + 4.7C, 55 + 9% RH...........83
22 Adults per 50 terminals, and the number of weevil lifestages per 10 fruits, 1 or 2
d before field releases of T eugenii at SWFREC, Immokalee, FL, 2005 ...............85
23 Parasitoid and pepper weevil adults recovered from 'Jalapefio' pepper following
field releases of T. eugenii at the SWFREC, Immokalee, FL, 2005 ......................85
Figure pge
1 Device for exposing different densities of weevil adults to 'Jalapefio' peppers.....24
2 Mean fecundity of adult pepper weevil females (n = 14) reared on 'Jalapefio'
peppers at 27 + 2 C and 14:10 (L:D) photoperiod. ............................................32
3 Longitudinal section of an infested 'Jalapefio' fruit showing a pepper weevil egg
and oviposition plug (ov-p). ..... ........................... ......................................48
4 Immature stages of T. eugenii: (A) Egg 22 h after oviposition, (B) First instar,
30-33 h after oviposition, with well developed mandibles. ....................................58
5 Larva of T. eugenii. (A) Emerging from the posterior end of the host, (B)
Beginning to feed on the host even before emerging completely..........................59
6 T. eugenii adults. (A) Female, (B) M ale. ...................................... ............... 60
7 Fecundity of T eugenii females at 27 + 2C either exposed to hosts changed
every 1.5 h (n=5) or exposed to hosts per 9 h (n=5). ...........................................62
8 Field cages used at the SWFREC. (A) General view of a unit containing two
cages. (B) Organdy partition and zipper between two experimental units .............75
9 Honey-wasp-refuge. (A) General view of the set up. (B) Using a unit as T
eug enii release point...................... ..................................... .. ........ ......77
10 Survival of T eugenii adults in plastic cups with and without honey, when the
cups were placed inside the canopy of plants, 25.4 + 4.90C, 67 + 19% RH ...........82
11 Daily temperatures and humidity during T. eugenii field releases on 'Jalapeio'
pepper at the SWFREC in Immokalee, FL, 2005. ...............................................84
Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
LIFE HISTORY OF Triaspis eugenii Wharton and Lopez-Martinez (HYMENOPTERA:
CONTROL OF PEPPER WEEVIL Anthonomus eugenii Cano (COLEOPTERA:
Esteban Rodriguez-Leyva
May 2006
Chair: Philip A. Stansly
Cochair: David J. Schuster
Major Department: Entomology and Nematology
The pepper weevil, Anthonomus eugenii Cano, is considered the most important
pest of peppers (Capsicum spp.) in Tropical and Subtropical America. Juvenile stages
develop inside buds and immature fruits, so that only adults are susceptible to insecticidal
control. No viable biological tactic has been developed to combat this pest but Triaspis
eugenii, a braconid recently collected in Mexico that attacks pepper weevil eggs, could
offer an important addition to its management. The parasitoid was collected from Nayarit,
Mexico, during 2003, and a rearing methodology was developed using pepper weevil
reared on immature 'Jalapefio' peppers. Low levels of parasitism and high levels of
superparasitism hampered the rearing and reliable estimation of parasitoid demography
until the weevil rearing system was improved. Confining individual mated females to a
single immature 'Jalapefio' fruit maximized weevil fecundity, and also the number of
oviposition punctures that were plugged by female. Weevils fed with pepper floral buds,
a high nitrogen source, and immature 'Jalapefios' laid 5.5 eggs per female per day over a
life span of 64.5 d at 27 2C. The net reproductive rate (Ro=158.1) and intrinsic rate of
increase (rm = 0.14) obtained in a life table study were higher than any previous report.
T. eugenii is a solitary egg-prepupal parasitoid of the pepper weevil. At 27 1C,
T eugenii eggs hatched at 23 1 h. Females developed in 16.6 0.9 d and males in 16.4
0.9 d. Females (n=10) laid 402 199 eggs of which less than 50% reached the adult
stage because of high levels of superparasitism in the laboratory. Ovipositing females
lived 16.5 3.02 d, and parasitoids died in fewer than 48 h without carbohydrates
(honey). Net reproductive rate (Ro) was estimated at 106 and 167, and intrinsic rate of
increase (rm) at 0.24 and 0.26 with and without superparasitism respectively.
The oviposition plug deposited by the pepper weevil played a decisive role in the
ability of T eugenii to find and to parasitize weevil eggs. Crowded rearing conditions
inhibited deposition of oviposition plugs and consequently efficiency of parasitism.
Wasps were able to find hosts in field cages, and semiochemicals from weevil-damaged
plants appeared to play a role in host patch location. However, T eugenii did not stay
more than 120 min in the patch regardless of the presence of weevil damaged plants.
Superparasitism was less frequent in the field cages (28%) compared to the laboratory
(55-64%). The relatively short duration of foraging on large patch size compared to the
rearing environment might explain why.
T. eugenii was recovered in low numbers from field releases in spring 2005 at
Immokalee, FL, and at least one generation was completed in the field. No wasps have
been recovered after that although sampling has been limited. Even if T. eugenii does not
establish in Florida, its host specificity, its ability to parasitize weevil eggs, and its almost
double intrinsic rate of increase, compared to the pest, make this parasitoid an excellent
prospect for biological control by augmentation against the pepper weevil.
The pepper weevil, Anthonomus eugenii Cano (Coleoptera: Curculionidae), is
considered one of the most important insect pests of all cultivated varieties of chile =
pepper (Capsicum spp.) in the New World. Pepper weevil is considered a key pest of this
crop in the southern United States, Hawaii, Mexico, Central America, and Puerto Rico
(Elmore et al. 1934, Burke and Woodruff 1980, Abreu and Cruz 1985, Riley and King
1994, Coto 1996, Arcos et al. 1998).
Pepper weevil biology, control tactics, distribution, and sampling methods have
been studied (Elmore et al. 1934, Wilson 1986, Patrock and Schuster 1992, Riley et al.
1992a,b, and Toapanta 2001). Nevertheless, insecticidal control is necessary once the
pest is present in pepper crops (Riley and King 1994, Riley and Sparks 1995, Arcos et al.
1998, Mariscal et al. 1998). Unfortunately, the use of insecticides as the principal method
of control can provoke many adverse effects such as marketing restrictions, exposure of
non-target organisms, environmental contamination, pesticide resistance, and secondary
pest outbreaks within the same crop (Doutt and Smith 1971, Van Driesche and Bellows
Biological control could be an alternative strategy to incorporate into the integrated
pest management (IPM) of pepper weevil (Riley and King 1994, Mariscal et al. 1998,
Schuster et al.1999). However, little is known of the natural enemies of this pest. Pepper
weevil arrived or was incidentally introduced from Mexico to the United States at the
beginning of the last century (Walker 1905, Pratt 1907). Early reports indicated two
species of ants, three pteromalids, one braconid, and one mite that attack this pest in the
United States (Pratt 1907, Pierce et al. 1912, Elmore et al. 1934, Cross and Chesnut
1971). However, the effect of those enemies on pepper weevil populations was not
considered important (Pratt 1907, Elmore et al. 1934, Elmore and Campbell 1954).
A few decades ago, Wilson (1986) reported on the ectoparasitoid Catolaccus
hunter Crawford (Hymenoptera: Pteromalidae) attacking pepper weevil in Florida. C.
hunter is the dominant parasitoid attacking this pest in Florida and Puerto Rico (Wilson
1986, Schuster et al. 1999), and one of the most abundant in some states of Mexico
(Mariscal et al. 1998, Schuster et al. 1999, Rodriguez et al. 2000).
C. hunter has been collected from a closely related pest of cotton, the cotton boll
weevil Anthonomus grandis Boheman (Coleoptera: Curculionidae), in several countries
in Central and South America, Mexico, and the United States (Pierce et al. 1912,
Towsend 1912, Cross and Mitchell 1969). Biological studies showed that C. hunter has a
higher fecundity than that of the pepper weevil (Rodriguez et al. 2000, Seal et al. 2002).
Those findings stimulated and supported the evaluation of C. hunter for controlling this
pest (Schuster et al. 1999, Rodriguez et al. 2000).
Releasing C. hunter in field plots in Mexico and Florida produced equivocal
results; in Mexico this parasitoid was not effective for combating pepper weevil on bell
pepper (Corrales 2002). However, some augmentative releases of this parasitoid prior to
and during the pepper season suggested that this parasitoid might have potential to reduce
weevil damage (D. Schuster, UFL, personal communication). These contradictory results
may be due, in part, to the fact that C. hunter attacks the 3rd instar host that is usually
inaccessible deep within the pepper fruit. Therefore, augmentative releases of C. hunter
prior to the crop cycle could reduce pepper weevil populations in alternative host plants
like nightshade, while releases in early infestations of pepper weevil, or in small fruited
varieties, might be effective because floral buds and small fruits allow C. hunter to reach
its host. Once the pepper fruits increase in size, there is no way to expect parasitism. In
fact, Riley and Schuster (1992) indicated that C. hunter was not detected in fallen fruits
larger than 2.5 cm in diameter. According to this information, an effective biological
control agent for pepper weevil might be one that attacks earlier and more accessible life
The search for biological control agents in the native region of the target pest is one
of the first steps in a successful biological control program (DeBach 1964, Hagen and
Franz 1973, Huffaker and Messenger 1976). Mexico and Central America have been
indicated as a center of origin and domestication of Capsicum annuum L., the most
important cultivated species of chile = pepper (Vavilov 1951, MacNeish 1964, Pickersgill
1969, 1971, Pickersgill and Heiser 1971, Eshbaugh 1976, 1980, Loaiza-Figueroa et al.
1989). Furthermore, it was in Mexico that the pepper weevil was first reported as a
pepper pest (Cano and Alcaciol894).
Mariscal et al. (1998) collected nine different genera of hymenopteran parasitoids
from pepper weevil in Nayarit, Mexico, including a subsequently described species
Triaspis eugenii Wharton & Lopez-Martinez 2000 (Hymenoptera: Braconidae). Only
Mexico appears to have this diversity of parasitoids of pepper weevil, and additional
search would likely yield more potential species.
T. eugenii was the most abundant parasitoid emerging on pepper weevil in Nayarit.
Incidence of parasitism ranged from 18 to 40%, making it the most important parasitoid
of this pest in the field (Mariscal et al. 1998, Schuster et al. 1999, Toapanta 2001). This
level of parasitism in unsprayed fields suggests that T. eugenii could be a good candidate
for inclusion in an IPM program against pepper weevil in the United States, Mexico, and
Central America. Nevertheless, information on the biology and ecology of this braconid
is needed to evaluate its potential as a biological control agent.
Literature Review
Origin of Capsicum spp. and Distribution of Pepper Weevil
Central and southern Mexico and part of Central America, a region called
Mesoamerica according to anthropologists, has been indicated as a center of origin and
domestication of the most important cultivated species of chile = pepper, Capsicum
annuum L. (Vavilov 1951, MacNeish 1964, Pickersgill 1969, 1971, Eshbaugh 1976,
Loaiza-Figueroa et al. 1989, Conciella et al. 1990). The other four important species of
cultivated chiles (or peppers) are Capsicum frutescens "Tabasco"; C. chinense Jacquin
"Habanero or Scotch Bonnet"; C. pubescens Ruiz and Pavon "Manzano or Pera"; and C.
baccatum L. All are endemic to South America (Heiser and Smith 1951, Smith and
Heiser 1957, Pickersgill 1969, 1971, Pickersgill and Heiser 1971, Eshbaugh 1976,
McLeod et al. 1982).
A Mesoamerican origin of the pepper weevil, Anthonomus eugenii Cano
(Coleoptera: Curculionidae), is suggested by the following facts: a) the origin of the most
important species of cultivated pepper (C. annuum) is probably Mesoamerica; b) the
pepper weevil has not been reported from South America (Clark and Burke 1996); and c)
this insect was found for the first time damaging peppers in Central Mexico in
Guanajuato (Cano and Alcacio 1894).
A few years after the report from Guanajuato, Mexico, the pepper weevil was
reported from Texas (Walker 1905). According to Pratt (1907) this insect was introduced
from Mexico on infested fruit shipments. After affecting pepper crops in Texas, the insect
was reported from California in 1923, New Mexico and Arizona in 1927, and Hawaii in
1933 (Elmore et al. 1934). It was reported from the west coast of Florida in 1935 (Goff
and Wilson 1937), and the east coast in 1972 (Genung and Ozaki 1972). During 1935-
1972, when high levels of organochlorine insecticides were used on the east coast, there
were no records of pepper weevil in the zone, but in 1972 it was a widespread pest
(Burke and Woodruff 1980).
Presently, the pepper weevil is a pest in all pepper growing areas of the United
States including North Carolina and New Jersey (Burke and Woodruff 1980). It was once
reported in greenhouses in Canada, probably introduced on seedling plants from
California (Riley and King 1994). Mexico, Guatemala, El Salvador, Honduras,
Nicaragua, and more recently Puerto Rico, Costa Rica, Jamaica, and Panama have been
added to the list of countries where this insect is considered a serious problem on peppers
(Cano and Alcacio 1894, Elmore et al. 1934, Burke and Woodruff 1980, Abreu and Cruz
1985, Andrews et al. 1986, Riley and King 1994, Clark and Burke 1996, Coto 1996).
Economic Importance
The pepper weevil lays eggs, feeds, and develops completely inside the fruits,
which contribute to the difficulty of controlling the insect. The damage to flowers and/or
young pods causes abscission and diminishes yield up to 30 to 90% if treatment is not
implemented (Campbell 1924, Elmore et al. 1934, Goff and Wilson 1937, Velasco 1969,
Genung and Ozaki 1972, Riley and Sparks 1995). In 1990, losses in the United States due
to the pepper weevil were estimated at 23 million dollars on the 31,000 ha grown in
California, New Mexico, Florida and Texas (Riley and King 1994). By 2003, the growing
area of the crop in the United States had increased to close to 32,000 ha and the value of
production reached 550 million dollars (National Agriculture Statistics Service 2004).
Using the same proportion of damage (10%) estimated by Riley and King (1994), the
economic damage in 2003 could have reached more than 50 million dollars. In Mexico,
Central America, and the Caribbean islands, serious economic impact is indicated but not
quantified (Riley and King 1994). Additional costs include the many adverse affects of
pesticides such as exposure of non-target organisms, environmental contamination,
pesticide resistance, and secondary pest outbreaks within the same crop (Doutt and Smith
1971, Van Driesche and Bellows 1996).
The pepper weevil shares many similarities with its congener the cotton boll
weevil: Mesoamerica as the likely origin, the damage to their hosts by feeding and
developing inside the fruits, and the history of dispersal in the United States (Burke et al.
1986). However, the difference in acreage and, consequently, economic importance of
cotton and pepper in the United States, explains why pepper weevil has never received
the same attention as the boll weevil, and why even a hundred years after the original
detection, there is relatively little information about biogeography, evolution, and
biological control of the pepper weevil (Riley and King 1994).
The pepper weevil was described from specimens collected from Guanajuato,
Central Mexico, by Cano and Alcacio (1894). The author mentioned as a motive the
importance of the insect as a pest of pepper crops in that region. This weevil belongs to
the subfamily Anthonominae and shares many characteristics with the other 330
described species of the genus Anthonomus (Burke 1976, Anderson 1992, Clark and
Burke 1996): basically robust and convex body, elongation of anterior part of the head to
form a rostrum longer than broad, reduced mandibles on the tip of this rostrum, and flat
scales like hair covering elytra, scutellum, and to some extent other parts of the body
(Burke 1976).
Detailed descriptions of immature lifestages and adults have been written by
Elmore et al. (1934), Burke (1968), and Clark and Burke (1996). Pepper weevil adults are
usually not longer that 3 mm, and males possess larger metatibial mucrones than females,
a characteristic useful to determine sex (Eller 1995). As with boll weevils, pepper weevil
males have a notch visible on the 8th tergum of the abdomen, which females do not have
(Agee 1964, Sappington and Spurgeon 2000). However, the small size of the pepper
weevil makes the last characteristic difficult to see (Eller 1995).
Biology and Life History
The biology of the pepper weevil was first described by Cano and Alcacio (1894),
Walker (1905), and Pratt (1907); however, Elmore et al. (1934) made the first complete
biological study. The pepper weevil feeds and develops on several species of Solanaceae,
but it is only a pest on peppers, Capsicum spp. (Elmore et al. 1934, Wilson 1986, Patrock
and Schuster 1992). Adults feed on buds, flowers, fruits, and even leaves. Larvae feed
and develop completely inside floral buds and immature fruits. Premature abscission is a
consequence of feeding and developing inside buds and fruits resulting in loss of
production (Cano and Alcacio 1894, Walker 1905, Pratt 1907, Elmore et al. 1934).
Even if there were no abscission of fruits, there could be loss, as the fruits have
larvae inside and are often spoiled. Actually, abscission of fruits could be considered an
advantage because fallen fruits can be gathered to avoid reinfestation of the crop
(Berdegue et al. 1994) and because fewer damaged fruits make it to the market.
There are three instars and the number of generations per year depends only of
temperature and availability of food resources (Elmore et al. 1934). Pepper weevil
develops within a wide range of temperature (10-30C), but the ideal range is from 27 to
30C (Toapanta et al. 2005).
Other reports that included information on generation time, oviposition, fecundity,
fertility, and host plant associations have advanced our understanding of the biology of
this species (Genung and Ozaki 1972, Burke and Woodruff 1980, Wilson 1986, Gordon
and Armstrong 1990, Patrock and Schuster 1992, Arcos et al. 1998). In addition, a life
table study by Toapanta et al. (2005) provided additional demographic parameters such
as net reproductive rate, intrinsic rate of increase, and finite rate of increase at different
temperatures. With this information the biology of the pepper weevil is reasonably well
known (Table 1), but is not complete. Various factors that were not included in fecundity
experiments could affect fitness of this species, including type and quality of food. It is
well known that the boll weevil needs to feed on floral buds, because of their high
nitrogen content, for ovarian development and oviposition (Hunter and Hinds 1905, Isley
1928). Other studies demonstrated that fecundity was optimal in boll weevil fed with a
diet containing high nitrogen content (10% cottonseed flour as the amino acid nitrogen
source) but drastic nitrogen reduction (2.5% cottonseed flour) reduced egg production
(Hilliard 1983). Oogenesis and oviposition in pepper weevil proceeds only on peppers
(almost any reference in Table 1). However, adults prefer to feed on floral buds instead of
fruits (Patrock and Schuster 1992), so the importance of this type of food and its effect on
fecundity should be considered for future studies.
Table 1. Biological data of pepper weevil, selected references until 2005
Parameter (average in days) Conditions Reference
Generation time
22.7, 13.9, 12.9
Longevity of adults
Oviposition period
76, 50, 52
Oviposition rate
(eggs/ female/ day)
1.9, 1.7, 3.1
Fecundity (eggs/female)
144, 85, 161
25.7-27.7C; 70% RH; on an
artificial diet
21.0, 27.0, and 300C, 60% RH;
14:10 (L:D), respectively
Laboratory reared
Laboratory reared
21.0, 27.0, and 300C, 60% RH;
14:10 (L:D), respectively
Laboratory reared
22-27 oC; 60-70% RH
21.0, 27.0, and 300C, 60% RH;
14:10 (L:D), respectively
22-27oC; 60-70% RH
21.0, 27.0, and 300C, 60% RH;
14:10 (L:D), respectively
Walker 1905
Elmore et al. 1934
Toba et al. 1969
Genung & Ozaki 1972
Wilson 1986
Toapanta et al. 2005
Elmore et al. 1934
Goff & Wilson 1937
Gordon & Armstrong 1990
Elmore et al. 1934
Goff & Wilson 1937
Toapanta et al. 2005
Elmore et al. 1934
Goff & Wilson 1937
Genung & Ozaki 1972
Wilson 1986
Gordon & Armstrong 1990
Toapanta et al. 2005
Elmore et al. 1934
Goff & Wilson 1937
Gordon & Armstrong 1990
Toapanta et al. 2005
Population values
a R =25.15, 11.76, 33.57 21.0, 27.0, and 300C, 60% RH; Toapanta et al. 2005
b rm= 0.06, 0.07, 0.11 14:10 (L:D), respectively
a Net reproductive rate (Ro); b Intrinsic rate of increase (rm); c Generation time (T)
Host Plant Association
Capsicum spp.
Host plants utilized by pepper weevil for reproduction are confined to the genera
Capsicum and Solanum, both in the family Solanaceae. All of the five species of pepper
grown as crops (C. annuum, C. frutescens, C. chinense, C. pubescens or C. baccatum) are
suitable for oviposition and development of the pepper weevil (Elmore et al. 1934,
Wilson 1986, Patrock and Schuster 1992). The majority of studies describing feeding,
oviposition, survival and reproduction have been developed in these hosts.
Pepper weevil adults feed on floral buds, flowers, fruits, and sometimes leaves of
pepper plants; they lay eggs inside floral buds and young pepper pods. For feeding,
females and males bore a small hole with the mandibles, which are located at the end of
the rostrum, to reach internal tissue (Walker 1905, Elmore et al. 1934).
For oviposition, females spend a short time in selecting a site on the fruits, after
which they bore a small hole with the mandibles, turn completely around and deposit an
individual egg into the hole. Once the egg is in the cavity, the female deposits a yellowish
or brownish substance that turns black on drying and seals the hole (=oviposition plug)
(Elmore et al. 1934, Goff and Wilson 1937, Genung and Ozaki 1972). After hatching, the
larva bores into the interior of the fruit until reaching the placenta and seeds. Once
development is completed, the insect pupates inside the fruit (Pratt 1907, Elmore et al.
1934, Goff and Wilson 1937, Burke and Woodruff 1980, Riley and Sparks 1995). The
particular feeding and reproduction behavior of this insect make it inaccessible to many
natural enemies, and also to most insecticides (Pratt 1907, Elmore et al. 1934, Elmore and
Campbell 1954).
The size, quality, and availability of buds, flowers, and fruits could be factors that
affect the selection of oviposition sites for pepper weevil (Walker 1905, Elmore et al.
1934, Riley and King 1994). More eggs were laid in fruits than in flowers (Patrock and
Schuster 1992). For feeding and oviposition, adults prefer small fresh fruits rather than
mature fruits (Walker 1905, Wilson 1986). According to Bruton et al. (1989), the pepper
weevil preferred developing bell pepper fruits from 1.3 to 5.0 cm in diameter rather than
smaller (less than 1.3 cm in diameter) or larger mature fruits (bigger than 5 cm in
diameter) for oviposition.
Solanum spp.
The suitability for feeding, oviposition and development of the pepper weevil has
been confirmed for at least 10 species of the genus Solanum (Table 2). Six additional
species of the family Solanaceae served as food, but were not suitable for oviposition in
non-choice tests (Wilson 1986, Patrock and Schuster 1992). Nevertheless, laboratory
results do not necessary apply to the field. For example, the pepper weevil was able to
develop in eggplant (S. melongena) in the laboratory, but rarely is observed to attack this
crop in the field.
The developmental time of pepper weevil reared on bell peppers was not different
from those reared in American black nightshade (S. americanum) or eastern black
nightshade (S. ptycanthum), but the dry weight of adults from pepper was greater than
those from the other hosts (Patrock and Schuster 1992). A hypothesis of restriction of
resources, where bigger fruits could offer more food for the developing larva, could help
to explain why pepper weevil females prefer fruits instead of flowers for oviposition. The
same hypothesis could explain the greater dry weight of pepper weevil adults reared from
bell pepper fruit than those from nightshade fruit (Patrock and Schuster 1992). The
qualitative differences in suitability among hosts of pepper weevil have not been
Table 2. Non Capsicum Solanaceae used by pepper weevil*
Plant species Feeding Oviposition Development
Solanum americanum Mill. L, F, Fr1 Fr S4
S. carolinense L. L, F, Fr F, Fr S
S. dimidiatum Fav. L, F, Fr F, Fr S
S. eleganifolium Cav. L, F, Fr F, Fr S
S. melongena L. L, F, Fr F, Fr S
S. pseudocapsicum L. L, F, Fr Fr S
S. pseudogracile Heiser L, F, Fr Fr S
S. ptycanthum Dun L, F, Fr Fr S
S. rostratum Dunal L, F F S
S. triquetrum Cav. L, F2 F2 S
S. tuberosum L. L, F2 N3
Datura stramonium L. L, F N
Lycopersicon esculentum Mill. L, F, Fr N
Nicotiana alata Link and Otto L, F N
Petunia parviflora Vilm F N
Physalis pubescens L. L, F, Fr N
*Source: Wilson (1986), Patrock and Schuster (1992)
L= Leaves, F = Flowers, Fr = Fruits. 2Fruit unavailable. 3No oviposition observed on flowers or fruits.
4Successful development.
Management Strategies
Host plant resistance
Host plant resistance is an important component of IPM (Painter 1951, NAS 1969).
Berdegue et al. (1994) indicated that some types of pepper exhibited less damage from
pepper weevil because of an escape mechanism -concentrated production before the
presence of the pest instead of antibiosis. Quifiones and Lujan (2002) indicated that
some 'Jalapefio' lines might have some tolerance mechanism against damage from this
pest. Seal and Bondari (1999) indicated that only two commercial varieties of peppers,
one of them Habanero, had lower rates of infestation by pepper weevil than the remaining
nine varieties tested in field evaluations. Unfortunately, there is not a single cultivated
variety known to provide any important resistance characteristic against this pest.
Cultural and chemical control
Current management practices against pepper weevil consist of a combination of
cultural and chemical control. According to Riley and King (1994), control practices
against pepper weevil focused on cultural control during almost the first 40 years
following introduction into the United States. The lack of other tactics was explained by
the difficulty of reaching the insect inside the fruit (Walker 1905). The inorganic
insecticides, available during those years, especially calcium arsenate, could not be used
because of human toxicity (Elmore et al. 1934, Elmore and Campbell 1954) and were
observed to provoke secondary pest problems in the crop (Folsom 1927, Elmore et al.
1934). Insecticides have improved in selectivity for other pests, but they have not
improved in efficacy against pre-imaginal stages of the pepper weevil.
Recommendations for avoiding damage included establishing a pepper free period
-some months if possible- to reduce populations by food deprivation. Other
recommended practices included 1) using weevil-free seedlings to establish new crops, 2)
removing alternative hosts inside and around the fields, 3) collecting and destroying
fallen fruits to reduce reinfestation, and 4) destroying crop residues immediately after
harvest (Cano and Alcacio 1894, Walker 1905, Pratt 1907, Elmore at al. 1934, Goff and
Wilson 1937, Watson and Lobdell 1939).
From the mid 1940's to 1980's, chemical control gained favor due to the
availability of synthetic insecticides (Elmore and Campbell 1954, Riley and King 1994).
Increasing knowledge of dispersal and sampling techniques (Andrews et al. 1986, Riley
et al. 1992a b, Riley and Schuster 1994) aided in managing this pest without calendar
spraying of pesticides (Riley et al. 1992a b, Riley and King 1994). When 5% of the
terminals were damaged (Cartwright at al. 1990) or when one adult pepper weevil was
present per 400 terminal buds (Riley and Schuster 1992 b), significant economic loss in
highly productive crops resulted. These criteria could serve as action thresholds.
Azadirachtin, bifenthrin, cyfluthrin, cryolite, esfenvalerate, oxamyl, permethrin,
acetamiprid, and thiamethoxam are some of the insecticides labeled for combating the
pepper weevil in Florida (Olson et al. 2005, P. Stansly, personal communication).
Biological control
Known natural enemies of the pepper weevil are summarized in Table 3. Although
biological control is usually considered an integral part of the IPM of any pest, natural
enemies of pepper weevil in Florida have not been shown to play an important role in
suppression of this pest (Elmore and Campbell 1954, Wilson 1986, Schuster et al. 1999).
For this reason, although efforts to introduce natural enemies against pepper weevil have
been considered, no specific parasitoid had been introduced and released for this pest in
the United States until the present study (Elmore et al. 1934, Elmore and Campbell 1954,
Clausen 1978, Schuster et al. 1999).
Entomopathogenic nematodes (Heterorhabditis sp. and Steinernema sp.) have
proved effective against certain insect pests that develop some part of their life cycle in
the soil (Glaser 1931, Bell at al. 2000). Studies in Texas have shown that S. riobravis can
kill boll weevil larvae inside abscised squares and bolls of cotton if applied to the soil
under ideal conditions of moisture (Cabanillas 2003). In a similar way, entomophagous
nematodes might attack the pepper weevil inside buds or pods that are on the ground, and
could thus diminish pest populations; however, there is little information in regard to
entomopathogenic fungi, as well as viruses or bacteria that might attack this pest. In a
recent work in Florida, a commercial formulation ofBeauveria bassiana (Balsamo) was
used against pepper weevil adults, but proved to be ineffective in the field (Schuster et al.
Three pteromalids, one braconid, two species of ants, and one mite were identified
attacking the pepper weevil in the United States (Pratt 1907, Pierce et al. 1912, Elmore et
al. 1934, Cross and Chesnut 1971). Nevertheless, their impact on the pest was not
considered important (Elmore and Campbell 1954, Genung and Ozaki 1972). The cited
natural enemies that are endemic to the United States would be unlikely to act effectively
against an exotic pest.
Before 1950, two attempts of classical biological control of this pest were made in
the United States. Eupelmus cushmani Crawford and Catolaccus (Heterolaccus) hunter
Crawford were collected from Guatemala and released in Hawaii during 1934-37, where
they established, but did not provide encouraging results (Clausen 1978). The other
attempt during 1942-1943 occurred in California with the release of Triaspis vestiticida
Viereck, a parasitoid ofAnthonomus vestitus Boheman, the Peruvian cotton boll weevil in
Peru (Berry 1947, Clausen 1978). However, the parasitoid was never recovered from
peppers (Clausen 1978). These three natural enemies were originally obtained for
biological control programs against the boll weevil (Anthonomus grandis Boheman) and
evaluated against pepper weevil in the hope that similarities between these two pests,
might make possible the use of the same natural enemies (Clausen 1978). Nevertheless,
there are no reports of E. cushmani or T. vestiticida ever parasitizing pepper weevil.
C. hunter has the widest distribution of any parasitoid of boll weevil or pepper
weevil in the United States, Mexico, and Central America (Pierce et al. 1912, Cross and
Mitchell 1969, Cross & Chesnut 1971, Mariscal et al. 1998, Schuster et al. 1999,
Rodriguez et al. 2000). It is the most important parasitoid attacking pepper weevil in
Florida (Wilson 1986, Schuster et al. 1999), and one of the most widely distributed in
Mexico (Mariscal et al. 1998, Schuster et al. 1999, Aguilar and Servin 2000, Rodriguez et
al. 2000). It is also present in Honduras (Riley and Schuster 1992) Costa Rica (Schuster
et al. 1999), and it is the only one known from Puerto Rico (Schuster et al. 1999).
C. hunter is a generalist that is known to attack at least 17 species of Curculionidae
and 2 of Bruchidae (Cross and Mitchell 1969, Cross and Chesnut 1971); it usually
develops on the last instar of its host (Wilson 1986, Cate et al. 1990, Rodriguez et al.
2000). The particular biology of this species was useful for mass rearing at a moderate
scale on the cowpea weevil, Callosobruchus maculatus F. (Rodriguez et al. 2002,
Vasquez et al. 2005). Those findings, and some biology studies that showed that C.
hunter has greater fecundity and intrinsic rate of increase than pepper weevil (Rodriguez
et al. 2000, Seal et al. 2002), stimulated and supported the plan of evaluating C. hunter
as a biological control agent. Releases of 1050 C. hunter per hectare in Sinaloa, Mexico,
were not effective in combating pepper weevil on bell pepper (Corrales 2002).
Nevertheless, in Florida results of weekly releases of the equivalent of 7900 C. hunter
per hectare suggested that this parasitoid has potential to reduce damage of pepper weevil
on peppers (D. Schuster, personal communication). One of the most important
disadvantages of using C. hunter to combat pepper weevil is the fact that it attacks the
3rd instar host, which is usually inaccessible deep within the fruit of commercial pepper
Table 3. Natural enemies of the pepper weevil, references until 2005
Natural enemies Reference
Hymenoptera: Formicidae
Solenopsis geminata Fab.
Tetramonium guinense Fab.
Passeriformes: Icteridae
Sturnella magna (Easter meadowlark bird)
Zatropis incertus Ashmead
(=Catolaccus incertus Ashmead)
Catolaccus hunter Crawford
Habrocytus pierce Crawford
Bracon mellitor Say
Triaspis eugenii Wharton & Lopez-Martinez
Urosigalphus sp.
Bracon sp.
Aliolus sp.
Euderus sp.
Syempiesis sp.
Eupelmus sp.
Eurytoma sp.
Pratt 1907, Hinds 1907
Wilson 1986
Genung & Ozaki 1972
Pierce 1907, Cross & Chesnut
Pierce et al. 1912, Cross &
Chesnut 1971, Mariscal et al. 1998
Cross & Chesnut 1971
Pratt 1907, Pierce et al. 1912,
Cross & Chesnut 1971
Mariscal et al. 1998, Wharton &
Lopez-Martinez 2000, Toapanta
Mariscal et al. 1998, Toapanta
Mariscal et al. 1998
Mariscal et al. 1998, Toapanta
Mariscal et al. 1998
Acarina: Pyemotidae
Pyemotes ventricosus Newport Pierce et al. 1912, Cross &
Chesnut 1971
Even though Mexico and Central America have been indicated as the center of
origin and domestication of the most important cultivated species of peppers (Vavilov
1951, Pickersgill 1969, 1971, Pickersgill and Heiser 1971, Eshbaugh 1976, Loaiza-
Figueroa et al. 1989, Conciella et al. 1990), and the pepper weevil was first described as a
pest of that crop in Mexico (Cano and Alcacio 1894), no significant surveys for natural
enemies were undertaken until 1997 (Mariscal et al. 1998). A survey by these authors in
the west central coastal state of Nayarit, Mexico, detected four species of Braconidae, one
of Pteromalidae, two of Eulophidae, and one each from Eupelmidae and Eurytomidae
(Table 3). Of these, the most abundant was a new species, later described as Triaspis
eugenii Wharton and Lopez-Martinez (Hymenoptera: Braconidae) (Wharton and Lopez-
Martinez 2000), which was reported to reach incidences of 18-40% parasitism under field
conditions (Mariscal et al. 1998, Schuster et al. 1999, Toapanta 2001). This is a solitary
parasitoid which was reported to parasitize the egg, and to complete its life cycle in less
than three weeks with a life span of less than four (Mariscal et al. 1998, Toapanta 2001,
Rodriguez et al. 2004).
The genus Triaspis Haliday (Hymenoptera: Braconidae) is placed in the tribe
Brachistini of the subfamily Helconinae (Martin 1956, Sharkey 1997). This genus could
be represented by 100 species in the New World, but just a few of them have been
described from this region (Sharkey 1997). In general, biology and life history of the
genus Triaspis is poorly known. Some species of Brachistini are egg-larval parasitoids of
weevils, bruchids, and anthribids, and the last larval instar has an ectoparasitic phase
(Clausen 1940, Berry 1947, Saw and Huddleston 1991, Sharkey 1997). In spite of the fact
that the biology of the genus Triaspis is not completely known, some species of this
genus have been used in biological control programs in North America.
T. thoracicus was imported to Canada and the United States to combat species of
Bruchidae, especially the pea weevil, Bruchuspisorum L. Apparently this species failed
to establish because it has a preference for laying eggs in plant tissue instead of dry seeds
(Martin 1956, Clausen 1978). Efforts to combat the cotton boll weevil in USA with
Triaspis vestiticida Viereck, were previously mentioned. T. vestiticida is a solitary
parasitoid which attacks the egg of the host (peruvian cotton boll weevil) and develops as
an endoparasitoid in early instars. At the end of the last instar, the larva emerges from the
host to feed as an ectoparasitoid, leaving only the host cephalic capsule (Berry 1947).
Although these habits may prevail throughout the tribe, more detailed studies of T
eugenii are necessary to understand its habits and potential use.
The high incidences of parasitism by T. eugenii under natural conditions in Nayarit,
Mexico and the recent information that confirms that this species attacks the weevil egg
make it a good candidate for biological control of pepper weevil. Attempts to use this
species for biological control will require more specific information on host/parasitoid
relationships. Therefore, the objectives of the present study were the following:
1. Clarify key aspects of the biology of the pepper weevil, including demographic
parameters, to improve rearing methodologies
2. Evaluate oviposition behavior and population dynamics of T. eugenii to optimize
rearing procedures and to assess control potential
3. Test the ability of T. eugenii to control pepper weevil
Adult pepper weevils, Anthonomus eugenii Cano, feed on floral buds, flowers,
fruits, and even pepper leaves, but the larvae feed and develop completely inside floral
buds and immature fruits (Cano and Alcacio 1894, Walker 1905, Pratt 1907, Elmore et al.
1934). When feeding in floral buds, weevil larvae damaged especially the anthers
(Walker 1905, Elmore et al. 1934, Patrock and Schuster 1992). During the oviposition
process on fruit, females spend a short time in selecting a site on the pod, usually close to
the calyx. They then bore into the pod, turn completely, and deposit an individual egg in
the hole. Once the egg is in the cavity, the female deposits a brownish substance that
turns black on drying and seals the hole (oviposition plug) (Walker 1905, Elmore et al.
1934, Goff and Wilson 1937). The pepper weevil laid more eggs in pepper fruits than in
flowers (Patrock and Schuster 1992), and adults preferred small and immature fruits to
mature fruits for feeding and oviposition (Elmore et al. 1934, Wilson 1986, Bruton et al.
Although the pepper weevil pepper weevil has been considered the major pest of
peppers for more than eight decades in Mexico and the United States (Cano and Alcacio
1894, Walker 1905, Pratt 1907, Elmore et al. 1934, Riley and King 1994), no artificial
rearing system has been developed to facilitate biological studies. Consequently, most
studies have depended on weevils emerging of infested peppers collected from the field
(Elmore et al. 1934, Wilson 1986, Patrock and Schuster 1992). An attempt to rear pepper
weevil on artificial diet can be excluded from this generalization (Toba et al. 1969).
These authors were able to rear 6 generations of weevils using diet. However, females
would not lay eggs in the diet, so the processes of collection and manipulation of eggs are
laborious and ineffective (Toapanta 2001).
Toapanta et al. (2005) described a methodology to produce weevils in peppers, but
these authors did not define the quality of fruits offered. A similar methodology with a
few variations is practiced at the Golf Coast Research and Education Center (GCREC),
Wimauma, FL. Both methodologies consist basically of exposing peppers for 48-72 h to
weevil adults in cages 50x40x40 cm. Fruits are subsequently removed and held in
ventilated plastic containers until the emergence of weevils. Toapanta (2001) used mainly
'Serrano' peppers, whereas 'Jalapefio' peppers from the supermarket were used at
GCREC. Toapanta (2001) also indicated that the largest number of weevils, 1.7 adults per
fruit, was obtained using 5 weevils per fruit. At GCREC a ratio of 15 adults per fruit is
used, although an evaluation of efficiency has not been conducted (D. Schuster, personal
communication). These methodologies have been useful to maintain dozens of
generations in the laboratory, but efficiency could be improved by considering factors
such as competition within fruits, fruit decay, and effect of food quality. Therefore,
modifying fruit quality and decreasing competition could improve the laboratory rearing
of the weevil.
Even though there is a basic understanding of the biology of pepper weevil, there
are no studies that refer to food quality and its effect on fitness of this species. For
example, there are no reports that explain why weevils prefer feeding on floral buds
instead of fruits. This might be related to food quality (nitrogen concentration or protein
content). That is suggested because the ovarian development and oviposition of the boll
weevil, Anthonomus grandis Boheman, are biological processes that depend upon the
presence of cotton squares, a high nitrogen content food (Hunter and Hinds 1905, Hunter
and Pierce 1912, Isley 1928). The pepper weevil does not need floral buds for ovarian
development as has been proved by authors who reared the weevil in the laboratory with
only peppers available for feeding and oviposition (Toapanta et al. 2005, D. Schuster,
personal communication). However, studies that include feeding on floral buds are
necessary to understand nutritional factors in this species.
The biology of the pepper weevil has been studied by several authors (as
summarized in Riley and King 1994), but most, including Toapanta et al. (2005),
reported a fecundity of no more than 150 or 160 eggs per female. Only Elmore et al.
(1934) reported a fecundity as high as 340 eggs per female. Possibly a better
understanding of the effects of food quality and the availability of immature fruits and
floral buds could allow a further realization of the reproductive potential of this species
and thereby improve the rearing system. The objectives of this chapter were to determine
the effects of crowding on oviposition and the effects of fruit quality on rearing of the
pepper weevil, and to provide demographic parameters when immature fruits and floral
buds are offered to adults.
Materials and Methods
Laboratory Weevil Colony
A colony of the pepper weevil was established in a laboratory at the Entomology
and Nematology Department in Gainesville, UFL, with 200-250 insects that emerged
from infested fruits collected during the Spring of 2003 at the Southwest Florida
Research and Educational Center (SWFREC), Immokalee, FL. Genetic enrichment was
provided by addition of insects collected periodically from the same site, particularly
during Spring 2004 and 2005. The insects were reared continuously on fresh peppers,
'Jalapefio M' (Harris Seeds Rochester, NY), and 'Jalapefio Mitla' (Otis S. Twilley Seed
Co. Hodges SC), but all the experiments were conducted with 'Mitla' fruits.
The colony was moved in April 2004 to a rearing room at the SWFREC maintained
at 27 + 20C, 60-70% RH, and 14:10 (L:D) h photoperiod, and the experiments with the
pepper weevil were conducted at those conditions, unless otherwise indicated. Pepper
fruits were exposed to unsexed adult weevils using a ratio of 10:1 insects per fruit. Plastic
jars 3.8 L (14x14x25 cm Rubbermaid Home Products Wooster, OH), with four lateral
holes 3 cm in diameter covered with polyethylene screen, were used as oviposition cages.
The cages had a knit sleeve (Kimberly-Clark Co. Roswell, GA) 50 cm long taped to the
opening of the jar to prevent escape of insects during manipulations. Each cage held ca.
100 unsexed weevil adults. Water was provided in a cotton wick placed in 28-mL plastic
cups, and lines of honey were dispensed every day on the upper side of the cage. Every
24 h, 10 small fresh 'Jalapefios' (ca. 5 cm long) were placed in the cages with the
weevils, and 10 fruits placed 24 h previously were removed. The fruits were then held in
plastic containers, 60-70 fruits each, until the emergence of adults. These containers
(33x21x11 cm Rubbermaid Home Products Wooster, OH) had six lateral holes 3 cm in
diameter covered with polyethylene screen to ensure ventilation. Weevils that emerged
within the same week were collected and confined in a new oviposition cage. Each cage
was used for four or five weeks, because fecundity was assumed to decline thereafter and
because many weevils died during daily manipulations.
Crowding Effects on Oviposition
Production of eggs and presence or absence of oviposition plugs was evaluated for
different numbers of mated females alone and for different numbers of males and females
together. Weevils were held in clear plastic cups 250 mL, each provided with a single
fresh 'Jalapefio' pepper (4.95 + 0.61 cm long, 1.86 + 0.24 cm wide, and 8.57 + 2.02 g
weight, n=49). Individual peppers were vertically oriented using adhesive poster putty
(Henkel Consumer Adhesives, Inc. OH) on the tip of the fruits. Water was provided by
daily saturating a cotton wick placed in the bottom of each cup (Fig. 1).
Figure 1. Device for exposing different densities of weevil adults to 'Jalapefio' peppers.
The treatments were 1 female, 1 female/ 1 male, 2 females, 2 females/ 2 males, 5
females, 5 females/ 5 males, and 10 females. The large metatibial mucrones of males
were used to sex weevils (Eller 1995). To facilitate sexing, groups of 10-12 weevil adults
were placed in Petri dishes over a white background and were observed through a
dissection microscope at 16X magnification. An average of 22 females and 16 males
could be sexed in 10 min using this method (n=4). These seven treatments were arranged
in a randomized complete block design with three replications. Because it is assumed that
fecundity of pepper weevil is higher in the first weeks of its life (Patrock and Schuster
1992) and because of the availability of insects, each block was set up at different time
using insects 7-10 days old. 'Jalapefio' fruits were replaced daily for 7 d and the number
of eggs per day, and number of eggs with or without an oviposition plug, was evaluated.
To compare the number of eggs per female per day, data were analyzed using an
analysis of variance (PROC ANOVA, SAS Institute 2000) and means were compared by
Least Significant Difference (LSD) (P < 0.05). Data were normalized prior to the analysis
using the square root transformation (PROC CAPABILITY, SAS Institute 2000), but
data are presented in the original scale. The presence or absence of oviposition plugs by
treatment was analyzed using a chi-square test (df = 1, P < 0.05) to determine differences
between observed and expected (1:1) values (PROC FREQ, SAS Institute 2000). When
one of the cells had expected counts less than 5, the Fisher's exact test to contrast the null
hypothesis was used (PROC FREQ, SAS Institute 2000).
Fruit Quality
The number of adult weevil progeny obtainable using the ratio of 10 unsexed
weevils per fruit was compared for two fruit qualities: immature green and green mature
(marketable fruits). Prior to evaluation oviposition cages (3.8 L) containing 100 + 10
unsexed weevils 1-5 d old were provided with 10 immature fruits and 5 or 10, depending
on availability, floral buds. The fruits and buds were replaced daily for 3 days. After that,
one cage was assigned to one of two treatments: 10 immature green fruits (4.75 + 0.61
cm long, 1.77 + 0.25 cm wide, and 7.66 + 2.01 g weight, n=37), harvested from potted
plants grown outdoors, or 10 mature green 'Jalapefio' fruits (6.67 + 0.77 cm long, 2.06 +
0.41 cm wide, and 14.96 + 3.58 g weight, n=37), bought at the supermarket. Both
qualities of fruit were washed with liquid dish detergent and rinsed up with tap water
before offering to the weevils. Fruits were replaced every 24 h (5:00 PM to 5:00 PM) and
exposed fruits were held until adult emergence in the ventilated plastic containers used in
colony maintenance. Each cage of weevils was considered an experimental unit and was
evaluated for 22 days. Because of the availability of peppers three replicates were
conduced at different times during July and September of 2003. After checking the data
for normality (PROC CAPABILITY, SAS Institute 2000), statistical differences were
determined using the student's t-distribution (PROC TTEST, SAS Institute 2000).
Adult Feeding and Oviposition
One pepper weevil female and one male both 18 h old or younger were placed in
individual 250 mL plastic cups. A lid closed each cup and a 3 cm diameter organdy-
sealed hole provided ventilation (Fig 1). Each weevil pair was considered an
experimental unit and assigned to one of two treatments: a) peppers only or b) peppers
plus floral buds. Every day during the first 7 days of life, each weevil pair was provided a
cotton wick saturated in water, as well as either a single young pepper (length 3.23 +
0.40, width 1.07 + 0.15, n = 15) or a single young pepper plus three floral buds before
anthesis. The peppers were vertically oriented in the cups, as indicated above, and floral
buds were placed in the bottom of the cups. Each treatment had 8 replicates. Because
there were not enough floral buds for doing all replicates at the same time, the replicates
were performed in groups at different times (3, 3, and 2). After 24 h of exposure, food
items were replaced and observed under a stereoscopic microscope to evaluate number of
feeding and/or oviposition punctures. Punctures were dissected to determine the presence
or absence of eggs. Before the analysis, the data were normalized by the arcsine square
root transformation (PROC CAPABILITY, SAS Institute 2000), but means are presented
in the original scale. Statistical differences were determined using the student's t-test
(PROC TTEST, SAS Institute 2000). Results from weevils that received both floral buds
and fruits in the same cup were further analyzed to compare the preference of feeding and
ovipositing in floral buds or fruits using student's t-test (PROC TTEST, SAS Institute
Life Table
Fourteen weevil pairs 18 h old or younger were caged individually in plastic cups
as previously described. Males were removed after 7 d to avoid possible interference with
oviposition behavior (Elmore et al. 1934). A male was subsequently introduced into each
cage for 2 d every 4 wk for mating. The females, or pairs when the males were present,
received one fresh 'Jalapefio' pepper daily (length 3.23 + 0.40, width 1.07 + 0.15, n =
25). The pepper was removed and dissected to count the number of eggs. Two floral buds
were offered every day in addition to the fruits for the lifetime of the female, except
during week 2-4 due to short supply. The procedures were followed until death of the
The sex ratio of the species was estimated from field and laboratory samples, and
survivorship was evaluated from a sample of 102 peppers with only one weevil egg per
fruit from the laboratory colony. In addition, fertility was estimated from a sample of 171
eggs laid in immature peppers from the same colony. Previous observations indicated that
an oviposition plug ensured the presence of an egg in 91% of all cases (SE = 0.018, n =
216). Therefore, removing oviposition plugs to check for the presence of eggs was not
necessary. A permanent marker was used to mark the oviposition plugs and the fruits
were dissected 4 d later to look for larvae.
Using LIFETABLE.SAS (Maia et al. 2000), the whole data set was used to
estimate net reproductive rate (Ro), defined as the number of females produced by one
adult female during its mean life span; generation time (T), the period between the birth
of parents and the birth of the offspring; doubling time (Dt), the time necessary to double
the initial population; intrinsic rate of increase (rm), the potential growth of a population
under given conditions; and finite rate of increase (k), the daily rate of increase of each
cohort (Birch 1948, Maia et al. 2000, Southwood and Henderson 2000, Toapanta et al.
2005). Data reported by Elmore et al. (1934) and Toapanta et al. (2005) were subjected to
the same analysis and compared to results obtained in the present study.
Nitrogen Content of Fruits and Buds
The nitrogen content of anthers, pericarp and a mix of placenta and seeds of three
maturation stages of 'Jalapefios Mitla' was evaluated. These maturation stages were
classified according to size and appearance as: a) immature green, b) mature green, c)
mature red (Table 4). Four samples were collected from plants 2-4 months old which
were grown in a greenhouse. During each sampling, 70-90 floral buds just prior
dehiscence and 6-7 peppers of each maturation stage were collected. Anthers were
removed with forceps, and fruits were dissected to separate pericarp from placenta and
seed. Because of possible variation among harvests (June and July 2005), each sampling
was considered as a block.
Table 4. Characteristics of 'Jalapefio Mitla' used for nitrogen content determinations
Pepper quality (n=10) Length, cm Width, cm Wall thickness
Immature Green 4.24 + 0.24 1.12 + 0.14 0.18 + 0.02
Mature Green' 6.76 + 0.60 2.42 + 0.24 0.39 + 0.03
Mature Red 7.72 + 0.67 2.57 + 0.20 0.41 + 0.03
1Similar to fresh market quality
The samples of each botanical structure were placed in individual paper bags, dried
at 50 C for three days, and ground to a powder. Nitrogen content was determined by the
soil laboratory at the SWFREC using the alternative dry Micro-Dumas combustion
analysis for total nitrogen in solid phase samples. This method is based on transformation
to the gas phase by extremely rapid and complete flash combustion of the sample
material (Matejovic 1993, 1995, Anonymous 1997). There were four replications
(samples) in a randomized block design. The data were normalized by square root
transformation (Sokal and Rohlf 1969, Ott and Longnecker 2001) prior to ANOVA
(PROC ANOVA, SAS Institute 2000); however, the means and standard deviations are
presented in the original scale. Means were compared by the LSD test (LSD) (P < 0.05)
when the F value was significant.
Crowding Effects and Oviposition
The number of weevils per fruit had a significant influence on the numbers of eggs
per female per day (F = 12.47; df= 8,138; P < 0.0001). The largest number of eggs per
female per day was obtained with a single female per fruit followed by one female and
one male (Table 5). There were no differences among the remaining treatments (LSD test
P < 0.05). Increasing the number of weevils from 1 female to 2 females/2 males or even 5
females had no effect on the number of plugged eggs (Fisher's exact test P = 0.266 and
0.139, respectively). However, the percentage of unplugged eggs increased 5 or 6 fold at
densities of either 5 females/5 males or 10 females (Table 5). There was no differences
between these treatments (X2 = 2.61, P = 0.1064).
Table 5. Numbers of eggs per female per day for different numbers of females and males
per single 'Jalapefio' pepper fruit
Weevil density Eggs/female/day (Mean + SD)1 Unplugged eggs (%)
1 5.0 + 2.43 a 5 (4.76)
1 1 3.38 + 1.99b 2 (2.82)
2$ 2.05 +1.37c 1 (1.16)
2 92 1.97 +0.8c 3 (3.61)
5 $ 1.68 + 0.54 c 4 (2.26)
5 9 5 1.74 + 0.82 c 25 (13.66)
10 Y 1.57 + 0.48 c 30 (9.10)
Means with the same letter are not significantly different (LSD P < 0.05)
Fruit Quality
Immature green and green marketable fruits were both able to support the rearing
of pepper weevil, although there were significant differences. Using the 10 weevils per
fruit density 2.65 + 1.16 weevil adults emerged per immature fruit, compared to only 1.11
+ 0.81 weevils per marketable fruit (t = 8.81, df= 65, P < 0.0001).
Feeding and Oviposition in Peppers versus Floral Buds (7d)
When floral buds and peppers were offered at the same time, females fed and laid
eggs in both (Table 6). Nevertheless, weevils preferred feeding on floral buds instead of
fruits (t = 13.39, df = 55, P < 0.0001). In contrast, females preferred to lay eggs in pepper
fruits rather than floral buds (t = 9.59, df = 55, P < 0.0001).
Table 6. Feeding and oviposition of pepper weevil adults when given a choice of
'Jalapefio' pepper fruit or floral buds (7d)
Parameter Pepper fruits Floral buds t-test (P < 0.05)
Feeding punctures per day 3.27 + 4.99 18.75 + 8.64 *
Eggs per day 12.73 + 10.46 0.16+0.49 *
Oviposition with or without Floral Buds (7d)
Differences in oviposition were observed between females that were provided with
pepper fruits only, compared to those that were provided with both floral buds and fruits
(Table 7). Females provided with fruits and buds laid many more eggs the first day of
oviposition (t = 2.81, df = 14, P = 0.0261), and over the entire 7 d test period (t = 5.08, df
= 14, P = 0.0002) than females provided only with fruits. Pepper weevil females laid only
1.25% of eggs in the floral buds when both floral buds and fruits were provided at the
same time.
Table 7. Oviposition of pepper weevil females fed with 'Jalapefio' pepper fruits only or
with pepper fruits plus floral buds
Parameter Peppers Pepper plus floral t-test (P < 0.05)
Days to begin oviposition 2.87 + 0.99 2.62 + 0.52 ns
Eggs at first day of 3.50 + 1.41 11.87 + 8.30 *
Total number of eggs (7 d) 23.75 + 11.16 89.12 + 34.65 *
Life Table
The 14 females survived 28 to 103 d, with an average of 64.5 + 25.8 d. During this
time 8 of 14 females (57%) began oviposition on the second day, and 13 of 14 females
were depositing eggs by the third day (Fig. 2). The remaining female took seven days to
begin oviposition. Fecundity ranged from 0 to 32 eggs per female per day with an
average of 5.51 + 5.31 eggs per female per day, and declined to near zero by 86 d. The
fertility of eggs deposited in immature fruits was 97.66% (n = 171, SD = 0.15) and the
survivorship of all preimaginal stages was 89% (n = 102, SD = 0.31). These data and the
sex ratio of the species, that was not significantly different from 1:1 either in the field or
in the laboratory (X2 =0.95, P < 0.05, X2 =0.35, P < 0.05, respectively), were used to
estimate the demographic parameters of pepper weevil (Table 8).
0 12
1 6 11 16 21 26 31 36 41 46 51 56 61 66 71 76 81 86 91 96101
Age in days
Figure 2. Mean fecundity of adult pepper weevil females (n = 14) reared on 'Jalapeio'
peppers at 27 + 2 C and 14:10 (L:D) photoperiod.
Table 8. Life table parameters for A. eugenii females (n =14) reared on 'Jalapefio'
peppers at 27 + 20C, and 14:10 (L:D) photoperiod
Parameter Value (+ 95% confidence limits)
Net reproductive rate (Ro)a 158.1 (115.1 201.1)
Intrinsic rate of increase (rm)b 0.14 (0.12 0.15)
Generation time (T)c 36.04 (31.26 41.22)
Doubling time (Dt)' 4.93 (4.42 5.49)
Finite rate of increase (X) d 1.15 (1.13 1.17)
aFemale/female, bloge (Ro)/T, cDay, dFemale/female/day.
Nitrogen Content of Fruits and Buds
Nitrogen content was different among botanical structures and maturation stages of
peppers (F = 41.61; df = 6, 27; P < 0.0001). Highest nitrogen content occurred in the
anthers of floral buds. An intermediate concentration occurred in placentas and seeds at
any maturation stage, and in pericarp of the immature green fruits. The lowest nitrogen
concentration was detected in pericarp of mature green and mature red fruits (Table 9).
Table 9. Nitrogen content (Mean + SD) of anthers in floral buds and 'Jalapefio' fruits at
three maturation stages
Maturation stage Botanical structure N content (%) dry weight
Floral buds Anthers 5.23 + 0.09 a
Immature green Placenta and seeds 3.39 + 0.04 b
Mature red Placenta and seeds 3.21 + 0.25 bc
Immature green Pericarp 3.19 + 0.38 bc
Mature green Placenta and seeds 2.96 + 0.09 c
Mature green Pericarp 2.25 + 0.42 d
Mature red Pericarp 2.01 + 0.35 d
Means followed by the same letter are not significantly different (LSD, P < 0.05)
Crowding Effects and Oviposition
Increasing density of female pepper weevils per fruit reduced oviposition and
increased the number of eggs lacking their normal oviposition plugs. An individual
female produced more eggs than a female and a male together, and both treatments had
higher number of eggs per female than two females together. The first comparison
confirmed a previous study by Elmore et al. (1934) that suggested that the presence of
males could inhibit oviposition behavior of females. However, the second comparison
indicated that inhibition of oviposition also could occur with the presence of more
females. Similar results with unsexed weevils were reported by Toapanta (2001), who
indicated that a ratio of five unsexed weevils per fruit were more efficient for producing
adults than larger ratios (10:1, 15:1, 20:1).
Crowding by either males or females interfered with the oviposition process. Males
may interfere by seeking to mate with females, and females may interfere by contending
with each other for oviposition resources. The final phase of oviposition is deposition of
the plug, so the same factors might have influenced the presence of eggs lacking
oviposition plugs.
Feeding could also contribute to the reduction of oviposition plugs by crowding.
Pepper weevil prefers both to oviposit and to feed close to the calyx (Walker 1905,
Elmore et al. 1934, Wilson 1986, Toapanta et al. 2005). More weevils per fruit would
mean more feeding punctures and, consequently, more mechanical damage to tissue close
to the calyx, including the plugs.
These results demonstrated why rearing the pepper weevil by using large numbers
of weevils per fruit is inefficient. A small number of insects per fresh 'Jalapefio', even a
single mated female, may be the most efficient combination. Additionally, this system
maximizes the number of oviposition plugs which may prevent dehydration of eggs as
well as exposure to opportunistic predators, such as mites or ants.
Fruit Quality
Quality of the host can exert an important influence on fecundity and survival of
insects (Awmack and Leather 2002). Exposing pepper weevil females to immature green
peppers resulted in more offspring than exposing females to marketable fruits (= mature
green). The nitrogen content in the pericarp of immature fruits was higher than in mature
green fruits (Table 9). Partitioning of nitrogen by fruit age and tissue could affect the
capacity of both larvae and adults to acquire nitrogen.
Eggs are laid generally in the pericarp of peppers (Walker 1905, Elmore et al. 1934,
Goff and Wilson 1937) due to the small size of the pepper weevil proboscis (Clark and
Burke 1996). Eggs laid in older fruits will produce larvae that have to bore through a
thicker pericarp, which is lower in nitrogen compared to an immature fruit (Tables 4, 9).
Additionally, the maturation process in peppers includes substituting soft tissues with
fiber, the clearest example being the seed coat on mature fruits (Bosland and Votava
1999). The high nitrogen content in mature seeds may not be accessible to the larva.
Thus, thickening of the pericarp and redistribution of nitrogen to seeds during maturation
of pepper fruits could explain why the pepper weevil prefers to feed and to oviposit on
immature fruits, and why the rearing process of weevils could be improved if immature
fruits are used.
Adult Feeding and Oviposition
Early observations of pepper weevil adults in the field indicated a feeding
preference for floral buds compared to fruits (Walker 1905, Elmore et al. 1934). These
observations were later corroborated with laboratory tests (Wilson 1986, Patrock and
Schuster 1992). The data presented here corroborate this behavior in 'Jalapefio' floral
buds and fruits, and demonstrate the likely cause. Many more feeding punctures were
made per floral bud compared to 'Jalapefio' fruit. Patrock and Schuster (1992) reported a
mean of 0.74 and 0.75 feeding punctures for floral bud and fruit, respectively, produced
by a single female. This contrasted with 3.27 feeding punctures in fruits against 18.75
feeding punctures on floral buds using a pair of weevils in this study. Patrock and
Schuster (1992) used 10-20 d old females in bell pepper fruits ('Early Calwonder') at 22
+ 4C, while 1-7 d old weevils in 'Jalapefio' fruits at 27 + 2C were used in the present
study. More information related to the quality of the varieties of peppers and the vigor of
the insects used could help to better understand those differences.
Patrock and Schuster (1992) indicated that a female laid more eggs on immature
fruits than floral buds (0.96 and 0.36 eggs, respectively), because fruits offered more
resources to support larvae. Data collected here (0.16 eggs per floral bud and 12.73 eggs
per fruit) followed the same trend, although the number of eggs laid per fruit was 10
times greater.
The seven day oviposition experiment provided information on the ability of floral
buds to increase fecundity. The preoviposition period was not affected by access to floral
buds, and a similar value (2 or 3 days) was reported by Elmore et al. (1934), Wilson
(1986), Gordon and Armstrong (1990), and Toapanta et al. (2005). Nevertheless, the
number of eggs at the first day of oviposition (3.5 vs 11.9) and the total number of eggs
during the first seven days after emergence (23.7 vs 89.1) was always higher for females
that received anthers. The total number of eggs deposited in the first seven days of life of
females provided with immature 'Jalapefio' fruits and floral buds was greater than any
number reported before for this species in previous works (Elmore et al. 1934, Wilson
1986, Gordon and Armstrong 1990, Patrock and Schuster 1992, Toapanta et al. 2005),
probably because of feeding on anthers. An effect from feeding on floral buds (= cotton
squares) has been reported for the boll weevil, which needs to feed on floral buds for the
ovaries to develop (Hunter and Hinds, 1905, Hunter and Pierce 1912, Isley 1928, 1932).
The pepper weevil is able to obtain sufficient nutrients for egg production from
pepper fruits alone, as the majority of references in Table 1 indicate, and it is common
practice to rear the weevils on a strict diet of fruit. The present comparison was
conducted for only 7 days, so it is not possible to say that total fecundity would be lower
on a strict fruit diet. Possibly, the resulting nitrogen deficiency could be compensated by
increasing consumption, as has been observed for other species including Celerio
euphorbiae L. (Lepidoptera: Sphingidae), and Pieris rapae L. (Lepidoptera: Pieridae)
(House 1965, Slansky and Feeny 1977, Awmack and Leather 2002).
In addition to nitrogen content, a proper balance of amino acids is critical for
growth and reproduction in insects (Linding 1984, Weis and Berenbaum 1989, Nation
2002). Therefore, the increased fecundity observed for the pepper weevil could also be
due to the balance of amino acids that might be present in floral buds. Suitable diets for
the boll weevil were developed only after developing a basic knowledge of the proteins
and free amino acids in anthers of young cotton flowers (Earle et al. 1966, Hilliard 1983).
Further studies that identify the proteins and amino acids of anthers of peppers could be
useful in the development of a better artificial diet for pepper weevil adults.
Life Table
Pepper weevils laid a mean of 341 eggs per female with an average of 4.7 eggs per
day over an average life span of 78.7 d (Elmore et al. 1934). No temperature was
recorded in that study. Goff and Wilson (1937) reported a fecundity of 198 eggs with an
average of 6 eggs per day. Gordon and Armstrong (1990) reported 253.6 eggs over a life
span of 31.7 d, with an average of 8 eggs per day at 22 to 27C. Recently, Toapanta et al.
(2005) reported a wide range of fecundity for this species according to the number of
fruits offered to the weevils and temperature. Using eight females per group, these
authors reported 3.1 eggs per day at 300C and 158 eggs per female in single peppers over
51 days. Offered 3 peppers per day at 270C, females were able to lay 3.9 eggs per day or
281 eggs over 72 d. The fecundity of pepper weevil females fed with floral buds observed
in this study was 355 eggs per female, with an average of 5.5 eggs per day and a life span
of 64.5 d.
During the adult stage of insects, the reproductive potential is fully realized only if
the females have an adequate supply of energy for surviving and of nutrients for egg
production (Hilliard 1983, Awmack and Leather 2002). The biology of the pepper weevil
has been studied for more than a hundred years, but just three studies (Elmore et al. 1934,
Gordon and Armstrong 1990, and the present) provided floral buds along with fruits to
females when estimating fecundity. Not surprisingly, these studies reported the highest
fecundity for this species. Fecundity is the major component determining population
dynamics of a species and, along with survivorship, influences all demographic
parameters (Birch 1948, Southwood and Henderson 2000). Consequently, the
demographic parameters based on fecundity were higher for data reported by Elmore et
al. (1934) and the present study (Table 10).
Table 10. Life table parameters for the pepper weevil
Parameter Elmore et al. (1934)' Toapanta et al. This study
Net reproductive rate (Ro) 153.45 33.57 158.10
Intrinsic rate of increase (rm) 0.09 0.11 0.14
Generation time (T) 53.22 32.39 36.04
Doubling time (Dt) 7.33 6.35 4.93
Finite rate of increase (k) 1.10 1.11 1.15
'The sex ratio and survivorship used to do the statistical analysis were 0.5 and 0.9, respectively.
No temperature data provided.
The Ro value for pepper weevil females fed with immature 'Jalapefio Mitla' and
floral buds was 4.6 times greater than the highest value reported by Toapanta et al.
(2005). These authors fed the insects only with 'Serrano' or 'Jalapefio' peppers, but no
floral buds. Furthermore, the condition or quality of fruits was not indicated. The
Generation time (T), and Doubling time (Dt) reported by Elmore et al. (1934) were larger
than those observed in this study. This could be a consequence of distributing the eggs
evenly over the entire oviposition period. It was not possible to know the true distribution
of eggs over time to make a better estimation. Therefore, both parameters might be
overestimated. If the generation time of this study is used with the approximation
indicated by Birch (1948) to calculate the intrinsic rate of increase (rm = loge Ro / T), the
rm for Elmore et al. (1934) data set is similar to this study (rm = 153.45/36.04 = 0.14).
The difference in results in this study versus Elmore's data are due to generation
time, with fecundity (as indicated by Ro) being the same. These values represented the
potential of this species when feeding on floral buds and immature fruits, a situation that
would occur in the field. It might explain in part the success of this species in infesting a
young pepper crop, when floral buds and immature fruits are abundant. These
demographic parameters could be useful for: a) comparisons of different rearing
conditions, b) comparisons of population dynamics with natural enemies, and c)
predictions of the potential of biological control agents to control this pest.
LIFE HISTORY OF Triaspis eugenii Wharton and Lopez-Martinez (HYMENOPTERA:
The pepper weevil, Anthonomus eugenii Cano (Coleoptera: Curculionidae), is
considered one of the key pests of peppers in the United States, Mexico, Central
America, and some Caribbean Islands (Elmore et al. 1934, Riley and King 1994,
Mariscal et al.1998, Schuster et al.1999). Adults lay eggs in floral buds and immature
peppers, and larvae develop inside beyond the reach of insecticides. Consequently,
chemical control targets only adults. An action threshold of a single adult weevil per 400
pepper terminals has been proposed (Riley et al. 1992 a,b). This low action threshold, and
the use of insecticides as the principal method of control, can result in adverse effects
including marketing restrictions, exposure of non-target organisms, environmental
contamination, pesticide resistance, and secondary pest outbreaks (NAS 1969, Luckmann
and Metcalf 1982, Doutt and Smith 1971). Unfortunately, no other viable control strategy
for the pepper weevil has been developed during the one hundred years that have elapsed
since the pest was first reported in Mexico (Cano and Alcacio 1894) and the United
States (Walker 1905, Pratt 1907).
Biological control could be an alternative tactic to incorporate into integrated pest
management (IPM) of the pepper weevil; however, sufficiently effective natural enemies
have yet to be discovered. One of the natural enemies of pepper weevil which has
received attention as a potential biological control agent is the generalist and
cosmopolitan parasitoid Catolaccus hunter Crawford (Hymenoptera: Pteromalidae). This
ectoparasitoid of the last instar of the pepper weevil has a greater fecundity than its host
(Rodriguez et al. 2000, Seal et al. 2002). It has been recorded as one of the most common
parasitoids of pepper weevil in some states of Mexico (Mariscal et al. 1998, Rodriguez et
al. 2000), Central America, and Florida (Cross and Mitchell 1969, Wilson 1986, Riley
and Schuster 1992, Schuster et al. 1999). However, releasing the equivalent of 1150 C.
hunter adults per hectare per week in field plots at Sinaloa, Mexico, was not effective
against this pest (Corrales 2002). On the other hand, results of releasing 7900 C. hunter
per hectare, prior to and during the early pepper season, indicated that this parasitoid
could reduce damage by the pepper weevil in Florida (D. Schuster, personal
communication). Divergent results may be due, in part, to the fact that C. hunter attacks
the 3rd instar host that is inaccessible deep within the pepper fruit. Augmentative releases
of this parasitoid prior to the crop cycle on the alternative host plant nightshade, which
has small fruits, could reduce pepper weevil populations. Releases early in the crop cycle
when fruits are small, or in small fruited varieties, might be effective because the host is
more accessible to C. hunter. Unfortunately, once the fruits increase in size the effect of
the parasitoid is limited. In fact, Riley and Schuster (1992) indicated that C. hunter was
not detected in fallen fruits larger than 2.5 cm in diameter. Therefore, it is desirable to
find species that may be effective as biological control agents of this pest, preferably
those that attack earlier and more accessible life stages.
A candidate parasitoid was recently reported in Mexico, where pepper, pepper
weevil, and parasitoids likely evolved. This species is considered the most important
parasitoid of the pepper weevil in Nayarit, Mexico, where it causes the highest incidence
of parasitism on pepper weevil (from 18 to 40%) in field samples (Mariscal et al. 1998,
Schuster et al. 1999, Toapanta 2001). This solitary endoparasitic wasp was described in
2000 as Triaspis eugenii Wharton & Lopez-Martinez (Hymenoptera: Braconidae).
However, there is still little information available on its biology and behavior (Sharkey
1997, Mariscal et al. 1998, Toapanta 2001).
This Chapter focuses on the biology and life history of T. eugenii. Initial efforts to
maintain the parasitoid colony were hampered by low levels of parasitism and high levels
of superparasitism. These factors limited reproductive potential and, thus, reliable
estimates of demographic parameters. In addition, inefficient rearing demanded excessive
resources (peppers and hosts). Preliminary observations in the laboratory suggested two
possible causes for low levels of parasitism: 1) a limited acceptable range of host-age for
parasitizing, and 2) the inability of females to find or recognize the host. One hypothesis
to explain this latter difficulty was that exposing pepper fruit to too many weevil adults in
the colony resulted in fewer eggs sealed with an oviposition plug (Chapter 2). The lack of
acceptable hosts could then result in either retention of eggs or in excessive
superparasitism. These questions required a solution before demographic parameters
could be reliably estimated and before the potential of the parasitoid as a biological
control agent could be evaluated. The objectives of this study then were to determine the
host lifestage suitable for parasitization by T. eugenii, to determine the parasitoid's ability
to recognize and to parasitize plugged and unplugged host eggs, to describe the life cycle
of the parasitoid, and to estimate demographic parameters for this species.
Materials and Methods
Based on results of Mariscal et al. (1988) and Toapanta (2001), two surveys for T.
eugenii were conducted in pepper growing regions at Nayarit, Mexico. In April and May
of 2003, sampling was conducted in Puerta de Mangos, Caiada del Tabaco, Villa Juarez,
Los Corchos, Los Medina, and Palma Grande, all located in Santiago Ixcuintla county,
Nayarit, between 21 36' and 22 17' north latitude, and between 104 53' and 1050 40'
west longitude (INEGI 1999).
Hurricane Kenna made land fall in San Blas on October 25, 2002, just 30 km from
Santiago Ixcuintla and many pepper fields were destroyed. Late replanting resulted in
delayed harvest, with the result that fields included in the first survey in April 2003 were
still being managed for weevils with insecticides. During the second survey in May 2003,
the majority of peppers were collected from abandoned pepper crops. Pepper fruits with
signs of damage of the weevil (including presence of feeding and/or oviposition
punctures) were collected from the plants and/or from the ground. A random sample
(n=57) of these fruits was dissected to confirm weevil presence. Around 25 kg of fruit or
about 3,300 peppers were collected in each survey. All were Capsicum annuum L. The
dominant variety was 'Serrano' but local cultivars such as 'Serranillo', 'Caloro', and a
few 'Jalapefio' were also collected. Fruits were held in plastic nets of 2 kg in the field and
later at room temperature to avoid water condensation. Within 3 d the plastic nets
containing the peppers were placed in individual Ziploc plastic bags (S.C. Johnson &
Son, Inc. WI) provided with a paper towel to absorb moisture, and transported by air to
the United States in two insulated coolers provided with ice substitute (Rubbermaid Inc.,
Wooster, OH). The material was allowed to enter in the United States under the permit
46799 of the Animal and Plant Health Inspection Service (APHIS), USDA.
Upon arrival in Miami, the material was brought immediately to the quarantine
facilities at the Florida Department of Agriculture and Consumer Services (FDACS),
Division of Plant Industry (DPI), in Gainesville, Florida. In the presence of the quarantine
officer, the peppers were distributed into lidded plastic containers (30x23x10 cm). Each
container had 4 lateral holes, 5 cm in diameter, covered with fine mesh screen for
ventilation. A paper towel was placed at the bottom of each container to absorb excess
moisture. These emergence containers were held in the maximum security room in
quarantine at 27 + 30C, 50 to 70% RH, and 12:12 (L:D) h photoperiod. All the
parasitoids except T. eugenii were preserved in 70% ethanol and labeled and identified
with the aid of available literature. Identifications of braconids and chalcids were verified
by R. Wharton and R. Lomeli, respectively, both of Texas A&M University, College
Station. Voucher specimens were deposited at the Florida State Collection of Arthropods
at DPI.
Parasitoid Rearing
Wasps were held in 3.8 L plastic cages (Chapter 2) with the lateral holes sealed
with organdy. Parasitoids had free access to water in a cotton wick placed in 28-mL
plastic cups, and to lines of honey dispensed every day on the upper side of the cage. In
addition, a 7-8 cm long pepper flush (of leaves and floral buds) was inserted in a 28 mL
plastic cup filled with water and placed in each cage to simulate natural host finding
conditions. The flush had been exposed for 48 h to three pepper weevil adults.
Pepper weevil hosts were obtained from a colony held in Florida Reach-In
Incubation chambers (Walker et al. 1993) maintained at 27 + 1IC and 70-80% RH, and
14:10 (L:D) h photoperiod. Immature 'Jalapefio' fruits (4.7 + 0.77 cm length, 1.56 + 0.27
cm width, and 5.57 + 1.07 g weight, n = 25) were exposed to weevils for 24 h at a ratio of
10 weevils per fruit. Under these conditions, pepper weevil eggs take 2.9 + 0.1 d to hatch,
and an additional 1.7 + 0.1 d to reach the second instar (Toapanta et al. 2005).
Accordingly, two age ranges of eggs (2-24 h and 26-48 h after weevil exposure) and first
or early second instars (4 to 5 d after infestation), were offered to the wasps. Each cage
contained one or two T. eugenii females. At least two pepper fruits of each age category
were offered daily to the wasps beginning the second day after emergence. Following
exposure, fruits were held in ventilated containers until emergence of insects.
When it became apparent that parasitoids were only recovered from peppers that
had weevil eggs, the exposure period of fruits to weevils in the colony was limited to 24
h. Fruits were replaced between 4:00 to 6:00 PM, held at 22 + 20C until 10:00 AM the
next day, exposed to parasitoids for 24 h, and then held as above.
After the second generation of parasitoids, 10-15 females and 10-12 males were
included per cage to maintain a ratio of one parasitoid female per infested fruit per day.
Each cage was used 10-12 d. Females emerging from one cage were used to mate with
males emerging from a different cage to prevent inbreeding within the colony. Using this
rearing system, 80-100 females and 100 males were held each generation until the colony
could be moved from quarantine during October and November, 2003. The colony was
then held at the Entomology and Nematology Department, University of Florida,
Gainesville, at 27 + 1C and 70-80% RH, and 14:10 (L:D) h photoperiod for five months.
The colony was moved in April 2004 to the South West Florida Research and Education
Center (SWFREC) at Immokalee FL, and was maintained in a rearing room at 27 + 20C,
60-70% RH, and 14:10 (L:D) h photoperiod. Experiments were conducted under these
environmental conditions, unless otherwise indicated. 'Jalapefio Mitla' was the variety
used most in rearing the colony and in conducting experiments, but when this cultivar
was not available, 'Jalapefio Ixtapa' was used.
Host-Age Range
Choice tests were conducted during December 2003 to compare the acceptance of
eggs either 24-40 h or 48-64 h old. 'Jalapefio' fruits 5.25 + 0.53 cm length, 1.68 + 0.15
cm width, and 7.41 + 0.70 g weight, (n = 15), were exposed the required time to 10
weevil females per fruit. Fruits with eggs were offered to the parasitoids for 8 h.
A no-choice test was conducted during June 2005 and included eggs either 3.5,
27.5, 51.5 or 75.5 + 3.5 h old. 'Jalapefio' fruits (4.73 + 0.68 cm length, 1.48 + 0.26 cm
width, and 5.26 + 0.67 g weight, n = 10) were exposed seven hours to female weevils at a
density of one weevil per fruit. Fruits with eggs of the first age class were used without
storage while fruits in the other age classes were held at 22 + 20C and 50-60% RH for 24-
72 h before being offered to T. eugenii. Infested fruits were exposed to parasitoids for 2
h. Three randomly selected weevil eggs were removed under a dissecting microscope
from peppers in each age class to observe development. Heat units for each age,
including the time exposed to the wasp, were proportionally estimated using the average
temperature for each period. Heat units = (maximum temperature + minimum
temperature/2) threshold temperature. The threshold temperature was considered to be
For the choice test, an experimental unit was considered a group of 10 infested
peppers, five for each egg age class, offered at the same time to 20 T. eugenii females.
Five replicates were run. For the no-choice test, the experimental unit was five infested
peppers of each egg age class, which were offered simultaneously to 10 T eugenii
females, there were three replicates. Because parasitoid females were used only once in
each replicate in both assays and many parasitoids were needed, replicates were carried
out in different days. Parasitoids were 5-10 d old and they were randomly taken from the
colony in any replicate. Fruits were removed after exposure, and held in ventilated
containers until the emergence of insects. Chi-square analysis (PROC FREQ, SAS
Institute 2000) was used to test if parasitism was independent of age.
Role of the Oviposition Plug in Host Location
Choice and no-choice tests were conducted during May and June 2004 to determine
the importance of the oviposition plug to T. eugenii for finding and parasitizing host eggs.
Previous observations showed that the oviposition plug indicated the presence of an egg
in 91% of all cases (Chapter 2). Host eggs for these tests were obtained from groups of
three weevil females, 7-10 d old, placed in 250 mL plastic cups. Each group was
provided with a 'Jalapefio' pepper (4.65 + 0.48 cm length, 1.93 + 0.29 cm width, and
7.40 + 1.47 g weight, n=50) for 24 h. Subsequently, fruits were held at room temperature
(22 + 20C and 60-70% RH), before exposure to the parasitoid the next day. Only pepper
fruits with 10 eggs were used. Excess plugs and eggs (Fig. 3) were removed from the
fruits using fine-tipped forceps.
Each experimental unit was considered as one or two pepper fruits, depending on
the test, offered to an individual T. eugenii female 4-6 d old. Subject parasitoids were
randomly selected from the colony and used only once. Each female was placed
individually in 1.9 L plastic containers (12x11x15 cm) with two males to ensure mating.
Water and honey were provided as previously described. Infested fruits were exposed to
the parasitoids for 24 h.
Figure 3. Longitudinal section of an infested 'Jalapefio' fruit showing a pepper weevil
egg and oviposition plug (ov-p).
Choice tests were conducted by offering two pepper fruits (10 weevil eggs each) to
individual T eugenii females. The oviposition plugs were removed from half of the eggs
of each pepper 24 h before the test. Plugs were removed under a stereoscopic microscope
using forceps, and a cotton swab was used to clean any visible residue. No-choice tests
were conducted by offering a single pepper fruit with ten weevil eggs, either with or
without oviposition plugs. Weevil eggs were later removed, placed in a drop of tap water
on a microscope slide (4 or 5 at once), and crushed under a glass cover slide. A binocular
composed microscope was then used to observe parasitoid eggs (100X magnification).
Fourteen replicates were run for each treatment, and data were analyzed using chi-square
tests to determine the differences between observed and expected values (PROC FREQ,
SAS Institute 2000).
Effect of Weevil: Fruit Ratio on Parasitoid Production
Cages (3.8 L) containing 20-30 T. eugenii females and 20-25 males, 1-3 d old, were
used as the experimental unit. The percentage of parasitism was compared between
'Jalapefio' fruits exposed 24 h to unsexed weevils (ratio 10:1 fruit), or exposed 24 h to
mated females (ratio 1:1 fruit). Daily during 10 consecutive days 13 infested fruits (4.44
+ 0.72 cm length, 1.47 + 0.23 cm width, and 4.83 + 1.10 g weight, n = 30) of each pepper
weevil ratio, exposed to weevil adults as above, were placed in the corresponding
parasitoid cage. Fruits were exposed for 24 h to the parasitoids the first and the last day,
but only 2.5 h the remaining 8 d. Fruits were removed and held in plastic containers until
emergence of insects. Three replications were run for each treatment during September
and October, 2004. Data were evaluated for normality (PROC CAPABILITY, SAS
Institute 2000) before the analysis, and statistical differences were determined using
student's t-distribution (PROC TTEST, SAS Institute 2000).
Life Cycle
'Jalapefio' pepper fruits containing pepper weevil eggs 18-40 h old were obtained
by exposing the fruits 24 h to a density of 10 weevils per fruit (5.29 + 0.80 cm length,
1.67 + 0.24 cm width, and 5.90 + 1.40 g weight, n = 10). An experimental unit consisted
of eight infested fruits exposed 2 h to T. eugenii in cages each containing 15 females and
20 males 5-7 d old. Fruits were then removed and incubated in ventilated containers at 27
+ 1IC, 50-60% RH, and 14:10 (L:D) photoperiod. Eight experimental units were run
concurrently. In addition, 10 infested pepper fruits from the same batch, but not exposed
to the parasitoids, served as control for the whole experiment.
To determine incubation time of T. eugenii 40 to 60 weevil eggs, usually one egg
per fruit of each experimental unit, were removed and dissected from fruits at 18, 20, 22,
and 24 h after parasitoid exposure. In addition, ten to 15 control eggs were dissected each
time to confirm the absence of parasitoids. Length and greatest width of parasitoid eggs
18-20 h old (n=33) were measured using a binocular microscope fitted with an ocular
micrometer calibrated with a stage micrometer (parallel lines at 10 [a).
The remaining pepper fruits (64 exposed and 10 unexposed) were held under
conditions described above for 9 days after parasitoid exposure. Peppers were then
dissected and all prepupae were removed, together with the completed or partially
completed pupal cell, and held in individual polystyrene cells (Multiwell, 24 well plates
with lid, Becton Dickinson and Co.). Cells were filled with paper towel packed into the
cells with no water to support the pupal cells, which were observed every 4 h during the
first 3 d, and then twice a day (9:00 AM and 6:00 PM) afterwards until the emergence of
adults. The Multiwell cells with the host and parasitoids were maintained at 27 + 1IC,
50-60% RH, and 14:10 (L:D) photoperiod, but the observations were made at room
temperature (220C).
Twenty unexposed and 160 exposed pepper weevil larvae were isolated as above,
but only data from those parasitoids that were observed emerging from the host or
spinning their cocoons and successfully reaching the adult stage are reported.
Development times were calculated for the egg, endoparasitic and ectoparasitic larval
stages, prepupa (spinning cocoon), and pupa. Stages were considered completed when
50% plus 1 individuals reached the next stage. The prepupal stage was considered to
occur from the cessation of larval feeding through the completion of the cocoon. Means
and standard deviations were calculated for each stage.
During September to December 2004, 10 adult T eugenii females 18 h old or
younger were placed individually in 3.8 L plastic containers. Three males were included
in each cage to ensure mating. Each cage was provided daily with 'Jalapefio' fruits
previously exposed for 24 h to pepper weevil females at the density of one pepper weevil
per fruit (4.60 + 0.45 cm length, 1.94 + 0.30 cm width, and 7.37 + 1.52 g weight, n = 46).
Weevil adults were provided water and honey as previously described. Five of the T
eugenii females were each provided every day from 9:00 AM to 6:00 PM with 12 fresh
peppers, each containing 4-8 pepper weevil eggs. The other five females were each
provided with two peppers fruits replaced every 1.5 h during the same period, so that the
total number exposed was also 12 fruits per female. Every day, half of the pepper weevil
eggs of each fruit were removed and dissected to count the number of T. eugenii eggs per
host using the methodology described above. The remaining eggs and fruits were held
until the emergence of T eugenii to estimate survival and sex ratio. The program
LIFETABLE.SAS (Maia et al. 2000) was used to estimate net reproductive rate (Ro),
generation time (T), doubling time (Dt), intrinsic rate of increase (rm), and finite rate of
increase (k) (Chapter 3) (Birch 1948, Maia et al. 2000, Southwood and Henderson 2000).
Frequency of superparasitism between treatments was compared using chi-square
analysis (PROC FREQ, SAS Institute 2000).
Adult Longevity
T. eugenii adults 12 h old or less were caged in two 1.8 L containers as follows: 1)
10 females and 10 males provided with water only, and 2) 10 females and 10 males
provided with honey and water. The wasps were observed twice a day (9:00 AM and 7:00
PM) and mortality was recorded until all died. The two treatments were replicated three
times. A comparison of longevity was also made between these females and ovipositing
females from the fecundity experiment. The Student's t test (PROC TTEST, SAS
Institute 2000) was used to make all comparisons.
About 50 kg of pepper fruits infested by pepper weevil larvae were collected under
natural conditions from Nayarit, Mexico. Weevil and parasitoid recovery varied with
location (Table 11), although fruits from all sites were severely infested. Although a
mean of 1.40 + 0.97 weevil per fruit was observed in the field from a random sample of
57 fruits, only an average of 0.73 weevils per fruit emerged in quarantine. Two genera of
Braconidae (including Triaspis eugenii) and one each of Pteromalidae, Eurytomidae and
Eupelmidae were recovered, accounting for only 2.85% of total parasitism (Table 11).
The dominant species recovered from both collecting trips was C. hunter, which
represented about 83% of emerged parasitoids.
Table 11. A. eugenii and parasitoids emerged from approximately 50 kg of infested hot peppers collected in the state of Nayarit,
Mexico, April and May, 2003.
Location (sites visited) Anthonomus eugenii Catolaccus hunter Triaspis eugenii Eurytoma sp. Eupelmus sp. Bracon sp.
Cafiada del Tabaco (3)
Los Corchos (2)
Puerta de Mangos (1)
Cafiada del Tabaco (1)
Los Corchos (5)
Los Medina(3)
Palma grande (1)
Villa Juarez (1)
A p r il 2 0 0 3
1 2
1 1
4 1 1 1
4 5 2 6 1 1 0
Host-Age Range
Heat units were calculated for each age class to precisely determine which ones
could be parasitized (Tablel2, 13). T eugenii preferred younger eggs to older eggs in
choice tests (X2 = 5.88, df = 1, P= 0.015) (Table 12). Nevertheless, T eugenii was
equally able to parasitize eggs over four age classes when given no choice (X2 = 1.53, df
= 1, P = 0.674) (Table 13). The eyes and mandibles of pepper weevil larvae were visible
through the chorion in the most mature eggs before wasp exposure.
Table 12. Parasitism of pepper weevil eggs by T eugenii when given the choice of two
ages of eggs
Host maturity Parasitism
Heat units' Age (h) Not parasitized Parasitized
Number Percentage2
12.66 28.66 24 48 51 64 55.6
23.66 39.66 48 72 98 68 40.9
'Accumulated heat units based on 11 C threshold temperature
2X2 = 5.88, df= 1, P = 0.015
Table 13. Parasitism of pepper weevil eggs by T eugenii when given no choice of egg
Host maturity Parasitism
Heat units1 Age (h) Not parasitized Parasitized
Number Percentage2
0.10-4.66 0 7 6 13 68.4
12.45-15.66 24 31 10 29 74.3
23.45-26.66 48 55 16 26 61.9
34.45-37.66 72 79 16 30 65.2
'Accumulated heat units based on 11 C threshold temperature
2X2 = 1.53, df= 1, P = 0.674
Role of the Oviposition Plug in Host Location
Parasitism of pepper weevil eggs by T eugenii was clearly affected by the presence
or absence of the oviposition plugs (Table 14). Over two and a half more eggs were
parasitized when covered normally by an oviposition plug, compared to eggs over which
the plugs were removed in choice tests.
The predominance of parasitism in plugged eggs was just as evident under no
choice conditions (Table 15). Given no choice, T eugenii parasitized three times more
eggs covered normally by an oviposition plug, compared to unplugged eggs.
Table 14. Parasitism of pepper weevil eggs by T eugenii when given the choice of eggs
covered with an oviposition plug and eggs with no plug
Oviposition plug Parasitism
Not parasitize Parasitized Percentage1
Normal 69 71 50.7
Removed 114 26 18.6
'X2 = 31.94, df= 1, P < 0.0001
Table 15. Parasitism of pepper weevil eggs by T eugenii when given no choice of eggs
covered with an oviposition plug and eggs with no plug
Oviposition plug Parasitism
Not parasitize Parasitized Percentage1
Normal 51 89 63.6
Removed 111 29 20.7
'X2 = 52.73, df= 1, P < 0.0001
Effect of Weevil: Fruit Ratio on Parasitoid Production
When T eugenii females were exposed to 'Jalapefio' pepper fruits infested with
pepper weevil eggs by mated weevil females at a ratio of one weevil per fruit more
parasitoids (t= 5.26, df= 58, P < 0.001) and fewer weevil adults (t= 5.26, df= 58, P <
0.001) emerged per fruit, compared to when T eugenii were exposed to fruits infested by
unsexed weevils at a ratio of 10 weevils per fruit (Table 16). Percentage of arasitism was
similarly affected (t= 4.40, df = 58, P < 0.0001). Therefore, infesting pepper fruits with
one weevil female per fruit made more efficient use of weevils, weevil eggs, and fruits
than infesting pepper fruits with 10 weevils per fruit.
Table 16. T eugenii produced with two pepper weevil densities
Weevil adult density Insects per fruit Parasitism (%)
T. eugenii Weevils
Unsexed weevils (10/ fruit) 0.81 + 0.26 0.94 + 0.35 45.5 + 7.1
Mated weevil females (1/ fruit) 1.25 + 0.48 0.52 + 0.30 71.2 + 12.3
Life Cycle
The eggs of T eugenii were translucent and elongated with no evident surface
sculpturing. They were 246 + 33.6 [tm long by 73.4 + 16 [tm wide. The length included a
small pedicel of 31.2 + 5.7 utm long (Fig 4A). The majority of the eggs hatched 23.0 + 1
h after the 2 h exposure to T eugenii (Table 17). Emergence was completed (41 larvae of
42 eggs) after 25 + 1 h. After emergence and for a few more hours, first instar T. eugenii
were translucent, except for the large sclerotized head and large mandibles. The segments
of the body were nearly of equal size, except for the last one which was rounded and
slightly larger than the rest (Fig 4B). Some of the larvae observed on the microscope
slides appeared to be attacking unhatched parasitoid eggs within the host-egg.
The endoparasitic phase lasted almost 10 d and comprised more than half of the
total parasitoid developmental time (Table 17). Parasitized pepper weevil larvae, even
late 3rd instars, appeared to develop and to molt normally and had no evidence of
decreased activity, compared to the observed control. The parasitoid larva emerged from
the pepper weevil prepupa, usually after the completion of the pupal cell. The parasitoid
larva emerged through a hole made in the distal end of the abdomen of the host. Bending
the base of the head forward, it began to feed immediately on the weevil prepupa (Figure
5A). The last few abdominal segments of the parasitoid remained in the host at the
beginning of the ectoparasitic phase (Figure 5B). In about 8 h the parasitoid completely
consumed the host, except for the cephalic capsule (Table 17). The parasitoid then spun
its cocoon and pupated inside the pupal cell of the host, where it remained for 6-7 d.
Some parasitoid larvae removed from the pupal cell of the host and placed directly into
the polystyrene cells made partial cocoons; however, they failed to complete the cocoon,
and eventually died. Possibly, the lack of the pupal cell of the host caused dehydration.
The developmental times of males and females did not differ (Table 17), but it is
common to see in the laboratory that males emerge before females.
Table 17. Developmental times of T. eugenii in days (Mean SD) at 27 1C
Sex Egg Parasitic development Prepupa1 Pupa Total
Endo Ecto 9 or o 9 n= 25 9 n= 25
n= 197 9 n= 8 0 n= 9 n=14 6 n= 35 6 n= 35
Female 0.96 + 0.2 9.87 0.30 0.27 0.22 0.40 + 0.17 6.63 + 0.82 16.63 0.88
Male 0.42 + 0.18 6.40 + 0.52 16.36 + 0.88
Spinning cocoon
Adults emerged by chewing through the cocoon then through the fruit wall. The
emergence from infested peppers could be noticed as a small rounded hole. Adults were
no longer than 2.4 mm and completely black. The only evident difference between sexes
observed was the presence of the ovipositor in the female (Figure 6 C, D).
Figure 4. Immature stages of T eugenii: (A) Egg 22 h after oviposition, (B) First instar,
30-33 h after oviposition, with well developed mandibles.
Figure 5. Larva of T. eugenii. (A) Emerging from the posterior end of the host, (B)
Beginning to feed on the host even before emerging completely.
Figure 6. T. eugenii adults. (A) Female, (B) Male.
~ ~i~i~iiil
~ ~ii~iliiil
The preoviposition period was shorter than 24 h for six often females, between 24-
48 h for three females, and between 48-72 h for one female. Little difference in the
oviposition pattern was observed between host exposure treatments (Fig. 7). Maximum
fecundity was reached on the 4th day after emergence and then gradually declined,
reaching zero by day 18. Females deposited 95% of their eggs by day 12 when hosts were
changed every 1.5 h, and 92% when hosts were left for 9 h. During that period, females
laid 29.63 + 9.8 and 33.13 + 12.2 eggs per day, respectively, for the 1.5 h and 9 h host
exposure periods. All females died by day 21 (Figure 7).
When hosts were changed every 1.5 h, females produced an average of 372.40 +
223.14 eggs, compared to 433.20 193.80 when hosts were left for 9 h; however, the
difference was not significant (t= 0.5, df = 164, P = 0.901).
Superparasitism was common with both treatments, although the proportion of
superparasitized hosts was significantly greater when T eugenii females were exposed to
infested fruits for 9 h, compared to when females were exposed to infested fruits every
1.5 h (64.4% vs 55.3%, X2= 7.95, df = 1, P = 0.0048). Superparasitism ranged from two
to nine eggs, and averaged 2.24 + 1.34 for the 9 h exposure period compared to 2.10 +
1.40 for the 1.5 h exposure treatment. These values were not significantly different (t=
1.76, df = 924, P = 0.0779).
5\ \ Host changed every 1.5 h
35 -/ \
\ --*-- Host available per 9 h
15 \
0 / \
0 ..--------------------'- ^ *-. a--. --, -, -
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
Age in days
Figure 7. Fecundity of T. eugenii females at 27 + 2C either exposed to hosts changed
every 1.5 h (n=5) or exposed to hosts per 9 h (n=5).
The proportion of superparasitism and the numbers of emerged T. eugenii and
pepper weevils suggested strongly that more than half of the host eggs were not
dissected. Consequently, the proportion of superparasitism from the dissected host eggs,
and the survivorship of pepper weevil (90%) estimated in Chapter 2, were used to
estimate survivorship of the parasitoid. The estimate was made using the proportion of
superparasitism observed from the dissected hosts each day. Demographic parameters for
T. eugenii were estimated for the 9 h and 1.5 h treatments (Table 18). Values were not
significantly different between the two host exposure intervals according to Student's t-
test comparison, P < 0.05 (Maia et al. 2000).
Adult Longevity
Males and mated females with access only to water lived 2.3 + 0.7 d and 2.3 + 0.6
d, respectively, with no significant difference between the sexes (t=0.15, df= 58,
P=0.881). Mated females provided with honey lived significantly longer (24.7 + 10.6 d)
that did males (15.8 + 9.2 d) (t=3.47, df = 58, P < 0.001). Finally, females laying eggs
and provided with honey lived 16.5 + 3 d, which was significantly less than the 24.7 +
10.6 d for females not laying eggs (t=3.26, df = 38, P < 0.002).
Table 18. Demographic parameters and confidence limits ( 95%) for T. eugenii reared
on pepper weevil eggs in 'Jalapefio' fruits when parasitoid females were
offered hosts for different exposure intervals at 27 + 2C
Parameter1 Host changed every 1.5 h Host available for 9 h
Net reproductive rate (Ro) 102. 83 106.15
(8.47- 197.18) (26.89- 185.40)
Generation time in days 19.22 19.95
Intrinsic rate of increase 0.24 0.24
(rm) (0.20 0.29) (0.20 0.28)
Doubling time (Dt) 2.82 2.92
(2.24- 3.40) (2.37- 3.47)
Finite rate of increase (k) 1.28 1.27
(1.22 1.34) (1.21- 1.32)
Sex ratio 1:1 1:1
Difference between treatments was not significantly (Student's t-test for pairwise comparison,
P < 0.05, Maia et al. 2000).
The total number of parasitoids recovered from both trips was 139, most of which
emerged from weevil infested fruits collected in May. C. hunter and T. eugenii
represented 83% and 6.5%, respectively, but parasitism reached only 2.85%. These
numbers were lower than those previously reported from that region: 865 and 1 210
parasitoids (recovered of 45 and 80 kg of infested fruit by pepper weevil, respectively)
according to Mariscal et al. (1988) and Toapanta (2001). T eugenii was the dominant
species (84% and 88%, respectively), with levels of parasitism ranging from 29 to 40%,
The destruction by hurricane Kenna and subsequent reduced acreage of the
growing pepper area in Nayarit during 2003, the possible damage to alternative hosts by
the hurricane, and the intensified use of insecticides during late months in the season, are
possible explanations for low levels of parasitism of the pepper weevil observed during
the surveys in 2003 (2.85%) compared to previous reports (29-40%) (Mariscal et al.
1988, Toapanta 2001). Nevertheless, the 115 C. hunter observed in the present study was
not very different from 87 and 102 individuals observed in the previous studies. C.
hunter is a generalist ectoparasitoid of more than 17 species of Curculionidae and
Bruchidae (Cross and Mitchell 1969, Cross and Chesnut 1971). It can survive as an adult
for more than 40 days and, in addition to carbohydrates, can feed on host larvae or,
sometimes, pupae to survive (Cate et al. 1990, Rodriguez et al. 2000). Thus, C. hunter is
well equipped to survive unstable environmental conditions. In contrast, the pepper
weevil is the only known host of T. eugenii. It has not been observed to host feed and
cannot survive as long as C. hunter, especially without a carbohydrate source. Thus, T
eugenii would have fewer resources to draw upon under rapidly changing conditions.
Host-Age Range
During choice tests, T. eugenii preferred younger eggs to older eggs. Age may be
related to host suitability (Godfray 1994). Because eggs may lack a cellular defensive
response to foreign intrusion (Askew 1971) or because, with the exception of eggs in
diapause, eggs rapidly change from storage nutrients to chemically more complex
embryonic tissue (Sander et al. 1985), parasitoids may prefer young eggs. These
explanations have been supported by authors who found that parasitism by some egg
parasitoids declines as the host ages (Strand and Vinson 1983a, b, Ruberson et al. 1987,
Reznik and Umarova 1990, Castillo et al. 2005). Nevertheless, this information has been
supported mainly in observations with parasitoids from Trichrogrammatidae, Scelionidae,
Mymaridae, and Eulophidae. They are idiobionts that complete their development in the
eggs, and they may have differences with parasitoids which develop as koinobionts, such
as egg-larval or egg-pupal parasitoids (Strand 1989, Vinson 1998, Godfray 1994, Quicke
In contrast to many idiobiont egg parasitoids, egg-larval or egg-pupal parasitoids
may lay eggs in a more advanced stage of host development (Clausen cited by Quicke
1997, Abe 2001, Quimio and Walter 2001, Kaeslin et al. 2005). Strand and Pech (1995)
suggested that egg parasitoids may evade the immune response of the host by
preferentially ovipositing in pregastrula embryos in which the immune system has not yet
become functional. However, Kaeslin et al. (2005) observed that all stages of Spodoptera
littoralis (Boisduval) eggs can be parasitized by Chelonus inanitus (L.), because the
behavior of the parasitoid larva or adult assure that the larva will be located in the
haemocoel of the host. When freshly deposited host eggs are parasitized, the parasitoid
hatches in the yolk and enters the host embryo either after waiting or immediately
through the dorsal opening. When host eggs are parasitized at 1 or 2 d old, the host
embryo is covered by an embryonic cuticle through which the hatching parasitoid larva
bores with its abdominal tip. When old eggs (2.5-3.5 d) are parasitized, the female
parasitoid oviposits directly into the haemocoel of the host embryo.
T. eugenii was able to parasitize young and old eggs, an ability which is shared
with other koinobiont species that attack eggs of their hosts but later emerge from larvae
or pupae (Clausen cited by Quicke 1997, Abe 2001, Quimio and Walter 2001, Kaeslin et
al. 2005). The physiological interactions between host and parasitoid are not completely
understood, but the ability to use either young or mature hosts represents a desirable
characteristic that increases probability of locating an appropriate host.
Role of the Oviposition Plug in Host Location
Host habitat location by parasitoids often involves chemical cues, as well as
physical and electromagnetic signals (Quicke 1997, Vinson 1976, 1998, Turlings et al.
1998, Havill and Raffa 2000). These signals include odors emanating from host frass,
symbiotic fungi, or host-damaged plants (Quicke 1997, Vinson 1976, 1998, Turlings et
al. 1998). Parasitoids attacking concealed larvae, such as fruit flies or wood borers, can
locate their hosts by detection of movement transmitted as vibrations in the substrate
(Godfray 1994, Quicke 1997). Such stimuli are not available to parasitoids of concealed
eggs. Such parasitoids could rely on olfactory signals from chemicals such as kairomones
of the host (e.g. accessory gland secretions), or from tactile and chemical signals
emanating from mechanical damage such as oviposition wounds (Strand 1989, Godfray
1994). Once a probable site of a host has been located, parasitoids respond to less volatile
substances, or to tactile cues where closer antennation and the ovipositor might play more
important roles (Godfray 1994, Quicke 1997).
This study demonstrated that the host oviposition plug is important for parasitoid
host location. A high proportion, around 80%, of pepper weevil eggs were not parasitized
by T eugenii when the oviposition plug was removed. The oviposition plug, then,
provided means for the parasitoid to detect and, subsequently, to parasitize its host. It is
possible that the antennae were used to detect host kairomones found in the plugs,
because drumming was observed to increase as soon as the female was close to the plugs.
The response of some parasitoids to these substances has been demonstrated in more than
one species (Quicke 1997, Turlings et al. 1990, 1998, Vinson 1998). However, the design
of the present study did not permit the evaluation of other sources of stimuli, such as
physical or visual characteristics of the plugs, which may be important in the host
detection and oviposition by T eugenii. More studies need to be done in this area.
When the pepper weevils were reared under crowded conditions, the proportion of
eggs lacking ovipositions plugs increased (Chapter 2). For this reason, it was more
efficient to rear the pepper weevil by exposing pepper fruit to one female weevil per fruit
rather than 10 unsexed weevils per fruit. Additionally, less feeding damage on pepper
fruits by weevils helped to diminish the number of rotten fruits in the colony (E.
Rodriguez, unpublished data).
Life Cycle
T. eugenii is a member of the tribe Brachistini in the subfamily Helconinae and the
family Braconidae. The members of Brachistini are cosmopolitan and are known as egg-
larval endoparasitoids of different families of Curculionidae and Bruchidae (Beirne 1946,
Martin 1956, Shaw and Huddleston 1991, Sharkey 1997). Additionally, there is at least
one species, Nealiolus curculionis (Fitch), which has been reported parasitizing early
larvae of the sunflower stem weevil, Cylindrocopturus adspersus (LeConte) (Charlet and
Seiler 1994, Charlet et al. 2002). Early reports confirmed and described the endo- and
ectoparasitic phases of the immature stages of some Triaspis species, such as T
vestiticida and T caudatus (Berry 1948, Obrtel 1960); however, few species of this genus
have been studied in detail (Sharkey 1997).
Females of T eugenii were able to lay eggs from the first day of life. This behavior
is characteristic of many koinobiont parasitic wasps which do not need to consume
protein-rich host hemolymph to initiate oogenesis (Quicke 1997). The parasitoid egg was
deposited in the host egg, where it hatched in less than 24 h. No parasitoids were
recovered from larvae (4-5 d after infestation). The endoparasitic phase thus initiated in
the egg and continued until the prepupal stage of the host. Only one parasitoid emerged
from each host egg. The first larval instar possesses large mandibles. The presence of
such mandibles in the early instars of solitary parasitoids has been suggested as useful for
physical defense against either the same or different species in case of superparasitism or
multiple parasitism, respectively (Clausen 1940, Godfray 1994, Quicke 1997, Ming et al.
2003). In contrast, physiological suppression by asphyxiation may be a common
mechanism by which established larvae eliminate younger competitors (Fisher 1971,
Godfray 1994, Quicke 1997).
Egg-larval parasitoids occur in several subfamilies of Braconidae (Alysiinae,
Helconinae, Ichneutinae), but members of the subfamily Cheloninae more often use this
strategy (Clausen 1940, Shaw 1997). The potential as biological control agents of some
members of this subfamily could be one reason for the availability of information on
species such as Chelonus sp. nr. curvimaculatus Cameron, an egg-larval parasitoid of the
pink bollworm, Pectinophora gossypiella (Saunders) (Hentz et al. 1997), and Chelonus
inanitus, an egg-larval parasitoid of Spodoptera littoralis (Kaeslin et al. 2005). The
information reported in the present study confirms similarities in the life cycle of T
eugenii with that of these species of Cheloninae, and with that of Triaspis vestiticida
(Berry 1947). In addition, the present study describes life table parameters of T. eugenii,
information which is needed to establish the potential of this species.
Potential of T. eugenii as a Biological Agent of the Pepper Weevil
Despite the fact that five species and at least five other genera of parasitoids have
been collected from the pepper weevil (Pratt 1907, Pierce et al. 1912, Elmore et al. 1934,
Cross and Chesnut 1971, Mariscal et al. 1998, Toapanta 2001), T. eugenii and
Urosigalphus sp. are the only known species that parasitize eggs of this pest.
Urosigalphus sp. (14 females and 9 males) was collected by P. Stansly during July 2003
in Oaxaca, Mexico, and was held in quarantine facilities and reared in the same
conditions that T eugenii. However, the parasitoids produced only males in its offspring,
making it impossible to establish a colony (E. Rodriguez, unpublished data).
The egg of the pepper weevil could be considered the stage more ecologically
susceptible to be parasitized, because it is defenseless and is deposited close to the
surface in the pepper fruits. Therefore, the ability to parasitize pepper weevil eggs could
be one of the most important biological advantages of T. eugenii. Because T. eugenii can
reach the host before the larva migrates deep inside into the fruit, it could attack the
pepper weevil in any cultivar, regardless of fruit size.
The reproductive capacity of T eugenii is another important biological
characteristic of the parasitoid. Females were able to produce an average of 402.8 + 199.5
eggs. Females laid more than 90% of their eggs during the first 12 days of life, depositing
31.4 + 22.6 eggs per female per day.
Superparasitism was common, although the proportion of superparasitized hosts
observed was greater where all infested fruits were left for 9 h rather than being changed
at 1.5 h intervals (64% versus 55%). T eugenii lays eggs individually in only 28 sec (E.
Rodriguez, unpublished data). The short time required for oviposition coupled with the
longer exposure period may explain the higher rate of superparasitism. However, it does
not explain the apparent inability of the parasitoid to distinguish parasitized hosts, even in
the presence of nonparasitized hosts.
Decreasing exposure time from 9 h to 1.5 h reduced superparasitism. Further
reduction of exposure time would be impractical within the context in the rearing system
developed here. Additional manipulations may be necessary such as more hosts per
female, fewer hosts per fruit, and larger cage size. On the other hand, superparasitism is
relatively common in laboratory conditions in other rearing systems of parasitoids, such
as Chelonus sp. nr. curvimaculatus (Hentz et al. 1997), or Catolaccus grandis Burks
(Morales-Ramos and Cate 1993); however, superparasitism does not occur often in the
field according to the same authors. Given these considerations, the reproductive
potentials of the pest and parasitoid were compared using fecundity and demographic
parameters of the pepper weevil (Chapter 2) and of T. eugenii using the data set of the
combined 10 T. eugenii females. It was assumed that superparasitism does not occur
under field conditions (Table 19).
The fecundity of the pepper weevil (355.28 + 167.51) and that of T eugenii (402.8
+ 199.55) with the same sex ratio (0.5) and survivorship (0.9) indicated no differences in
the net reproductive rate (Ro) (P = 0.82). Nevertheless, the remaining parameters favored
T. eugenii (P < 0.00001). The intrinsic rate of increase (rm), which might be one of the
most important parameters to compare populations because it combines Ro and
generation time (T) (rm = loge Ro / T, Birch 1948, Southwood and Henderson 2000), was
almost twice as great for T eugenii as for the pepper weevil. Therefore, the important
pest/parasitoid difference was generation time.
Table 19. Life table parameters (+ 95% confidence limits) estimated for 10 T. eugenii and
14 pepper weevil adults at 27 + 2C
Parameter Pepper weevil' T. eugenii1
Net reproductive rate (Ro) 158.10 167.1
(115.1 201.1) (92.7- 241.3)
Intrinsic rate of increase (rm) 0.14 0.26
(0.13 -0.16) (0.24 0.28)
Generation time (T) 35.4 19.6
(30.4 40.4) (19.0 20.2)
Doubling time (Dt) 4.8 2.6
(4.3 5.4) (2.4- 2.8)
Finite rate of increase (k) 1.2 1.3
(1.1- 1.2) (1.2- 1.3)
1The sex ratio and survivorship used to do the statistical analysis were 0.5 and 0.9, respectively
T. eugenii has been cited as the most abundant parasitoid of the pepper weevil in
field samples from Nayarit, Mexico, a region where the pepper weevil occurs naturally
(Mariscal et al 1988, Schuster et al. 1999, Toapanta 2001). It parasitizes the egg of its
host and has almost twice the potential for increase than the pepper weevil. These
attributes make T eugenii an excellent candidate for biological control of the pepper
RELEASE AND RECOVERY OF Triaspis eugenii Wharton and Lopez-Martinez
The pepper weevil is considered one of the key pests in peppers (Capsicum spp.) in
many growing regions of the United States, Mexico, and Central America, and only
cultural and chemical tactics are presently available for its control (Elmore et al. 1934,
Riley and King 1994, Mariscal et al. 1998, Schuster et al. 1999). Since the pepper weevil
was reported in Mexico (Cano and Alcacio 1894) and in the United States (Walker 1905,
Pratt 1907, Elmore et al. 1934), no viable biological control strategy has been developed
to manage the pest.
Some reasons for the lack of successful biological control of the pepper weevil may
include: (1) a paucity of natural enemies recorded from this pest (Pratt 1907, Pierce et al.
1912, Elmore et al. 1934, Cross and Chesnut 1971, Clausen 1978), (2) limited impact of
those parasitoids (Elmore and Campbell 1954, Genung and Ozaki 1972), (3)
incompatibility of natural enemies with insecticides used to control pepper weevil, and
(4) limited biological control research because of the relatively small economic
importance of pepper compared with other crops (Riley and King 1994).
Recently Mariscal et al. (1998) identified nine different species of parasitoids
attacking pepper weevil larvae in the state of Nayarit, Mexico, which is located in the
central pacific coast and which is a likely center of origin of the pest. The most abundant
of these parasitoids was the braconid Triaspis eugenii Wharton and Lopez-Martinez,
which attained 18-40% parasitism in the field (Mariscal et al. 1998, Schuster et al.1999,
Toapanta 2001). These relatively high levels of parasitism, complemented with the
continual presence of T. eugenii during surveys between 1997 and 2003, and with its
ability to attack pepper weevil eggs, renewed interest in biological control to combat the
pepper weevil (Mariscal et al. 1998, Schuster et al. 1999, Toapanta 2001).
T. eugenii is a solitary egg-prepupal parasitoid of the pepper weevil. Eggs are
deposited into pepper weevil eggs, and hatching larvae feed as endoparasitoids; however,
larvae later emerge from the host prepupa and complete development as ectoparasitoids
(Chapter 3). The habit of attacking the egg, located close to the fruit surface, rather than
the larva that burrows deep into the fruit, in addition to its reproductive superiority to
pepper weevil, make T eugenii a good candidate for biological control of the pest.
Nevertheless, the reproductive potential in the field could be influenced by many factors,
including the ability of the parasitoid to locate host and food, and the influence of
environmental conditions. Therefore, the evaluation of field releases is an indispensable
step in assessing the effectiveness of the parasitoid as a biological control agent.
T. eugenii cannot survive for more than two or three days at 27 + 1IC without a
carbohydrate supplement such as honey. Under field conditions, many parasitoids use
nectar or honeydew for this purpose (House 1977, Powell 1989, Baggen and Gurr 1998).
It cannot be assumed that these sources would always be adequate in or near every
pepper crop. Many parasitoids and predators also use plant volatiles to locate the host
plants of their hosts or preys (Vinson 1976, Alphen and Vet 1989, Godfray 1994, Quicke
1997). The volatiles often are released from damaged plants (Turlings et al. 1990, 1995,
1998, Powell et al. 1998, Takabayashi et al. 1998, Havill and Raffa 2000, James and
Grasswitz 2005). There is no information about the importance of weevil damage in host
location or foraging behavior of T. eugenii within a patch. This information could be used
to improve the effectiveness of establishing the parasitoid in the field. The objectives of
these studies were to assess the ability of T. eugenii to survive and parasitize the pepper
weevil in large field cages and in the open field, and to evaluate the effect of honey
supplements and weevil damage on this ability.
Materials and Methods
Pepper weevil and T. eugenii adults were obtained from colonies maintained at the
SWFREC in Immokalee, FL, as described in Chapters 2 and 3. Mated weevil females, 8
to 10 d old, and parasitoids 3 d old or younger were randomly chosen from the colonies
for release into field cages. Parasitoids that were released in the open field were 4 d old or
Field Cages
Experiments were conducted in two screenhouses 7.3 x 3.6 x 3.65 m, with sides of
50 x 24 mesh screen (266 x 818 [tm openings) and roofs of polyethylene (Figure 8A).
Each screenhouse was divided into 2 large cages 3.65 m in length by an organdy cloth
partition. A zipper was sewn into the partition to serve as a door. Each cage represented
an experimental unit (Figure 8B). Temperature and humidity were recorded during assays
using HOBO data loggers (Onset Computer Corporation, Bourne, MA).
Figure 8. Field cages used at the SWFREC. (A) General view of a unit containing two
cages. (B) Organdy partition and zipper between two experimental units.
Effect of Honey Supplement
Five pots 7.8 L, each containing two 'Jalapefio' pepper plants, were placed in each
cage. Plants were 20.6 + 5.2 cm tall (n=10) and were spaced 35 cm apart. At the time of
the evaluations, plants had 4.0 + 2.4 fruits smaller than 4 cm in length. To provide natural
host finding conditions for T eugenii, two pepper weevil adults were confined in organdy
sleeves on floral buds on each of two plants (in the same pot) per cage two days before
experiments commenced.
Honey was offered in lines and replaced every other day on two plastic yellow
cards (5 x 8 cm) which were protected from sunlight by a cork roof (15 x 22 cm)
supported by wire mesh (Figure 9A). A pepper flush damaged by pepper weevil adults
was placed in a 28 mL plastic cup filled with water (Chapter 3) and attached to the wire
under the cork roof to attract parasitoids to the source of carbohydrates (Figure 9B). One
of these devices (wasp refuge) was suspended 30 cm above the plants in each cage.
The experiment had two treatments, a refuge with honey and a refuge without
honey, which were replicated four times. Because replications were developed at
different times, each screenhouse containing both treatments was considered a block. The
two weevil damaged plants per cage of each block were replaced with newly damaged
plants prior to the release of T. eugenii. Using a fine brush, 10 T. eugenii females 3 d old
or younger were placed onto the pepper flush of the refuge of each cage (Figure 9B),
which was located 1.6 m from the infested fruits. Refuges and pepper flushes were
observed for 5 sec at 1, 2, 3, 8, 24, and 48 h after release to check for the presence of
parasitoids. Parasitism was evaluated daily in each cage for 3 d by hanging two infested
fruits per each of the damaged plants. The fruits contained a total of 20 weevil eggs 24-40
h old.
Figure 9. Honey-wasp-refuge. (A) General view of the set up. (B) Using a unit as T
eugenii release point.
After 8 hours (9:00 AM to 5:00 PM), the exposed fruits were removed and
dissected in the laboratory to assess the percentage of parasitism and superparasitism
(Chapter 3). A new group of infested fruits was hung in the same location at the same
time the next morning. Each block was replicated four times and each cage received the
alternative treatment for the following replication to nullify an influence of cage
orientation. Chi-square analysis (PROC FREQ, SAS Institute 2000) was used to test
whether parasitism was independent of the presence of food (honey).
Survival of T. eugenii was evaluated using females confined in 250 mL plastic cups
that had 3 cm diameter organdy sealed holes in the tops for ventilation (Chapter 2, Fig.
1). Cups were placed inside the canopy of potted plants to avoid direct sunlight. Five T
eugenii females 3 d old or younger per cup represented an experimental unit. Treatments
were cups without honey or cups with honey streaked every 2 or 3 d on the internal side
of the cups. Survival was checked every 1, 3, 9, 24, and 32 h, and then was checked each
24 h thereafter until all parasitoids were dead. Five replicates were performed in a single
block. Student's t-test (PROC TTEST, SAS Institute 2000) was used to evaluate
differences between treatments.
Effect of Host-Damaged Plants
Twenty 7.8 L pots, each containing two 'Jalapefio' pepper plants were placed in
each cage. The pots were spaced 30 cm apart and plants were 40 + 14.3 cm tall with 15 +
7.8 fruits less than 4 cm long at the beginning of evaluations. Treatments in this assay
were non-damaged plants and weevil damaged plants replicated five times. Because
replications were performed at different times, each screenhouse containing both
treatments was considered a block. Plants were noticeably larger and more mature during
the last two blocks, and the presence of open flowers which could be a source of nectar
was estimated from five plants per cage. At the same time, plants infested with aphids
and leafminers in these two blocks were redistributed among cages to maintain a
homogenous environment. At the end of each replication, plants used to support fruits
with hosts were replaced, and cages received the alternative treatment. A "Jalapefio' plant
was damaged by using an organdy sleeve to confine three mated females (8-10 d old) on
one terminal with floral buds 48 h prior to releasing T. eugenii. The damaged plant was
used to hang infested fruits with pepper weevil eggs daily as above. The same procedure
without weevils was followed for the control. Five T eugenii females 3 d old or younger
were then introduced into each cage, using a fine brush to place the parasitoids onto the
foliage of the potted plants furthest (3.6 m) from infested fruits. Previous observations
indicated that this number of wasps was sufficient to detect a response.
Parasitism was evaluated daily in each cage for two days by hanging five
'Jalapefio' pepper fruits containing a total of 25 pepper weevil eggs, 24-40 h old, in a
random pattern on the previously infested plant or in the previously designated plant,
which was placed in an equivalent position among the 20 plants, in undamaged
treatments. Infested fruits were held for 9 h as in the previous experiment and dissected to
check parasitism. In addition, these fruits were observed for 20 sec at 10, 20, 40, 60, 120,
and 180 min after releasing the parasitoids. Cage walls were also observed for parasitoids
for 30 sec at 1, 2, 3, 8, 24, 32, and 48 h after release. A chi-square test (PROC FREQ,
SAS Institute 2000) was used to compare parasitism in cages with and without weevil
damaged plants. The numbers of parasitoids observed in infested fruits were compared in
cages with and without weevil damaged plants using student's t test (PROC TTEST, SAS
Institute 2000). Before analysis, data were transformed using the square root of y + 0.5 to
satisfy assumptions of normality (Sokal and Rohf 1969). Student's t test was applied also
to data on wasps observed on cage walls after verifying normality of the data (PROC
CAPABILITY, SAS Institute 2000). All means are reported in the original scale.
Field Release and Establishment
On June 17, 2004, 100 T eugenii females and 70 males 4 d old or younger were
released at the SWFREC in Immokalee, FL. Parasitoids were released from 7:30 to 8:30
AM using a fine brush to place them on the foliage of pepper plants that were at the end
of the fruiting cycle. Parasitoids were released on six 100 m rows of 'Jalapefio' pepper.
Three hundred randomly selected terminals were observed to assess the adult pepper
weevil population. No flowers or buds were seen and fruits were large (7.68 + 1.46 cm
length, 2.77 + 0.73 cm width, n=10) and mature. Fruits (n=10) were dissected to estimate
the infestation of weevil larvae. The crop was destroyed a week after the release. Before
eliminating plants, 6 kg of fruits were collected and held in an organdy fabric cage (60 x
60 x 60 cm) at 27 + 2C to allow weevil or parasitoid adults to emerge. The number of
collected fruits was estimated by weighting 20 fruits.
During January to April of 2005, T eugenii was released five times on 90 m row of
approximately 400 'Jalapefio' plants of different ages maintained at the SWFREC (Table
20). Parasitoids 4 d old or younger were placed with a fine brush on floral buds, or
foliage, in groups of 5-10 females at 10 m intervals along the row.
Table 20. Releases of T eugenii during 2005 at the SWFREC in Immokalee, FL.
Date T. eugenii
January 10 100 80
February 8 80 40
February 18 35
March 25 60 50
April 20 80 60
Total 355 230
Before each release, 10 immature pepper fruits with weevil feeding punctures were
collected and dissected to confirm the presence of weevil eggs. The pepper weevil
infestation was also estimated by checking 50 terminals of pepper plants just prior to each
parasitoid release. Fruits were harvested every 1- 2 wk, and peppers with weevil feeding
punctures or chlorotic calyxes were held in the laboratory until emergence of weevil or
parasitoid adults. On March 25, three honey-wasp-refuges were established in the field
and 60 parasitoid females and 50 males, 4 d old or younger, were released at those points
at 5:00 PM. Five second observations at those honey-wasp-refuges were made at 1, 15,
and 24 h after releasing in each refuge to check for the presence of parasitoids.
During the summer of 2005 at the SWFREC, 10 potted pepper plants 5-6 months
old and with mature fruits present were transplanted 10-20 m from a 'Jalapefio' plot
severely infested by the pepper weevil during the previous spring. These plants were
watered and fertilized during that season. In August 8, 2005, 70 fruits from those plants,
and 15 more from a greenhouse nearby, were collected by P. Stansly. Fruits were held at
27 + 2C until the emergence of weevil or parasitoid adults. During the fall of 2005,
experimental 'Jalapefio' peppers were destroyed at the SWFREC by hurricane Wilma. No
peppers were grown in the field during spring of 2006 at the SWFREC, but 25-30 potted
(7.8 L) 'Jalapefio' plants were kept outdoors during spring 2006 and any pepper fruit with
feeding punctures or chlorotic calyxes were collected to attempt further recovery of T.
Effect of Honey Supplement
T. eugenii found and parasitized pepper weevil eggs in the presence or absence of
honey in the wasp-refuge during the first day of release. Eggs from the second and third
days were not parasitized. No difference in parasitism was found between refuges with
honey (94.3%) or without honey (82.3%) (X2 = 2.29, df = 1, P = 0.1299). After releases,
some parasitoids remained a few minutes in the wasp-refuge searching the pepper
flushes, whether honey was present or not, but soon left. No parasitoids were
subsequently observed at any of the refuges.
Parasitoid females in cups with honey survived longer (3.65 + 2.06 d) than
parasitoids without honey (0.62 + 0.56 d) (t = 7.11, df = 24, P < 0.0001). In the presence
of honey, more than 50% of the females were alive at day four, and the last females died
at day seven. In contrast, females without honey died in an average of 1.3 days (Fig. 10).
5 --Honey
S- -A-- No honey
1 \
0.04 0.13 0.37 1 1.3 2 3 4 5 6 78
Figure 10. Survival of T. eugenii adults in plastic cups with and without honey, when the
cups were placed inside the canopy of pepper plants, 25.4 + 4.90C, 67 + 19%
Effect of Host-Damaged Plants
T. eugenii females were able to find and to parasitize pepper weevil eggs in pepper
fruits hung in damaged and undamaged 'Jalapefio' plants; however, the level of
parasitism was higher in fruits hung in damaged plants (57.6%) than that in fruits hung in
undamaged plants (26.4%) (X2 = 24.97, df = 1, P = 0.001). No parasitism was observed
on eggs in fruits hung in cages a day after the release of parasitoids.
Overall, the numbers of T eugenii adults observed on fruits hung on plants
damaged by the pepper weevil decreased with time (Table 21). More T. eugenii females
were observed on fruits hung in plants damaged by the pepper weevil 10 and 20 min after
release, compared to the number observed on fruits hung on undamaged plants (t= 2.45,
df = 8, P = 0.040). Fewer parasitoids were observed at 40 and 60 min after release, with
no differences between treatments. No T. eugenii adults were observed on fruits hung on
undamaged plants after 60 min, or hung on any plant after 120 min. However, parasitoids
were seen on the upper wall of cages during the entire 48 h observation period. The
number decreased with time; 3.4 + 0.9 versus 3.6 + 0.5, respectively for damaged and
undamaged plants at 1 h; 2.2 + 0.8 versus 2.0 + 1.2, respectively at 24 h; and 0.4 + 0.5
versus 0.6 + 0.9 respectively at 48 h. There were not differences between treatments at
any time (t-test, P < 0.05).
Table 21. Number of T eugenii adults released in field cages and subsequently observed
on pepper fruits (n=5) hung on pepper plants either damaged or undamaged by
the pepper weevil, 28.2 + 4.7C, 55 + 9% RH
Time after releases Adults observed on fruits of pepper plants t-test
(min) damaged by weevil undamaged (P < 0.05)
10 2.0 + 1.22 0.4 + 0.55 *
20 2.0 + 1.22 0.4 + 0.55 *
40 1.4 + 0.89 0.8 + 0.84 ns
60 0.6 + 0.89 0.6 + 0.89 ns
120 0.2 + 0.45 0
180 0 0
Field Release and Establishment
At the time of the first release of parasitoids on 17 June 2004, 3.3 weevil adults
were observed per 100 terminals, and 1.1 + 0.57 weevil larvae were seen per fruit (n=10),
although no weevil eggs were detected. An estimated of 731 pepper fruits were collected
before the crop was destroyed, from which 380 adult weevils but no wasps emerged.
Environmental conditions during T eugenii releases at SWFREC during 2005 are
shown in Figure 11. Pepper weevil adults were found in the crop in excess of one adult
per 100 hundred terminals at the time of four of the five releases of T eugenii, and more
larvae than eggs were detected in the fruits (Table 22). Overall, the number of fruits
damaged by pepper weevil larvae and the number of emerged weevil adults increased
through time (Table 22). No T eugenii adults were observed in honey-wasp-refuges
following releases of the parasitoid during March 25.
Month 2005
Figure 11. Daily temperatures and humidity during T. eugenii field releases on 'Jalapefio'
pepper at the SWFREC in Immokalee, FL, 2005.
T. eugenii emerged for the first time on April 14 from infested fruits collected on
April 6. Infested fruits were then collected almost every week, and parasitoids were
recovered in low numbers through May 2 (Table 23). A few Catolaccus hunter adults
also emerged. T. eugenii and C. hunter together caused 6.7% parasitism. The pepper row
was eliminated during the last week of May, 2005.
Table 22. Adults per 50 terminals, and the number of weevil lifestages per 10 fruits, 1 or
2 d before field releases of T. eugenii on 'Jalapefio' pepper plots at SWFREC,
Immokalee, FL, 2005
T. eugenii release No. of pepper weevils
(date) Adults Eggs Larvae Emerged adults
January 10 0 0 0 0
February 8 2 0 12 12
February 18 1 0 2 148
March 25 3 2 5 300
April 20 1 2 7
Table 23. Parasitoid and pepper weevil adults recovered from 'Jalapefio' pepper
following field releases of T. eugenii at the SWFREC, Immokalee, FL, 2005
Emergence Triaspis eugenii Catolaccus hunter Pepper weevil
April 1 180
April 14 6 10 4 1 147
April 19 1 1
April 25 5 8 2 535
April 30 20 5
May 2 9 5 206
Total 41 28 7 1 1068
Pepper weevil adults emerged from the field sample and greenhouse samples
collected by P. Stansly during the summer 2005 at the rate 0.9 and 0.53 weevils per fruit,
respectively. However, no T. eugenii emerged. No pepper weevils were detected in the
potted 'Jalapefio' plants established at the SWFREC by the time sampling was terminated
on April 14, 2006.
Effect of Honey Supplement
T. eugenii searched the pepper flushes offered in the wasp refuges for a few
minutes but left, apparently not to return. Parasitism did not differ between honey and no
honey treatments, and was observed only during the first day of exposure.
As expected, survival of T eugenii adults was greater when adults were caged in
plastic cups with honey. Nevertheless, longevity of adults was drastically reduced to 3.7 d
in the plastic cups, compared to 16.5 d for ovipositing females in the laboratory at 27 +
1C (Chapter 3). The difference on survivorship may be due to heat stress that might have
occurred in the cups or the field cages.
It is known that floral and extrafloral nectars are indispensable sources of
carbohydrates for most adults of Hymenoptera (House 1977, Powell 1989, Baggen and
Gurr 1998, Kogan et al. 1999). Availability of these foods will affect longevity,
fecundity, and parasitism (Powell 1989, Idris and Grafius 1995, Baggen and Gurr 1998).
It is expected that efficiency, biological potential and survival of parasitoids that are
dependent upon these sources of energy to survive could be limited in monocultures that
provide those resources only for a limited time (Altieri 1992, Kogan et al. 1999). In the
field cages, T. eugenii in the plastic cups died in less than 48 h without a carbohydrate
source. Offering honey in the wasp-refuges did not appear to increase foraging time.
However, it is possible that food was not a limiting factor in the field cages. Parasitoids
might have used honey or left the patch before any carbohydrate was required.