CLASSICAL BIOLOGZCAL CONTROL OF THE TARNISHED PLANT BUG, LYGUS
LDIEOLARIS, IN ONTARIO, USING IMPORTED BRACONID WASPS,
PERISTENUS SPP.
A Thesis
Presented to
The Faculty of Graduate Studies
of
The University of Guelph
by SIMON LACHANCE
In partial fulfiilment of requirements
for the degree of
Doctor of Philosophy
June, 2000
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CLASSICAL BIOLOGICAL CONTROL OF TME TARNISHED PLANT BUG, LYGUS
LNEOLA'S, IN ONTARIO, USING IMPORTED BRACONID WASPS,
PERZ'STENUS SPP.
Simon Lachance Advisor: University of Guelph, 2000 Professor M. K. Sears
The tarnished plant bug, Lygus lineolaris (Palisot de Beauvois) is an important native agricultural Pest in Canada. Braconid parasitoids of the genus Peristenus introduced fiom Europe could help to reduce Lygus populations. Native parasitoids of mirids and their potential interactions with the introduced parasitoids were investigated before planned releases. Peak densities of nymphs of L. Zineolaris and AdeZphocoris
IineoZatus (Goeze) were observed in mid-June and at the end of Jdy/early August.
Annual rates of parasitism (May-Sept.) varied between 3.7% and 7.7% for L. lineolaris nymphs and aduits, and A. ZineoZatus nymphs, and varied with the time of sampling.
Parasitism rates were much higher for another mirid, Leptopterna dolabruta (Linnaeus).
Peaks of parasitism corresponded to peak presence of L. lineolaris and A. Zineolatus nymphs. Parasitism rates on alfalfa were, in general, lower than those found on other host-plants. Five species of native parasitic Hymenoptera fiom L. lineolaris have been coIIected in the Guelph area: Perisïenus pallipes (Curtis), Per istenus pseudopaZlipes
(Loan), Leiophron lygivorus Coan), Leiophron soZidaginis Loan, and Leiophron sp. near brevipetiolatus Loan. Laboratory experiments determined a threshold of development of 9.6"C for L. lineolaris, and ernergence thresholds for the European parasitoid P. digoneutis Loan and
P. sSrgicus Loan. Accurnulated degree-days in the field above the thresholds, and predicted occurrence of parasitoids and various stages of L. Zineoluris indicated a deIay of almost 30 days between initial parasite emergence and the appearance of L. heolaris nympb. Predictions of L. Zineoluris appearance fiom laboratory studies do not seem to reflect peak collections of nymphs in the field.
In-host compatibility and competitiveness of the exotic multivoltine parasitoids,
P. sSlgicus and P. digoneutis, and the native parasitoids, L. Zygïvorus, P. pallipes and P. pseudopaIZipes, showed that > 92% of the parasitoid attacks on plant bug nymphs resulted in oviposition and development of larvae. P. digoneuris and P. sîygicus appear to be superior in-host cornpetitors compared with the three North Arnerican parasitoids.
In the laboratory, most P. st;vgicus were induced into diapause when transferred to short day photopenod pior to 6.5 days after oviposition at 22OC. Only a slight increase in diapause induction was observed for L. lygivorus when reared at short day photoperiod.
Both P. digoneutis and P. stygicus show potentid for biological control of Lygus bugs in
Canada. 1 would like to thank my supervisor, Dr. Mark K. Sears, for his helpful advice and criticism, and his constant availability.
1also wish to gratefully acknowledge the help of Dr. Bruce Broadbent and Jay
Whistlecraft, fkom Agriculture and Agrî-Food Canada, London; Drs. Peter Mason and
Henri Goulet, frorn AAFC, Ottawa; and Dr. Ulrich Kuhlmann fiom CAB1 Bioscience
Switzerland, who al1 have participated closely in my research.
Many close fkiends fiom Environmental Biology have made my graduate studies enjoyable and interesting: Tracey Baute, Parry Schnick, Diane Stanley-Hom, Kurt
Randall, Claudia Sheedy, Jim Comgan, Jamie Heal, and others.
The hancial support for this research was provided by the Fonds pour la formation de Chercheurs et l'Aide à la Recherche (FCAR) fiom the Québec govemment. Financial support fiom the Entomological Society of Canada in the form of a research grant to travel to CAB1 Bioscience in Switzerland was also greatly appreciated. Table of Contents
Acknowledgement ...... i .. Table of Contents ...... II
List of Tables ...... v
List of Figures ...... vïi
List of Agpendices ...... ix
Chapter 1. Literature Review
1.1 Introduction ......
1.2 Palaearctic species ...... 4
1.2.1 Peristenus digoneutis Loan ...... 5
1.2.2 Peristenus swcus Loan ...... 8
1.2.3 Perisfenus rubricollis (Thompson) ...... 10
1.2.4 Peristenus conradi Marsh ...... 11
1.2.5 Peristenus adelphocoridis Loan ...... 12
1.3 Nearctic species ...... 13
1.3.1 Leiophron lygivorus (L, oan) ...... 13
1.3.2 Leiophron uniformis (Gahan) ...... 14
1.3.3 Peristenus pseudopallipes (Loan) ...... 16
1.3.4 Peristenus howardi Shaw ...... 17
1.4 Holarctic species ...... 18
1 .4.1 Peristenus pallipes (Curtis) ...... 18
1.5 Overview of parasitism rates and potential for biocontrol ...... 22
1.6 General objectives ...... 28 Chapter 2. Native Species of Euphorine Parasitoids of the Tarnished Plant Bug. Lygur
ZineoIaris (Hemiptera: Miridae) in Southern Ontario. Canada
2.1 Abstract ...... 29
2.2 Introduction ...... 31
2.3 Materials and Methods ...... ~...... 32
2.4 Results ...... 34
2.4.1 Mirid species and densiîy ...... ~...... 34
2.4.2 Parasitism rates ...... 41
2.4.3 Parasitoid identifications ...... ,, ..... ,...... 51
2.5 Discussion ...... 54
Chapter 3. Synchrony Between the Tarnished Plant Bug, Lygus lineolaris, and two
Exotic Braconid Parasites. Peristenus spp .
3.1 Abstract ......
3 -2 Introduction ......
3.3 Materials and Methods ...... 3.3.1 Diapausing plant bugs and parasitoids ......
3-3 -2 Assessment of synchrony ......
3 -3-3 Assessrnent of field synchrony ......
3.4 Results ......
3.5 Discussion ......
Chapter 4. Interspecific Competition Between Exotic and Native Parasitoids of the
Tamished Plant Bug. Lygus Iineolaris (Palisot de Beauvois)
4.1 Abstract ...... 79
4.2 Introduction ...... 81 4.3 Materials and Methods ...... 83
4.4 Results ...... 85
4.5 Discussion ...... 92
Chapter 5. Diapause Induction in the Parasitoids Peristenus stygicus and Leiophron Iygvorus (Hymenoptera: Braconidae: Euphorinae)
5.1 Abstract ...... 97
5 -2 Introduction ...... 98
5.3 Materials and Methods ...... 100
5.3.1 Diapause induction ...... 100
5.3.2 Stage of diapause induction in Peristenus sfygicus ...... 101
5.4 Resdts ...... 102
5.4.1 Parasitoid survival ...... 102
5 .4.2 Diapause induction ...... 104
5.4.3 Stage of diapause induction for Peristenus stygicus ...... 104
5.4 Discussion ...... 111
6 . General conclusion ...... 114
7 . References ...... 122
8 . Appendix ...... 133 List of Tables
Table
Overall parasitism of mirids collected fiom dfalfa fields and weedy fields
in the Guelph area, 1997 - 1999, Ontario, Canada ...... 44
Overall parasitism of minds in alfalfa fields vs. weedy fields in the
Guelph area, 1997 - 1999, Ontario, Canada ...... 46
Maximum parasitism rates of mirids (No. dissected >IO) collected in the
Guelph area, 1997 - 1999, Ontario, Canada ...... 50
Parasitoid emergence from three mirid species collected in 1999 and
held at 26°C &er diapause...... 53
Cumulative number of days for development of Lygus Zineolaris at various
temperature after diapause ...... 63
Emergence of Peristenus species following 3,6 or 9 months in diapause ...... 64
Longevity of Peristenus stygïcus at various temperature following 3,6 or
9 months in diapause ...... 65
Day-degree requirements for emergence of parasitoids and appearance
of plant bug nymphs in the laboratory, and predicted dates using average
field temperatures (1 997-1 999) at the Guelph Turfgrass hstitute ...... 7 1
Parasitism rates of nymphs of Lygus Zineolaris after a single attack by each
of the five different species of parasitoids ...... 86
Percent emergence of 5 species of adult parasitoids fiom the host
Lygus lineolaris afier a single attack by a female wasp ...... 87 Emergence and mortality of parasitoids foIlowing parasitism of a Lygus
lineolaris nymph with 2 different species at 24 hours interval ...... 89
Length of surviving Iarva of Peristenus stygicus in the host Lygus lineolaris
seven days after parasitism ...... 90
Percent emergence of non-diapausing and diapausing individuals of the
parasitoids Peristenus sîygims and Leiophron Zygivorus from nymphs of
Lygus lineolaris ...... 1 03
5.2A Percent emergence of Peristenus sîygicus fkom nymphs of lygirs lineolaris
after transfer fiom long day photoperiod (16hL: 8hD) to short day photoperiod
(12hL: 12hD) at daiIy intervals foIlowing parasitism ...... 105
5.2B Percent emergence of Leiophron lyg»oms from nymphs of Lygus lineolaris
after transfer from long day photoperiod (lm:8hD) to short day photoperiod
(12hL: 12hD) at daily intervals following parasitism ...... 106
5.2 Stage and length of parasitoid larvae of Peristenus stygicus developing in
nymphs of Lygus lineolaris at daily intervals following parasitism ...... 110 List of Figures
Figure
2.1A Weekly parasitism of Lygus Zineolaris, nine fields pooled (4 alfalfa fields,
5 weedy fields), Guelph 1998 ...... 3 5
2.1B. Weekly parasitism of Lygus lineolaris, seven fields pooled (3 alfilfa fields,
4 weedy fields), Guelph 1999 ...... 3 6
2.2A Weekly parasitism of Lygus lineolaris, four aifalfa fields pooled,
Guelph 1998 ...... 37
2.2B Weekly parasitism of Lygus lineolaris, three aEalfa fields pooled,
Guelph 1999 ...... 3 8
2.3A Weekly parasitism of Lygus Zineolaris, five weedy fields pooled,
Guelph 1998 ...... 39
2.3B Weekly parasitism of Lygus lineolaris, four weedy fields pooled,
Guelph 1999 ...... 40
2.4A Weekly parasitism of Ade[phocoris lineolatus, nine fields pooled
(4 alfalfa fields, 5 weedy fields), Guelph 1998 ...... 42
2.4B Weekly parasitism of A. lineolatus and L. dolabrata, seven fields pooled
(3 aifalfa fields, 4 weedy fields), Guelph 1999 ...... 43
2.5A Parasitism of Lygus lineolaris, weedy hay field, Stone Rd.,
Guelph 1998 ...... 47
2.5B Parasitism of Lygus Zineolaris, weedy hay field, Stone Rd.,
Guelph 1999 ...... 48
vii Temperature thresholds of development for Lygus Zineolaris ...... 66
Thresholds of emergence for Peristems stygms and P. digoneutis &er
diapause periods of 3,6 and 9 mon& ...... 67
Comparison of laboratory emergence of parasitoids (3,6 or 9 months in diapause)
and appearance of early nymphs of Lygus Zineolaris after diapause ...... 69
Cumulative emergence of Peristenus digoneutis and P. stygcus above their
developmental threshold, dera diapause penod of 3,6 or 9 months ...... 70
Cumulative development of Lygus lineolaris above developmental
...... threshold of 9.6"C ...... 73
Day-degree accumulation above thresholds, calculated fiom 1997- 1999
average temperature (soil or grass), and predicted dates of appearance (arrows)
of L. lineularis, P. stygicus and P. digoneutis fiorn laboratory study ...... 74
Emergence of non-diapaused Peristenus stygz'cus after tram fer to short day
photoperiod (l2hL: 12h.D) following parasitism ...... 107
Emergence of non-diapaused Leiophron Zygivorus after transfer to short day photoperiod (l2hL: 12hD) following parasitism ...... 108 List of Appendices
Appendix
1.1 Parasitism of Lygus Zineolaris, Alfalfa-Hay field, Concession 2,
Guelph 1998 ...... 134
1.2 Parasitism of Lygus Zineolaris, Alfilfa, Wellington Rd. 29,
Guelph 1998 ...... 135
1.3 Parasitism of Lygus Zineolaris, Aifâlfa, Jones Baseline,
Guelph 1998 ...... 136
1.4 Parasitism of Lygus Zineolaris, Alfalfa-Hay field, WeIlington Rd. 29,
Guelph 1998 ...... 137
1.5 Parasitism of Lygus lineolaris, Vetch, Stone Rd.,
Guelph 1998 ...... 138
1.6 Parasitism of Lygus Zineolaris, Weeds, Student Housing,
Guelph 1998 ...... 139
1.7 Parasitism of Lygus lineolaris, Clover and weeds, Stone Rd.,
Guelph 1998 ...... 140
1.8 Parasitisrn of Lygus Zineolaris, Echium sp. and weeds, Jones Baseline,
Guelph 1998 ...... - 14 1
1.9 Parasitism of Lygus Zineolaris, Alfalfa-Hay field, Concession 2,
Guelph 1999 ...... 142
1.10 Parasitism of Lygus Zineolaris, Alfalfa, Wellington Rd. 29,
Guelph 1999 ...... 143 Appendix
1.1 1 Parasitism of Lygus lineolaris, Malfa, Jones Baseline,
Guelph 1999 ...... - - ...... - .- .- - - - -.. - .- .- .. - - ..-
1.12 Parasitism of Lygus ZineoIaris, Vetch, Stone Rd.,
Guelph 1999 .. . . . - ...... -. .. . -...... -. -. -...... - .- - - ..
1.13 Parasitism of Lygus lineolaris, Weeds, Student Housing,
Guelph 1999 .. - - ...... - .. . -. . . . . -...... - - -.
1.14 Parasitism of Lygus lineoloris, Weeds, Bovey Bldg.,
Guelph 1999 .. - ...... - -.. -. -. . . -. -. . ...
1.15 Parasitism of Ade[phocoris ZineoZatzis, AlfalfaHay field, Concession 2,
Guelph 1998 ...... - ...... -. . . -. -. . . . . -......
1.16 Parasitism of Adelphocoris Zineolatus, Alfâlfa, Wellington Rd. 29,
Guelph 1998 ...... - .. . - -...... - .-. . . -...... - - ... . . -......
1.17 Parasitism of AdeIphocoris ZineoZatus, Alfalfa, Jones Baseline,
Guelph 1998 .. . -...... - ......
1.1 8 Parasitism of AdeZphocoris lineolatus, Alfalfa-Hay field, Wellington Rd. 29,
Guelph 1998 ...... -....-...--...... *....
1.19 Parasitism of Adelphocoris lineolatus, Weedy hay field, Stone Rd.,
Guelph 1998 ...... -. . .. -. ..
1-20 Parasitism of Adelphocoris lineolatus, Vetch, Stone Rd.,
Guelph 1998 ...... -...... -...... -...... -. Appendix
1.2 1 Parasitism of Adelphocoris lineolatus, Weeds, Student Housing, .
Guelph 1 998 ...... 154
1.22. Parasitism of Adelphocoris lineolutus, Clover, Stone Rd.,
Guelph 1998 ...... 155
1-23 Parasitisrn of Adelphocoris ZineoZatus, Echium sp. and weeds, Jones Baseline,
Guelph 1998 ...... 156
1-24 Parasitism of Adeiphocoris lineolatus, Alfalfa-Hay field, Concession 2,
Guelph 1999 ...... ,...... 157
1.25 Parasitism of Adelphocoris Zineolatus, Alfalfa, Wellington Rd. 29,
Guelph 1999 ...... 158
1 -26 Parasitism of Adelphocoris Zineolatus, Alfalfa, Jones Baseline,
Guelph 1999 ...... , ...... 159
1.27 Parasitism of Adelphocoris lineolatus, Weedy hay field, Stone Rd.,
Guelph 1999 ...... 160
1.28 Parasitism of Adelphocoris Zineolatus, Vetch, Stone Rd.,
Guelph 1999 ...... 161
1.29 Parasitism of Adelphocoris lineolatus, Weeds, Sîudent Housing,
Guelph 1999 ...... 162
1.30 Parasitism of Adelphocoris Zineolatus, Weeds, Bovey Bldg.,
Guelph 1999 ...... 163
1.3 1 Parasitism of Leptopterna dolabrata, Alfalfa-Hay field, Concession 2,
Guelph 1999 ...... 164 Appendix
1.32 Parasitism ofLeptoptema doZabrata. Weedy hay field. Stone Rd.,
Guelph 1999 ...... 165
1.33 Parasitism of Leptopterna doZabrata. Vetch. Stone Rd.,
Guelph 1999 ...... 166
1.34 Parasitism ofLeptopterna dolabrata. Weeds. Student Housing.
Guelph 1999 ...... 167
1.35 Parasitism of Leptopterna dolabrata. Weeds. Bovey Bldg.,
Guelph 1999 ...... 168 Chapter 1. Literature Review
1.1 Introduction
Species of Lygus and Adelphocoris bugs (Herniptera: Miridae) are important
agricultural pests in North America In eastem North America, the Tamished Plant Bug,
Lygus lineolaris (Palisot de Beauvois), is the dominant species, while in western United
States and Canada several species including L. hesperus Knight, L. elisus Van Duzee, L.
lineolaris and L. boreulis (Kelton) are the more common mirid pests (Clancy 1968,
Graham et al. 1986, Schwartz and Foottit 1992). These pests feed mostly on developing parts of plants (Scott 1987) and cause early drop of flowers and fitand distortion of
fniit or seeds, which are unacceptable damages in crop production (Graham et al. 1986,
Butts and Lamb, 199 1b).
Alfaif* strawberries, apples, canola and various vegetable crops are commonly damaged by Lygus pests in Canada. Damage is ofien underestimated or unnoticed because the bugs also cause indirect effects by sucking the Sap of plants (Day 1987, Scott
1987). As adults, these highly mobile insects cm rapidly migrate fiom wild to cultivated plants and severely decrease the value of crops. Alfalfa cuttuig, for example, often results in migration of rnirids to adjacent crop plants. Lygur lineolmis feeds on a wide range of weeds (Snodgrass et al. 1984) and ofien occurs in very high densities in weeds surrounding agricultural fields (Cleveland 1982). Lygus lineolaris and the majority of other species of the genus Lygus overwinter in the adult stage.
The alfalfa plant bug, Adelphocoris lineolatzis (Goeze), is a Palearctic species that becarne established in North America at the begimhg of the century in eastem Canada
1 (Knight 192l), whereas the mpid plant bug, A. rapidus (Say), is a Nearctic species. Both
Adelphocoris plant bugs are rnostly pests of seed and forage alfia(Craig 1963, Craig and Loan 1987, Day 1987). Adelphocorfi lineolatus and A. rapidus oveminter in the egg stage and therefore their nymphs will appear earlier in the spring than Lygus nymphs.
Although in northeastern North America their damage is usually minimal, they sometimes can reach high numbers and cause the same type of damage as Lygus bugs.
Severe economic losses and a paucity of alternative control methods have directed researchers to look for the complementary action of biocontrol agents to reduce Lygus and Adelphocoris populations. Egg parasitoids in the parasitic wasp family Mymaridae have been studied (see Jackson and Graham 1983, Norton et al. 1992, Sohati et al- 1992) and are available cornmercially for Lygus control, pnmarily in greenhouses. However, most of the research on biocontrol agents has been carried out with parasitoids of rnirid nyrnphs.
Nyrnphal parasitoids of the sub'family Euphorinae (Hymenoptera: Braconidae) are associated with plant bugs of the family Miridae (Hemiptera) (Loan 1974b, Marsh 1979,
Loan 1980, Loan and Shaw 1987, Dolling, 1991), although some British species have been reported to attack Psocoptera (see Loan 1974b, Marsh 1979) and one African species was reared fkom a Lygaeid (Nixon 1946). Parasitoids discussed in this review belong to two braconid genera, Leiophron Nees and Peristenus Foerster. Nineteen
Nearctic species of Leiophron and twenty-four of Peristenus are known and described
(Marsh 1979).
Female parasitoids lay a single egg in the hemolyrnph of usually a second or third instar nymph. The larva develops within the nymph and the mature lama exits from the late instar nymph or teneral adult to pupate in the soi1 (Loan 1965, 1974b, 1980, Loan and Bilewicz-Pawinska 1973, Bilewicz-Pawioska and Pankanin 1974). In the case of
superparasitism, only one lama will develop and survive in the host @ilewicz-Pawulska
1974b). Teratocytes are almost always associated with Euphorinae parasitoids, and are
observed afier the parasitoid egg hatches in the host's hemolymph (Brindey 1939, Loan
1965, Bilewicz-Pawinska and Pankanin 1974, Lim and Stewart 1976a). Brindley (1 939)
observed 4 distinguishable instars for P. paZZ@es. Loan (1965), as well as Bilewicz-
Pawinska and Pankanin (1974), reported five instars deveioping within the host nympb
but did not explain how they were determined. More recent studies descnbed 3 instars
for the parasitoid P. digoneutis (Carignan et al. 1995) and for P. pseudopdlipes (Lim and
Stewart 1976a). Mthough unlikely, it is possible that the number of instars varies among
species, and a thorough study of the developrnentai biology and morphology of Leiophron
and Peristentis species would be useful. The host may survive for a few hours to a few
days derthe lama emerges (Brindley 1939, Lirri and Stewart 1976%Carignan et al.
1995). Most species of Euphorine parasitoids are univolthe and the adult wasp will
emerge fiom the cocoon the next spring, after 8- 10 months in diapause. A few species are
dtivoltine and a second generation, or occasionally a third, will follow later in the
summer, usually in late July-August.
The first records of Euphorine parasitoids of Lygus and Adelphocoris were reported
by Hey (1933), Muesebeck (1936) and Brindley (1939). Since 1965, numerous studies on
parasitoids of Lygus and Adelphocoris have been carried out, mostly in Europe arid North
America. European species include Perisrenus paZZipes Curtis, P. stygicus (Loan), P.
digoneutis Loan, P. rubricoZZis (Thompson), P. adelphocoridis Loan and P. con&
Marsh, mostly attaclcing Lygus rugulipennis Poppius and Adelphocoris lineolatus.
Native North Amencan species include P. pallipes (origin uncertain), P. pseudopaZlipes (Loan), P. howardi Shaw, Leiophron uniformis (Gahan) and L. wvorus Loan, mostly
found and collected fkom Lygus lineolaris, L. hesperzis, L elisus and first genedon A.
lineolatus. For a complete identification key to Nearctic and Holarctic species and keys
to known Palaearctic species of Lygus, see Schwartz and Foottitt (1998). For
identification of nymphal parasitoids, see Loan (1 974%b), Loan and Bilewicz-Pawinska
(1973) and Loan and Shaw (1987). Five European parasitoids have been released in
Noah Amenca, but only P. conradi and P. digoneutis have become established to date
(Day et al. 1990, Day et al. 1992). Programs are in progress for additional introductions
(Kuhlmann et al. 1998)-
Prior to introduction of any insect natural enemy, an effort should be made to
accumulate dl available information on its origin, distribution, biology, natural enemies
and impact in its area of ongin (FA0 1997). To assess possible impact, host specificity
of candidate biological control agents must be known, dius basic life history information
about the parasitoids of plant bugs is required (Kuhlmann et al. 1998). The fist part of
this introduction reviews the literature on nymphd parasitoids that attack Lygus and
Adelphocoris species, and can serve as a bais for Merresearch on candidate
parasitoids for release and establishment. This information is then discussed with respect
to the biological control of plant bug pests.
1.2 Palaearctic species
In Europe, three species of Peristenus, P. digoneuiïs, P. rubricollis and P. smcus attack Lygus spp. (Bilewicz-Pawinska 1982) and three species, P. adelphocoridis, P. pallipes and P. rubricollis are associated with A. lineolatus. Day et al. (1 992) described an additional species, P. conradi Marsh, fiom A. Zineollatus, thought to have been introduced unlaiowingly with other European species into Delaware and New Jersey at the beginning of the 1980's. Peristenus adelphocoridis and P. palllïpes are sibling species that are morphologicalIy difEcult to separate (Loan 1979) and this species complex also includes the Nearctic P. pseudopallipes (Loan and Shaw 1987). Peristenus digoneutis, P. rubricollis and P. conradi form another cornplex of species that are difficult to separate morphologicaUy (Loan and Bilewicz-Pawinska 1973, Day et al. 1992). A review of the systematics of Holarctic Peristenus is needed to facilitate sound ecologicai research.
The most comprehensive ecologïcal studies of the parasitoids of Lyps and
Adelphocoris were compteted in Poland in the 1960's and 70's (E3ilewicz-Pawinska
1977b, 1982). In Poland, Lygus rugulipennis is the most common Lygus bug encountered, and the first generation is more intensively parasitized in rye than in aifdfa
(Bilewicz-Pawuiska 1977~).Overall parasitism of L. rugul@ennis populations in Poland did not exceed 25%, and never exceeded 10% on A. lineolatus (Bilewicz-Pawinska
1977b). Studies in Fidand showed that parasitism by braconids of plant bugs on wheat is iower than in Poland, never exceeding 13-6% (Bilewicz-Pawinska and Varis 1985).
1-2.1 Peristenus digoneutis Loan
This bivoltine species is the most common in Europe where it attacks Lygus yugdipennis (Bilewicz-Pawinska 1976% 1982) and A. lineolatus (Coulson 1987). The biology of P. digoneutis, rates of parasitism and its host-plant habitats have been studied intensively in Poland by Loan and Bilewicz-Pawinska (1973) and Bilewicz-Pawinska
(1 977% 1982) and its developmental biology and morphology in Canada by Carignan et al- (1995). This species was found to have the greatest geographical and ecological distribution and to be the most common Peristenus species attacking mirids in Poland
(Bilewicz-Pawinska 1976% 1977a). Parasitism rates on lucerne (alf&a), xye, potatoes, wheat and barley are between 2-34% for L. ruplipennis (Bilewicz-Pawinska 1 976%
1977% 1982). This species also has been found on clover and maize fields (E3ilewicz-
Pawinska 1982). On potatoes, it is the most common Peristenus species found (Bilewicz-
Pawinska 1976b, 1978).
Although recorded fiom other cereal crops, P. digoneutis has never been recorded fiom oat fields in Poland (Bilewicz-Pawinska 1982). Bilewicz-Pawinska (1977~)stated that this species is adapted to warmer climates, as it was found mostly in southem Poland.
After a diapause of about 8 months (Bilewicz-Pawinska 1974% 1977c, 1 978), it emerges a few days earlier in the spring than the other parasitoids in Europe (Bilewicz-Pawinska
1969, 1974% 1978). Peristenus digoneutis apparently reacts to increases in temperature faster than P. stygcus (Bilewicz-Pawinska 1982, Bilewicz-Pawinska and Varis 1990).
When moved to 21 OC &er winter diapause, P. digoneutis emerges in 2-22 days while P. stygicus takes Z 1-32 days to emerge. Unfortunately, a developmental threshold was not determined by Bilewicz-Pawinska (1982). Adult survival of 1st generation P. digoneutis in the Iaboratory was approxirnately 1 month when honey was provided, whereas the second generation wasps survived generally not longer than 2 1 days (Bilewicz-Pawinska
1974a). Development fiom egg to cocoon was about 21 days at 25°C (Hormchan 1977).
Adults of second generation P. digoneutis emerge fiom rnid-July to August in
Europe (Bilewicz-Pawinska 1974% 1982) and attack second generation L. rugulipennis nymphs on potatoes, lucerne and goldenrod Goan and Bilewicz-Pawinska 1973). Males of the parasitoid emerge a few days before the females (Bilewicz-Pawinska 1974a), and the overall emergence period may vary hmone crop to another. This is probably related to the sequences in which the host plants are attacked by the mirid nymphs (Bilewicz-
Pawinska 1982), thereby delaying or accelerating parasitoid egg laying.
In New Jersey and New York, this species was released in the late 1970's and early
1980's and is now established @ay et al. 1990, 1998). Parasitism rates of first generation
L. Zineolaris in al£alfa have increased from 1246% (native P. pulZipes only) to 30-50%
(P. palZ@es and P. digoneutis combined) (Day et al. 1990, Day 1996). Parasitism rates of second generation L. Zineolaris have increased fiom 8.2% (native L. uniformis only) to
29.2% (with P. digoneutis) (Day et aï. 1990). Peristenus digoneutis accounted for about
88% of al1 parasitized Tarnished plant bugs. Post-release sampling has shown a 75% decrease in Tarnished plant bug populations in alfalfa in the northeast States (Day 1996).
Peristenus digoneutis distribution has extended northward fier releases (Day et al 1990,
Day 1996) and has now reached southem Québec, Canada (Broadbent et al. 1999). It is now officially present in seven States (New York, New Jersey, Pennsylvania,
Massachusetts, New Hampshire, Vermont and Connecticut) in the USA, which approximates 115 000 ImiZ (Day et al. 1998). The parasitism rate due to P. digoneutis was 3.6 times higher in Lygus than in Adelphocoris and two specimens of P. digoneutis emerged fiom L. dolabrata (Linnaeus) @ay 1996). In Europe, although L. dolabrata was present in habitats where P. digoneutis occurred, no L. dolabratu yielded P. digoneutis
(Bilewicz-Pawinska 1982). 1.2.2 Peristenus stygicus Loan
This multivoltine European species, although widely distributed, seems to be less
abundant than P. digoneutis and P. rubricollis. It is more common in the southem part of
Europe, although it is not rare in the north (D. Coutinot, pers. comm. 1999). Bilewicz-
Pawinska (1977~)stated that this parasitoid, as well as P. digoneutis, would be adapted to
warmer conditions, given the fact that it was collected rnostly fiom southern Poland- It
has been reported to attack and develop in L. rugulipennis, TrigonotyIus coelestialium
(Kirkaldy) (Bilewicz-Pawinska 1982), Pol'erus unifasciatus (Fabncius) and A.
Zineolatus (Coulson 1987) in the field. P. stygicus also has been reared fkom Lygus sp.
and Adelphocoris sp. in southem France, Turkey (Butler and Wardecker 1974), Spain and
Greece (Coulson 1987). The range of crops fiom which P. stygicus has been recovered in
Poland is extensive, akhough this species has never been a dominant one in any of these
plants (Bilewicz-Pawlnska 1982). Parasitism rates recorded on L. mguZipennis in alfa1fat
rye, wheat, barley and oats have never been over 25% (Bilewicz-Pawinska 1977c, 19821,
although the fkst generation of T. coelestialiurn in wild grasses is attacked by P. sîygicus,
and parasitism rates can be as high as 60% (Bilewicz-Pawinska 1982). Peristenus swgicus was found only in smdnumbers parasitizing first generation L. ruguZipennis in
Poland (Bilewicz-Pawinska and Pankanin 1974). Most of the parasitoids found were P. rubricollis and P. digoneutis. The second generation of P. srygicus emerges in July and
August to parasitize second generation L. mguZipennis and T. coelestialium (Bilewicz-
Pawinska 1982).
Host-plants where parasitized plant bug nymphs have been collected are various, and include &alfa (Loan and BiIewicz-Pawinçka 1973, VanSteenwyck and Stem 1976, Bilewicz-Pawinska l982), rye and potatoes (Loan and Bilewicz-Pawins ka 1973,
Bilewicz-Pawinska 1978, 1982), wheat, badey, oats, grasses near cereals, clover, maize
(Silewicz-Pawinska 1982) and asparagus @rea et al. 1973).
Colonies of Lyps mgzdipennis @rea et al. 1973), L. hesperus (Butler and
Wardecker 1974, VanSteenwyk and Stern 1976) and L. ZirreoZaris (Broadbent 1976) have been maintained in the laboratory in order to study biology of P. stygims. At 22"C, the egg-lard period lasted approximately 14 days (range 12-16), and the pupal stage an additional 14 days (range 12-18) @rea et al. 1973), in accordance with Iater findings
Tom Butler and Wardecker (1974) and Hormchan (1977). The host becomes paralyzed for up to several minutes following oviposition by P. stygicus @rea et al. 1973).
Host-range testing in the laboratory has determined that this species will attack and completely develop in L. hesperus, L. 2ineoZari.s and P. basalis (Reuter) (Mirinae), L. gem inata (Johnston) (Orthoty linae) and P. seriatus (Reuter) (Phylinae) (Condit and Cate
1982). However, partial development was observed in the mirine Dicrooscytus sp.; only attacks but no development in M maculrpennis (Knight) (Phylinae) and one orthotyline species; and no attack was observed on the mùine Taediajohnstoni (Kmght), 2 species of bryocorine and 3 species of Iygaeid (Condit and Cate 1982).
Peristenus stygicus has been released in western Canada and USA (VanSteenwyk and Stern 1977, Craig and Loan 1984b, CouIson 1987). Success of ovemintering was observed in California and four peaks of P. sStgicus larvae occurred during the summer following the release, but no permanent establishment to control L. hesperus was achieved (VanSteenwyk and Stern 1977). Peristenus sîygicm might be unable to respond in a density-dependent manner and seemed to disperse poorly (VanSteenwyk and Stem
1977), although another reason might be that the parasitoid was not adapted to such a warm climate as the San Joaquin valley in California, Efforts to release P- sîygicus in cotton have aiso been attempted, but the failures indicate that this parasitoid is poorly adapted to cotton-growing regions (Schuster 1987). Attempts have aiso been made, although unsuccessfully to date, to release P. stygic21s in northeast USA (R. Fuester, pers. cornm., 1999). However, it seems to possess severai desirable qualities for release: facultative diapause; short developmental time; high level of parasitism; and ease of mass-rearing (Broadbent 1976).
1-2.3 Peristenus rubricollis (Thompson)
This univolthe species is present in Europe, and has been studied by Bilewicz-
Pawinska in Poland (Bilewicz-Pawinska 1974b, 1977b, 1982, Loan and Bilewicz-
Pawinska 1973). It has been found in northern and central Poland on cereal and lucerne crops (Bilewicz-Pawinska 1976% 1977b, 1982). Its recorded hosts are fist generation L. rugulipennis and A. Zineolatus (Bilewicz-Pawinska 1982, Craig and Loan 1987).
Parasitism rates of this species on L. rugulipennis in alfalfa, wheat, barley, and oats can
Vary fkom I to 85% (Loan and Bilewicz-Pawinska 1973, Bilewicz-Pawinska 1977b,
1982). The average rate of parasitism of nymphs on rye was about 30% during June and
July, and maximum parasitism was observed in the fist 10 days of July (Bilewicz-
Pawinska 1969). Mean percent parasitism of adult L. rugulipennis was lower, varying fiom 0.5 to 11.O % over a 3-year study (Bilewicz-Pawinska 1969). Parasitism rates on first generation A. lineolatus by P. rubricollis and P. pallipes were much lower, never greater than 10% (i3ilewicz-Pawinska 1977b). In Poland, fiom 1976-1979, P. rubricollis was the dominant species in cereals (rye, wheat, oat, barley), accounting for 4585% of al1 Peristenus species observed (Bilewicz-Pawinska 1982). The flight period of this braconid lasted about 4-6 weeks in the field (May to early June) (Bilewicz-Pawinska
1982).
Peristenus rubricollis seems adapted to lower temperature conditions (Bilewicz-
Pawinska 1977~)~as it was observed and collected mostly in northem and central Poland
(Bilewicz-Pawinska 1974% 1982). Mer a diapause of about 10 months, Perisfenus rubricollis emerged several days later (beginning 9-32 days after being rnoved fiom overwintering conditions to 2 1°C) than P. digoneutis (beginning 2-22 days) (Bilewicz-
Pawinska 1969, 1982) or P. pallipes (Bilewicz-Pawinska 197%). Development fiom egg to formation of the cocoon lasted about 47 days at 2 1°C, or 21 days at 2S°C (Hormchan
1977), which is much longer than development of P. st~@ins. Males exited their cocoons first and adults suMved for about a month after emergence (Bilewicz-Pawinska 1974a).
Maximum emergence in Poland always coincided with the last 10 days of May (Bilewicz-
Pawinska 1974a). Peristenus rubricollis was released in Arizona and Texas in the earl y
1970's (Coulson 1987) and in Saskatchewan in the 1980's (Craig and Loan l984a, 1987), but has not established.
1-2.4 Peristenus conradi Marsh
Reported by Day et al. (1992), as being established in Newark, Delaware, USA, following releases fiom European collections (France and Austria), the specific origin of
Peristenus conradi remains unclear. This univoltine species is part of the P. digoneutidP. rubricollis species group, but differs by the fact that it is deuterotokous, or nearly thelytokous @ay et al. 1992). Based on the rearing of field-collected hosts, the combined parasitism of P. conradi and P. pallees on A. lineolatw was 25%, compared with 8% by
P. pallipes alone (Day et al. 1992). In North America, ninety-five percent of P. conradi
emerged fiom A. lirzeolatus, 5% fiom L. lineolaris (Day et al. 1992). This species is now
officially established in 3 states (Delaware, New York and New Jersey) and in the
province of Québec (Broadbent et al. 1999), but a more extensive survey is necessary to
determine its complete distribution in North America (Day et al. 1998). Like other
univoltine Peristenus species, P- conradi overwinters in a cocoon and emerges in May
and June. Because P. conradi closely resembles P. rubricollis, studies previous to 1982
on the biology and distribution of the latter species may include P. conradi. Revision of
the taxonomy of the P. conradi/PPdigoneutid..- rubricollis complex wili facilitate studies
to clarify the biology and host range of each species.
1.2.5 Peristenus adeZphocoridis Loan
Peristenus adelphocoridis was described by Loan (1 979), and its reported hosts are
two species of Adelphocoris (Iineolatus and rapidus) breeding on Medicago sativa
Linnaeus. It has been collected in France and Denmark fiom parasitized plant bug
nyrnphs feeding on alfalfa (loan 1979). Larval instars emerged fiom the 5th instar of
Adelphocoris sp. in late June, and adults emerged fiom cocoons the following ~May(Loan
1979). It has been released in Saskatchewan, Canada, and in the United States (New
Jersey) to controI the alfalfa plant bug A. Zineolatus, but has not become established
(Craig and Loan 1984% Day et al. 1992). This species is part of the P. pallipes species group which also includes P. pseudopaZZ@es, and can easily be confused with the smaller
P. pallipes, which shares many sirnilarities (Loan 1979). The first record of P. adelphocoridis parasitking A. ZineoZahrs, although no official identification was made, might have been made by Bilewicz-Pawinska (1976a), who collected parasitized larvae of this mirid in alfalfa fields. Moreover, P. adelphocoridis keys out to P. pallipes in the key to European species of Peristenus (Loan 1974a).
1.3 Nearctic species
Four native species of parasitoids of Lygus and AdeZphocoris spp. are present in
North Amerka: P. pseudopallipes (Loan), P. howardi Shaw, Leiophron unformis
(Gahan) and L. Zygïvonis Loan. The status of an additional species, P. pallipes, is uncertain because this species is present in Europe, and might have been introdiiced approximately a hundred years ago with the mirid, Leptopterna doZab rata (Linnaeus)
(Day 1987). PeristenuspaZZipes might also be found to be a complex of 3 or more distinct species in North Amenca (H. Goulet, pers. cornm. 1999). Peristenus pseudopaZZipes is univoltine, and the two species of Leiophron are multivoltine.
1-3.1 Leiophron lygivorus (=Euphoriuna Zygivora) (Loan)
This species was described by Loan (1970). It has been captured only in Ontario,
Canada, parasitizing L. Zineolaris on Solidago canadensis (Loan 1970, 1974b, 1980).
The recorded hosts of this multivoltine parasitoid are L. Zineolaris and L. vanduzeei
(Knight) (Loan 1980), but it develops successfully on A. ZineoZatus in the laboratory
(Lachance, unpublished data). It has been reported to emerge from late instar and adult plant bugs prior to pupation (Loan 1970). Average parasitism by this species was 8% (combined with P. pseudopallipes) on goldenrod fiom a late summer generation of L.
ZineoZmis and L. vanduzeei (Loan 1980). Contrary to observations by Loan (1980), this parasitoid is multivo1tine (Lachance, unpuHished data).
It is possible that some of the earliest captures of L. Zygivorus were identified as
Leiophron uniformis, as Loan (1970) pointed out that "L. Zygivorur nuis to uniformis
Gahan in the key to Nearctic species of Euphoriana ..."
1-3 -2 Leiophron uniformis (Gahan)
Leiophron unz$iormis is common and widely distributed throughout North derka
(Loan 1974a), but was not recorded in Canada until 1999 (Broadbent et al. 2999).
Biology of L. unformis and its host insects have been studied by Debolt (1 98 1, 1989a. b).
Although not present in hi& numbers, it is the most common species of parasitoid on plant bugs-found in the southwestern United States (Clancy 1968, Graham et al. 1986).
Its host range includes major species of Lygus CL. elisus, L. hesperus, L. Zineolaris
(Clancy and Pierce l966), L. desertinus (Debolt l989a)I and Halticus bractatus Say, the latter thought to be its preferred host, although this parasitoid does not seem to have a rigid host association @ay and Saunders 1990).
Laboratory studies have shown that L. ZineoZaris does not seem to be an acceptable host, as only 6.7% of the nyrnphs attacked yielded a cocoon (Debolt 1989a). Low numbers of eggs laid per attack and a high encapsulation rate inside the host were observed. However, L. hesperus hosts are acceptable for L. uniformis developrnent, as the average percentage of parasitized nyrnphs yielding an adult parasite was 34% (600 exposed hosts) (Debolt 198 1). Rates of parasitism for this multivoltine species on Lyps spp. are usually low and sporadic, but can reach peaks of 5O-7O% (Clancy and Pierce
1966, Graham et al. 1986, Day et Saunders 1990).- Average parasitism rates of 1-3% and
2.9%, respectively, were found in Indiana for alfalfa fields and non-cultivated fields
containing Erigeron strigosus (Sillings and Broersma 1974). The maximum average
monthly parasitism of nymphs was 10.6% in alfaLfa and between 10-28.6% on
Chenopodium sp. inalfalfa(Grahamet al- 1986). Jacksonet al. (1998) reportedalow
level of parasitism of 0-5.6% in alfalfa in Arizona, and an average of 29% parasitism der
the release of >4000 L. uniformis (ca. 35% female) in enclosed areas. In Ontario,
Canada, parasitism rates varied fi-om O to 37.5% on weedy alfalfa (Broadbent et al. 1999).
Also after cage releases, L. uniformis searched and parasitized L. hesperus to a high level
(up to 97% parasitism at the higher release rate) in strawberries, demonstrating the
potential of the parasitoid for inundative releases morton et al. 1992). In alfalfi this
species has 3 generations a year on H bractatus, the garden fleahopper, and average
parasitism on nymphs was 49% @ay and Saundeus 1990).
Occasional emergence of parasitoid larvae fkom adult plant bugs has been observed
(Debolt 1981, Day and Saunders 1WO), most likeily as a result of late instars being
parasitized. Leiophron uniformis has emerged mostly fiom Lygus collected on aifalfa
(Clancy and Pierce 1966, Graham et al. 1986, Day et al. 1998) or weedy alfalfa
(Broadbent et al. 1999), but also on Chenopodim sp. (Clancy and Pierce 1966, Graham et al. 1986), grain sorghum, Sisymbrium irio L., Amaranthus palmeri Wats., Hyrnenothrix wislizeni Gray, HapIopapus tenuisectus (Greene) and guayule (Partheniurn argentatum
Gray) (Graham et al. 1986). 1-3.3 Peristenus pseudopallipes (.oan)
The northeastem North American species P. pseudopallipes is disiinguished fiom
P. pallbes by its late surnrner appearance. It has been recorded fkom Ontario and
Québec, Canada, adConnecticut, USA (Loan 1965,1970, 1980, Streams et al. 1968,
Shahj ahan 1974, Lim and Stewart 1976b). Peristenus pseudopallipes will parasitize second generation L. lineolaris and L vanduzeei nymphs, the only two hosts recorded in the field (Loan 1970, 1980).
In contrast to P. pallipes, most of the larvae will emerge fiom the late nymphal instars of their host rather than the adult (Lim and Stewart 1976b). Synchrony of the parasitoid with second generation nymphs of L- lineolaris in late July was appropriate
(Lim and Stewart 1976b), with the peak of P. pseudopallipes coinciding with the second peak of early instar Tarnished Plant Bug nymphs. In alfalfa, no flying adults of P. pseudopallipes were captured, although parasitism rates of various Lygus instars varied fkom O to 25%. Lim and Stewart (1 976b) assumed that the parasites were P. pseudopnIlipes, although no rearing or identification was done. It is possible that some of the parasitoids were actually L. Zygivorus or L. unformis. Peristenus pseudopaZlipes has been captured in alfalfa and fiom many weed species in fields, including Solidago canadensis Linnaeus (Loan 1970, 197413, 1980), Chenopodium sp., Arnmthus sp., Aster sp., Eupatorium sp. (Loan 1970) and Erigeron sp. (Streams et al. 1968, as P. pallipes,
Loan 1970, Shahjahan and Streams 1973), as well as fiom weedy areas around crops
(Lim and Stewart 1976b). However, parasitism rates are usually quite low. Parasitism rates in weeds (about 8- 15%) were higher than in alfalfa (1 -7%) (Lim and Stewart
1976b). Shahjahan and Streams (1973) reported that parasitism rates are much higher in stands of Erigeron sp. that are flowering (average 5 1.6%) versus floweriess plants
(average 9.8%). Adult wasps seem to be attracted by the flowering plants, the nectar probably serving as a food source (Shahjahan and Streams 1973, Shahjahan 1974).
Moreover, odors fiom Erigeron sp. attract significantly more female P. pseudopallipes than odors firom the weeds Daucus carota (Linnaeus) and Amaranthus retuofrexus
Linnaeus (Shahjahan 1974), and Erigeron flowers also permitted females to live longer.
Parasitism rates of reddish nymphs of L. lineularis on Erigeron canadensis L. were much higher than green nymphs on the plant. It is probable that the red nymphs do not blend well with their environment (the plant) and were detected more easily by female parasitoids (Shahjahan and Streams 1973).
1.3 -4 Peristenus howardi Shaw
Peristenus howardi was recently discovered in Idaho alfalfa fields @ay et al. 1999), parasitizing Lygus hesperus. This thelytokous species was parasitizing the two
generations of L. hesperus nymphs to a high degree: up to 8 1% in generation 1and 44-
5 1% in generation II. The adult is very similar to P. pseudopdipes and P. pallipes, and was dso able to attack and develop in L. lineolaris in laboratory studies @ay et al.
1999). A few specimens have also been recorded in Washington State (Mayer et al.
1998). 1.4 Holarctic species
Peristenus pallîpes is the only Holarctic species of Peristenus (there are 24 named
Nearctic and 20 named Palaearctic species) and was the fist descnbed species (by Curtis
1833) of the mirid parasitoid complex. More data have been accumulated on this species than any other Peristenus species. It is apparently widely distributed in North America and Europe, although new studies suggest that this ccspecies"is actually a complex of 2 or
3 distinct species (H. Goulet, pers. comm., 1999).
1-4.1 Peristenus pallipes (Curtis)
Peristenus pallipes may have been introduced to North America in the late 1800's or early 1900's with the mirid L. dolabrata (Day 1 987). Brindley (1 939) and Loan (1 965,
1974b) published the first papers on the biology and taxonomy of this species. It has the largest host-range of the parasitoid species complex in mirids. In North America, it has been recorded fkom Lygus lineolaris, L. hesperus, L. desertinus, L. borealis, AdeZpho cor is
Zineolatus, A. rapidus, Chlamydatus spp., Plagiognathus medicagus Arrand, Lepto terna dolabrata, Labops hirtus Knight, Capsur ater Linnaeus and Trigono~luscoelestialium.
Moreover, in Europe and UK, it attacks Lygus rugulipennis, L. pratensis (Linnaeus),
Calocoris norvegicus (Grnelin), A. lineolutus, T. coelestialiurn, L. dolabrata and
Notostira erratica (Linnaeus). As well, P. pallipes has been collected fiom hosts on many host-plants. Due to the dificulty in separakg the species of Peristenus, associations made before the revisionary work of Loan (1974% b, 1979) should be reviewed to confïrm them. This would include the association with Calocoris norvegicus by Brindley (1939), and the association with L. mguZipennis and L. pratenris by Clancy
and Pierce (1966). The associations by the latter authors should also be viewed with
caution because they did not indicate where the matend was collected, who verified the
identification of the pmitoids, or whether they were reared fiom a mixed population or pure populations of these Lygur host species. The detailed studies by Bilewicz-Pawinska
(1977b, 1982) yielded P. digoneutis, P. rubricollis and P. stygcus fiom L. rugulipennis while P. pallipes was reared fiom other mirids in the same habitat, including L. doZabrata, A. Zineolatus, T. coelestiaZiurn and N. erratica. It is therefore probable that in
Europe, Peristenus reared fkom Lygus spp. were erroneously identified as P. pallipes, when in fact they were P. rubricollis.
In North Amencan habitats characterized by the presence of Erigeron spp., Lygus bugs were more heavily parasitized (often between 30-60%) than bugs eom several other plants (Stream et al. 1968). In Indiana, Sillings and Broersma (1 974) observed a similar pattern of higher parasitism rates by P. paZZipes in habitats characterized by the presence of Erigeron srrigosus versus alfalfa (20.2% vs. 4.0%, respectively), although theu data most Likely included also collections of P. pseudopalZ@es. FLowers of Erigeron spp. have been shown to attract females of the parasitoid P. pseudopalZ@es, and to serve as a food source (Shahjahan 1974). Erigeron spp. are also among the favorite host-plants of the
Tamished Plant Bug, L. lineoZaris. In Québec, Canada, P. pallipes ernerged fiom weedy fields in early spring (mid to end of May), which is well before the appearance of Lygus lineolaris nymphs, the most common mirid pest (Lirn and Stewart 1976b). This parasitoid probably attacks alternative mirid hosts in May, before L. lineolaris nymphs appear in the field. Peak numbers of P. pallipes in alfalfa occurred approximately one month later than in adjacent stands of weeds, and parasitism was higher in alfalfa than in the weeds (Lim and Stewart 1976b). In different regions of the prairies of Alberta and
Saskatchewan, Canada, average parasitism was 22%, ranging fiom 3 to 49%, depending
on habitat and host species (L. hesperus, L. borealis, L. desertinus) (Loan and Craig
1976). This parasitoid seems to be much better adapted to the native mirids T.
coelestiaZium and L. doZabrata than to any other mind @ay et al. 19 92, Day 1999) -
Average parasitism rate on T. coelestialium was 39% fiom 1986 to 1989 on alfaifa in
New Jersey @ay et al. 19921, which is higher than what is usuaiiy observed with other
mirids. However, Loan (1980) clearly stated that P. pall@es was not attaclcing T.
coelestialium, contrary to the observations by Day et al. (1 992) and Bilewicz-Pawinska
(1982).
Parasitisrn of L. lineolaris, A. lineolatus, A. rapidus and L. dolabrata on forage
legumes in Ontario by P. pallipes in June was 62,49,42 and 42%, respectively, which is high for al1 of these hosts (Loan 1980). European studies by Bilewicz-Pawinska (1977~)
in Poland showed that parasitism of A. lineolatus by P. pallipes together with P. rubricoZZis never exceeded 10%. Peristenus rubricollis adults emerged a little earlier than P. pallipes adults from the host A. 2ineoZatu.s (Bilewicz-Pawinska 197%). In cereals,
90% of the braconids were reared fiom spring generation L. dolabrata and N. erratica, and 30% of those reared fiom T. coelestia2ium were P. pallipes (Bilewicz-Pawinska
1982).
Most individuals of this species will ernerge from adult hosts, due to slow development of Iarvae afler eclosion of the egg in the host (Loan 1965) or oviposition by the fernale wasp into older nymphs. Development of the parasitoid then progresses rapidly within the teneral adult Total developmental tirne is therefore longer than any other mirid parasitoid, and was about 24.5k2.4 days at 2S°C/16h photophase and 2O0C18h scotophase in the lab on L. Zineolaris (Lim and Stewart 1976a) nom eggs to emergence of the final nymphal instar. Development of P. paZZ@es in the field nom egg to emerged lama is about 5-6 weeks (Brindley 1939, Loan 1965). Spring adult emergence occurs afker a diapause period of 11 months (Bilewicz-Pawinska 1982). Despite many reports of larvae emerging fiom teneral adults fiom different locations, Loan and Craig (1976) reported that the majority of 5th instar parasitoid larvae (most likely third instar) emerged fkom 5th instar nymphs of L. borealis, L. hesperus and L. desertinus, and some parasitoid larvae emerged fiom 4th instar of L borealis. Parasitism rates of adult plant bugs are rarely over 40%, but have reached 62% on vetch in Mississippi (Scales 1973).
Peristenm pallipes is very similar to P. pseudopallipes, and the easiest way to differentiate thern is by the dBerence in their biology. Peristenus pallipes attacks first generation plant bug nymphs in the early summer, while P. pseudopallipes is found on the second generation later in the summer, usually August and early September (Loan
1970). 1.5 Ove~ewof parasitism rates and potential for biocontrol
Overall, mean parasitism rates (May-September) for species of Perirtentcs and
Leiophron recorded for most studies are often below 20%, despite the occasional 50-60% parasitism of single observations. However, highly variable results are obtained fkom year to year, crop to crop and parasitoid and mirid species studied. Day (1 987) indicated that in most samples taken fiom alfalfa in Europe, parasitism rates are consistently higher than in North America and suggested that this indicates a longer host/parasitoid association. It is likely that native Lygus bugs, being polyphagous, quickly adapted to feed on alfalfa, a plant introduced to North Amenca, while native and specialist parasitoids might not readily switch their searchhg behavior to locate hosts in habitats characterized by introduced plants.
In cereal crops in Poland, P. rubricollis is regarded as the dominant parasitoid species (48.85% of al1 Peristenus species), with P. digoneutis and P. stygicus ranked second and third, respectively (Bilewicz-Pawinska 1 982). The degree of parasitism was positively correlated with the density of plant bugs in cereal crops (Bilewicz-Pawinska
1982); populations of the most common species of plant bugs were parasitized at the highest level. This may indicate an evolutionary host-switch toward the more common species of mirids, or an increase in fiuictional response by the parasitoids. Contrary to what was stated by VanSteenwyk and Stern (1977) for mirids and parasitoids in
California, reduction of cereal mirid feeding populations by Peristenus species in Poland is density-dependent (Bilewicz-Pawinska 1982). The importance of parasitoids in regulating populations of A. lineolaris seems to be minor in Europe and in North
Amenca. Low parasitism rates are usudly observed, and almost exclusively the first generation of the alfdfa plant bug seems to be attacked and parasitized by bivoltine
Euphorine parasitoids. The reasons are dinicult to determine, as the four bivoitine/mdtivoltine species (P. stygrgras,P. digoneutis, L. lygivorus, L. uniformis) attack this mind in its fist generation. A strong preference for Lygus species or a lack O synchrony between these parasitoids and A. lineolah~~are possible explanations.
In selecting a parasitoid for classical biocontrol, we must know the dîfferent biological characteristics of the various parasitoids proposed for importation and release.
As well, information about the biology and ecology of the native naturd ene~.esof the target insect present in the proposed release area is essential. The host range of most
Peristenus and Leiophron species is fairly restricted (Loan 1980). Peristenus pallipes is the most polyphagous of al1 the species, with about 16 hosts recorded. However, as noted above, caution must be used when interpreting the validity of associations made prior to comprehensive taxonomie revision, particularly with cryptic species such as the P. ade[phocoridis/PppallipeslP. pseudopallipes and the P. conradi/P. ddigoneutislP. rubricoZlis species groups.
Complete development of P. sîyginrs has been observed within nine mirid species.
Al1 the other parasitoid species discussed in this paper have a host record of 2 to 4 species. However, data accurnulated over the last 30 years has focused more on certain species of parasitoids, in specific areas, and host-range data are incomplete. In case of its establishment in a new area, a more polyphagous parasitoid might not be as efficient to control a specific target mirid as another more specific parasitoid, and rnight also be a danger for non-target native mirid species.
Peristenus sfygicus seems to be a prornising agent for introduction into North
America, even given the past failures in establishment attempts. It possesses desirable characteristics such as facultative diapause, shoa developmental the, a high level of parasitism and ease of mass rearing (Broadbent 1976). It also seems to be present in low
densities all over Europe, which suggests a wide range of climatic adaptation. A multivoltine parasitoid species introduced into North Amenca has the potential to parasitize both generations of Lygus species and probably reduce their populations @ay
1987).
It is important to document previous introductions and/or establishment attempts and leam fiom these successes or failures. Peristenus digoneutis, for exarnple, has been released successfully and is reducing Lygus populations in the northeastem USA @ay et al. 1990, Day 1996). Failures have mostly been recorded in the case of very low numbers of parasitoids released (see Craig and Loan 1987), probably ofien in coojunction with a low ratio of females released. For more information on North American releases of introduced parasitoids, see Craig and Loan (1987) Day (1987) and Coulson (1987).
Rearing euphorine parasitoids is hampered by obligatory diapause in most species
@ay 1987) and low emergence rates in the laboratory (Whistlecraft et al. 2000). For example, ody 50-80 % of the parasitoids P. rubricollis, P. digoneutis, P. stenodemae
Loan and P. pallipes ernerged fiom their overwintering cocoons in the laboratory
(Bilewicz-Pawinska 1977d). As a general rule, fewer males are produced when females parasitize late instars (344th instars), but total adult parasitoid emergence is lower
(VanSteenwyk and Stem 1976, Honnchan 1977, Debolt 198 1). It is therefore difficult to shidy the parasitoids and build up numbers in the laboratory for later release or shiprnents to other codes. Many rearing attempts have ended with zero or very little emergence of adult wasps (Coulson 1987, Day 1987). There is an urgent need for Mercl~cation of the different species of
Peristenus and Leiophron. The species of parasitoids are not yet recognizable in their larval stages in the host. Only with a clear definition of species, carefiil host associations and a clear knowledge of life histones can we be sure of the species present in the field andbr which ones we plan to introduce. Advances in biomolecular and PCR techniques may soon permit an easy and efficient identification of lard and adult parasitoids
(Foottit 2000). These techniques should significmtly improve our efficiency at ident-g larval parasitoids nom surveys, as well as from interspecific cornpetition in the host. Given the univoltinism of many species of parasitoids and the need for a long diapause penod before any identification of emerging adults is possible, the time saving during surveys would be signincant. Accidental introduction of one species, not disthguished fiom those released, could also be detrimental to the success of the biological control program. Fortunately, accidental introduction of Peristenus conradi with releases of P. rubricollis (Day et al. 1992) did not seem to have any negative impact, and even appears to have been complementary.
Most sampling and foreign exploration that has been done in the last 30 years in search of parasitoids of mirids has been in alfalfa fields, which may have eliminated parasitoid species not adapted to this crop @ay 1987). Therefore, an effort shouId be undertaken to sample diverse habitats, because many Lygus bugs are polyphagous, following the flowering sequence of plants (Cleveland 1982). Sillings and Broesrma
(1974) found generally more parasites in non-cultivated areas venus cultivated ones.
Targeting the non-cropping area with imported parasitoids would potentially reduce populations of Lygus in cropping situations. A good starting point would be to assess the reduction of Lygus lineolaris densities in crops surrounding alfiilfa-fields in New York state, where introduced Pmdigoneutis has been shown to decrease L. Zineolaris popdations by up to 75% in alfalfa @ay 1996). Spring populations of Lygus often will occur in weeds surrounding crops (Stitt 1949, Malcolm 1953, Fye 1980, 1982). As well, classical biological control agents are most likely to become established and perform welI in undisturbed areas (van den Bosch et al. 1976, Mackauer 2989). The possible discovery of new species and the information gathered fiom such studies would be valuable.
In selecting a parasitoid for introduction to control native Lygus pests in North
America, one should look at parasitism rates in relation to habitat and host-plants of the plant bugs. If the goal of the parasitoid introduction is to decrease plant bug populations in a non-cropping area, a species that performs well in weedy or abandoned fields in the country of origin should be selected for importation. Weeds around crop fields might be a more suitable habitat for the parasitoids than alfalfa fields (Lirn and Stewart 1976b) or other crop fields, and may serve as a source of parasitoids (Bilewicz-Pawinska 1973).
Nectar sources (sugar sources) would likely attract the adult parasitoid in the vicinity of flowering weeds, as well as extending the survival and parasitism period of females (see
Streams et al. 1968, Shahjahan 1974, Shahjahan and Streams 1973). In Europe, the extent of parasitism in smaller (1 ha) rye fields was greater than in large fields (Bilewicz-
Pawinska 1977a). This might suggest that weedy crop borders were acting as a source of nectar and sugar and that wasps were not dispersing far into the field from these borders.
Permanence of the agricuitural habitat (no harvestulg) or the surroundings, and the fact that it would be less disturbed (no herbicides or fertilizer applied) would also benefit the course of the life cycle of parasitoids and their hosts.
Classical biological conh.01 programs for control of Lygus pests in North America involve a new association of host-parasitoid (Hokkanen and Pimentel 1984). The parasitoids to be selected in Europe for releases in North America have no previous
association with the principal mirid targeted, Lygus ZineoZ~ris.In such cases, certain
aspects of the biology of the natural enemies and their target host have to be assessed to
increase the probability of success. Such traits rnight include cornpetitive ability,
synchrony of host/parasitoid, behavior, and non-target effect S. Relatively few studies
have examined the developmental thresholds and heat unit requirements for both the host
and its natural enemies (Miller 1983), which can be very important in biological control
(Osborne 1981).
An important part of classical biological control of Lygus pests, and more so
because it involves the control of an indigenous pest by an imported parasitoid, is the
evaluation of the potential impact an agent would have on non-target species (Kuhlmann
et al. 1998). This would include the effects of interspecific cornpetition with native
parasitoids on the establishment of exotic mirid parasitoids. As well, host-specificity
tests of candidate biological control agents are essential. The host range of most nymphd . parasitoids of mirids is usually resûicted to the subfamily level (Condit and Cate 1982), and impacts on non-target insects should be minimal.
It is highly probable that new species of parasitoids of Lyps and Adelphocoris will be described in the near fiiture. Since the parasitoid species are fairly specific (Loan
1980), sarnpling for other mirid species may lead to the discovery of new parasitoids.
Some of these species might prove to be excellent candidates for classical biological control of rnirid pests in North America and should be investigated. 1.6 General Objectives
The general objectives of this study were to:
1) Collect, rear and ide&@ the native nymphal parasitoids of Lygus lineolaris and A.
ZirzeoZufus in southwestern Ontario, to serve as a benchmark for subsequent establishment
of imported parasitoids;
2) Determine the synchrony of parasitoid/host interactions for the two exotic parasitoids
P. stygicus and P. digoneutis and their new host L. lineolarïs;
3) Assess the outcome of interspecific cornpetition between native and European
parasitoids of L. lineolaris;
4) Study the biology of native and European parasitoids of L lineolaris, as welI as develop and improve rearing procedures. Chapter 2. Native Species of Euphorine Parasitoids of the Tarnished Plant Bug,
Lygus Cineof.(Hemiptera: Mindae) in Southern Ontario, Canada
2.1 Abstract
The Tamished Plant bug (TPB), Lygus lineolaris (Palisot de Beauvois), and the
ALfaKa Plant Bug (APB), Adelphocoris 2ineolatzl.r (Goeze), are important agrkdturd
pests in North America. Introducing exotic parasitoids may enhance control of these
pests, however, better knowledge of native parasitoids present in Canada is necessary
prior to any introduction. Plant bugs and their native parasitoids were collected on alfalfa
and various host plants, dissected to determine parasitism rates, and subsamples were
reared for parasitoid identification. Numbers of adults and nymphs of TPB and APB
collected varied greatly depending on the theof sarnpling and the plants sarnpled. Peak
densities of nymphal TPB and APB were usually observed in mid-June and end of
July/earIy August. Annual parasitism rates varied between 3.7% and 7.7% for L.
lineoluris nymphs and adults, and A. lineolatus nymphs. However, rates were much
higher for L. dolabrata nymphs, with an average of 50.9% in 1999. For A. lineolatus
addts, no parasites were found in 2823 individuals dissected over the three years of the
study. Peaks of parasitism rates corresponded to peaks in TPB and APB nymphs,
however, very low parasitism was observed for the second generation APB. Host-plants had a significant effect on parasitism rates. Rates in alfia were in general lower than that obtained fiom the other host-plants. Five species of native hymenopteran parasitoids have been collected in the Guelph area in southem Ontario: Perisrenus pallees (Curtis),
Per istenus pseudopalripes (Loan), Leiophron Zygivoris (Loan), Leiophron solidaginis Loan, and Leiophron sp. near brevipetiolatus Loan. Further taxonomie studies may indicate that the spring P. pallipes complex in fact contains two or three dinerent species. 2.2 Introduction
The Tarnished Plant Bug, (TPB), Lygus lineolaris (Palisot de Beauvois), and the
Alfalfa Plant Bug, (APB), Adelphocoris Zineolatus (Goeze), are important agricultural pests in North Amenca. The TPB is native to North Amerka, but the AF'B was accidentally introduced at the beginnllig of the 1900's in eastem Canada (Knight 192 1).
Severe economic losses may arise fkom these insect pests and few alternative control methods have proven effective. Introducing exotic parasitoids may enhance control, however, better knowiedge of native parasitoids in Canada is necessary prior to any introduction.
Surveys and evaiuations of native parasitoids of the TPB and the APB have been carried out in North America (Clancy 1968, Clancy and Pierce 1966, Loan 1965, 1970,
1974b, 1979, 1980, Graham et al. 1986, Day 1987). In Ontario, Canada, more than 15 species of nymphal parasitoids (family Braconidae) were collected and identified fiom various mind species fiom the Belleville area (Loan 1970, 1980). The percentage parasitism vmied between 16 and 64%, depending on the species involved and the season. Many of the mirid species sampled were not of economic importance, and were collected on weeds and various grasses.
Six species of nymphal parasitoids attacking L. lineolaris and A. lineolatus have been collected fkom eastern Canada (Loan 1970, 1980, Broadbent et al. 1999):
Peristenus pallipes (Curtis), P. pseudopalZïpes (Loan), P. digoneutis Loan, P. conradi
Marsh, Leiophron lygivorus Loan, and L. uniformis (Gahan). Two of them, P. digoneutis and P. conradi, are introduced European species and have been collected only fkom the province of Québec (Broadbent et al. 1999), having moved noah fkom their release point in New Jersey, USA. However, despite occasional reports of high rates of parasitism by the nymphal parasitoids present, TPB is not signincantly controlled by native species of parasitoids.
The objective of this study is to identifL plant bugs and their native parasitoids on different host plants, to serve as baseline data for subsequent establishment of one or more exotic parasitoids for control of the TPB and other mirid pests in southem Ontario.
2.3 Materials and Methods
Three locations in southern Ontario [Guelph, London (Dr. A. B. Broadbent, AAFC) and Vineland @on Marshall, AAFC)] were sampled for minds and parasitoids in 1997,
1998 and 1999. Only results of collections fkom the Guelph area are presented in the thesis. Results fiom 1997 will not be discussed in detail because sampling was Iess intensive, dissection of collected specimens was started in mid-June, and collection of A.
Zineolatus was also started in mid-June only.
Sampling was done weekly fiom May to September on various alfalfa fields and fallow fields in the Guelph region. Eight fields were sampled in 1997, nine in 1998, and seven in 1999. Alfalfa (or hay) was the main host plant sampled, as it is potentially the most important host-plant of introduced Peristenus species (Hymenoptera: Braconidae) and occupies a rather Large area (about 2.5 millions acres) in Ontario (OMAFRA 1999).
Other common host-plants sampled were wild mustard (Sinapis mensis L.), redroot pigweed (Amaranthus retroflexus L .), lad's-quarters (Chenopodium album L.), charnomille (Matricaria spp.), goldenrod (Solidago canadensis L.), Canada fleabane
(Erigeron canadensis L.), crown vetch (Vicia cracca L.), viper's bugloss (Echium
32 vulgare L.), wild carrot (Damscarota L.) and tirnothy (Phleurn prarense L.). Most fields sampled other than alfalfa contained a mixture of some of these host-plants.
Three mirid species [Lygus ZineoZaris, Adelphocoris lineulatus and Leptopterna dolabrata (Linnaeus)J, chosen due to their abundance and pest status, were sampled to study their parasitism rates. Leptopterna dolubrata was sampled only in 1999.
Sampling involved between two and three sets of 50 sweeps per field with a 40 cm diameter sweep net. Mirid species, stage, and number were recorded for each sample.
Dissection of late instar nymphs and adult plant bugs was done in order to establish percent parasitism. The mirids collected were dissected up to a maximum of 30 individuals of adults and nymptis per mirid species per 50 sweeps.
Rearing a subsample of mirids found in fields with higher rates of parasitism was also undertaken to obtain adult parasitoid wasps for species identification (identification of the larvae inside the host is not yet possible). Parasitized nymphs were reared with lettuce and beans in a screened-bottom, plexiglass tub (15 cm height X 9.5 cm diameter) with moist pupation medium (vermiculite) placed underneath in a 500 ml clear plastic container (Whïstlecraft et al. 2000). The screen prevented any predation by TPB of the pupating parasitoids. Cocoons obtained were subsequently transferred to 14°C for about
7 days, held at 7°C for an additional week, and then stored at SOC for a minimum of 5 months to terminate diapause. In order for adult wasps to emerge, the cocoons were transferred to 22°C after diapause.
For each year sampled, average parasitism rates per field per week were pooled. As well, parasitism rates fiom alfalfa and other mùid host-plants (mostly weeds) were determined. Annual parasitism rates do not partïcularly reflect the importance of date and host plants on parasitism, hence rates of parasitism were separated by host-plant habitat. 2.4 Results
2.4.1 Mirid species and density
Lygus lineolaris
Average numbers of adults and nymphs of the tamished plant bug collected per fifty sweeps varied greatly depending on the time of sampling and the plants sampled. Peak densities of nymphs were usually observed in mid-June and end of Julyheginning of
August (Figures 2.1A and 2.1B). Adult numbers fluctuated greatly over the spnng and summer months, mahgit dinicult to determine a peak period of adult density when data fiom al1 fields are pooled (Figures 2.1A and 2.1B).
In 1997, the mean number of TPB collected per 50 sweeps varied from O to 105 adults, and fiom O to 98 nymphs. In 1998, mean number of adults collected varied from O to 66 per 50 sweeps, and mean number of nymphs fiom O to 87.5 per 50 sweeps. In 1999, the mean number of TPB adults collected in al1 samples varied fiom O to 42.7 per 50 sweeps, and the mean number of nymphs fiom O to 84 per 50 sweeps. Over 95% of the
Lygus spp. collected were L. lineofaris, with an occasional L. plagiatus Uhler, L. rufidorsus (Kelton) and L. vanduzeei Knight found.
Mirid densities observed throughout the sampling penod (May-September) are presented by numbers collected in alfafa fields (Figures 2.2A and 2.2B) and other host- plants (mostly weeds) (Figures 2.3A and 2.3B). The values for mean addt densities in
1999 in alfalfa fields varied more than in weedy fields (Figures 2.2B and 2.3B), most likely due to the periodic cutting of alfalfa. 35 r . ' % par. adults 1
6 11 18 25 1 8 15 22 29 6 13 2027 3 10 17 24 31 7 14 May June July August Sept Week coilected Figure 2.1A. Weekly parasitism of Lygus Zineolaris ,nine fields pooled (4 alfalfa fields, 5 weedy fields), Guelph, 1998 35-7- ' / % par. adults -30 L par. nymphs j ; -Lygus aduIts 25 - : *L~~ nymphs E 1
18 25 1 8 15 22 29 6 13 20 27 3 10 17 24 31 7 14 May June July August Sept- Week collected Figure 2.1B. Weekiy parasitism of Lygus lineolaris , seven fields pooled (3 alfalfa fields, 4 weedy fields), Guelph, 1999 4 11 18 25 1 8 15 22 29 6 14 20 27 3 10 17 24 31 7 14 May June Ju1y August - Sept. Week collected Figure 2.2A. WeekIy parasitism of Lygus Zineolaris ,four alfalfa fields pooled, Guelph, 1998 16 - 1 1 I%par. adults 1 II
18 25 1 8 15 22 29 6 13 20 27 3 IO 17 24 31 7 14 May June July August Sept. Week collected Figure 2.2B. Weekly parasitism of Lygus lineolaris ,three alfalfa fields pooled, Guelph 1999 1 % par. adults '
6 11 18 25 1 8 15 22 29 6 13 20 27 3 10 17 24 31 7 14 May June July August Sept. Week collected Figure 2.3A. WeekIy parasitism of Lygus lineularis, five weedy fields pooled, GueIph 1998 18 25 1 8 15 22 28 6 13 20 27 3 10 17 24 3E 7 14 May June Jury August Sept. Week collected Figure 2.3B. Weekly parasitism of Lygus Zineoluris ,four weedy fields pooled, Guelph, 1999 Adelphocoris lineolahts
In 1998, densities ofA. Zineolatw per sample varied fkom O to 36.5 per 50 sweeps for the nymphs, and between O and 3 1 for the adults. Number of nymphs peaked in mid-
June in both 1998 and 1999, and again in late Julylearly August (Figures 2.4A and 2.4B).
In 1999, A. lineolatus densities were higher and varied fkom O to 84 per 50 sweeps and fiom O to 56.5 for the nymphs and adults, respectively. The density of adult and nymphal
Adelphocoris was higher in vetch, Vicia cracca L., than in any other host-plant sampled
(see Appendix 1.15 to 1.30).
Leptop terna dolabrata
Densities of L. dolabrata nymphs were recorded in 1999 ody. Peak density of this univoltine species was observed during the first week of June (Figure 2.4B), with an average of 8.9 nymphs collected per 50 sweeps. Numbers decreased thereafter until reaching zero on June 22.
2.4.2 Parasitism rates
Parasitism rates in 1997, 1998 and 1999 for each of the species sampled varied considerably (Table 2.1) (see Appendix for graphs of al1 fields sarnpled in 1998 - 1999).
Annual parasitism rates varied between 3.7% and 7.7% for L. lineolaris nymphs and adults, and A. lineolatus nymphs. However, rates were much higher for L. dolabrata nymphs, with an average of 50.9% in 1999 (only year recorded). For A. lineolatus adults, no parasites were found in 2823 individuals dissected over the three years of the study. / % par. Adelphocoris , 1 Adelphocoris nyrnphs
June July August Sept Week collected Figure 2.4A. Weekly parasitism of Adelphocoris lineolatus ,nine fields pooled (4 alfalfa fields, 5 weedy fields), Guelph, 1998 - 35 % par. Adelphocoris EZEii % par. Leptoptema - 30 Adelphocoris nymphs V1 Leptoterna nymp hs ; - 25 - O> 5 - 20 g \L. w 9 - 15 E= C,
18 25 1 8 15 22 29 6 13 20 27 3 10 17 24 31 7 14 May June Jul y August Sept. Week collected Figure 2.4B. Weekly parasitism of A. lineolatus and L. dolabrata, seven fields pooled (3 alfalfa fields, 4 weedy fields), Guelph, 1 999 Table 2.1 Overall parasitism of mirids collected fiom alfalfa fields and weedy fields in the Guelph area, 1997 - 1999, Ontario, Canada.
I,yglrs lineolaris adults Lygus lineolaris nymphs tidelptiocoris lineolalus nym phs Leptopïerna dolabratn nymphs Year No. No. No. % No. No. % No. No, % No. No, % fields dissected prirasitizcd parasitism dissected parasitized parasitism dissected parasitized parasitism dissected parasitized parnsitism 1997' 8 666 30 4.5 793 46 5.8 268 II 4.1 1998 9 2104 7 8 3.7 1360 86 6.3 983 5 8 5.9 1999 7 1713 101 5.9 932 36 3.9 1307 101 7.7 273 139 50.9 Total 24 4483 209 4.7 3085 168 5,4 2558 170 6,6 273 139 50,9 * Dissection started mid-lune LypIineolaris
Lygus Zineolaris was the predominant mirid species sampled. Parasitized nymphs of that species were collected fiom May 18 to August 17 in 1998 (Figure 2.1 A), and fiom
June 1 to August 17 in 1999 (Figure 2.1 B). Parasitized adults were found fiom June 1 to
August 24 in 1998 (Figure 2.1A), and fiom June 15 to August 10 in 1999 (Figure 2.1B).
Peaks of parasitism corresponded to peaks in Lygus nymphs, which were reached approximately mid-June and end of July/beginning of August, respectively (Figures 2.1A and 2.1B).
Host-plant had a significant effect on parasitism rates of TPB nymphs, but not of adults (P < 0.05) (Table 2.2). Rates of TPB fiom alfalfa were in general lower than those of TPB obtained Çom the other host-plants. Average parasitism rates in alfalfa for adults and nymphs were 2.8 and 2.0%, respectively, while they were 6.2 and 7.0% in other host- plants (Table 2.2). For both years, parasitized nymphs and adults were found earlier in the spring and more consistently throughout the season in fields containing various host- plants (Figures 2.3A and 2.3B), than in fields containing predominantly alfalfa (Figures
2.2A and 2.2B). Very few parasitized nymphs and adults were found in alfalfa in late siunmer.
Cutthg of a weedy hay field in mid-July 1998 decreased host densities to low levels, and prevented the appearance of a mid-summer population of parasitoids (Figure
2.5A). The sarne field in 1999 remained uncut and a large number of parasitized mirids were collected in July/August (Figure 2.5B). Table 2.2 Overall parasitism of mirids in alfalfa fields vs. weedy fields in the Guelph area, 1997 - 1999, Ontario, Canada. - Lygiis lineolaris adults Lygtis lineolaris nymphs Adelphocoris lineolattis nymphs Leptopterna dolabrata nyrnphs Host- Year No, No. No. % No. No. % No. No. % No. No. % plant fields dissected parasitized parasitism dissected parasitized parasitism dissected parasitized parasitism dissected parasitized parasitism Alfalfa 1997' 4 173 12 6.9 84 O O 62 2 3.2 1998 4 864 18 2.1 516 13 2.5 390 II 2.8 1999 3 980 27 28 364 6 1.6 384 38 9.9 19 1 5,3 Total Il 2017 57 2.8 ri 964 19 2.0 a 836 5 1 6,1 a 19 1 5.3
Total 13 2466 152 6.2 a 2121 149 7.0 b 1721 119 6,9 a 254 138 54.3 * Dissection started midJune Averages wilhin the same column followed by the same lettcr are not significantly different (P > 0.05; Tukey's HSD multiple comparisons test), - 60 I%par. aduIts 1
4 11 18 25 1 8 15 22 29 6 13 20 27 3 10 17 24 31 7 14 May June July August Sept. Week collected Figure 2.5A. Parasitism of Lygus lineo2ari.s , Weedy hay field, Stone Rd., Guelph 1998 - 60 I% par. adulü :
! Lygus adult
. -. 18 25 1 8 15 22 29 6 13 20 27 3 10 17 24 31 7 14 May June July August Sept. Week collected Figure 2.5B. Parasitism of Lygus Zineolaris , Weedy hay field, Stone Rd., Guelph, 1999 In 1997, the highest parasitism rate of Lygus nymphs was 55%, on a field of clover on August 26 (Table 2.3). The highest parasitism rate for adult TPB was 29.4%, in an alfalfa field on My 11. In 1998, the peak parasitism rate for adults (38.4%) was recorded
June 24 in a hay field and for nymphs (39.1%) on July 27 in a weedy field. For 2999, the peak parasitism rate was 38.8% and 19.0% for adults and nymphs, respectiveIy, both in a hay fieid (Table 2.3).
Adelphocoris lineolatus and Leptopterna doZabrata
Parasitized nymphs of Adelphocoris Zineolatus were collected from May 18 to July
6 in 1998 (some parasitized nymphs were also recovered on August 17), and fiom June 1 to June 29 in 1999 (Figures 2.4A and 2.4B). The spring generation of A. lineolatus appears to be the ody one attacked by parasitoids, and synchronisation of host/parasitioids was appropnate.
Parasitized nymphs of Leptopterna dolubrata were found fiom May 18 to June 15,
1999 (Figure 2.4B). Parasitism rates were much higher than for L. lineolaris and A. lineolatus (Figure 2.4B). This mirid species is univoltine, and appears early in the spring.
Other parusites
A few specimens of the hyperparasite Mesochorus spp. (Hymenoptera:
Ichneumonidae) emerged fiom the reared samples. Some parasitic nematodes were also found during dissection. These nematodes were almost exclusively dissected fiom mid to
Iate summer-collected adult TPB (July/August) . Table 2.3 Maximum parasitism rates of mirids (No. dissected > 10) collected in the Guelph area, 1997 - 1999, Ontario, Canada. Lygirs lineolaris adults Lygrrs lineolaris nymphs Adelpttocoris lineolatus nymphs Leptoplerna dolabruta nymphs
Year % Number Date Host- % Number Date Host- % Number Date Host- % Number Date Host- parasitisni dissected plants parasitism dissected plants parasitism dissected plants parnsitism dissected plants 1997 29.4 17 July 11 Alfalfa 55.0 20 Aug. 26 Clover 40.9 22 June 29 Hay 1998 38.5 26 une 24 Hay 39,l 23 lu627 Weeds 52,9 17 lune Il ~etbh 1999 38,8 49 Junc23 Hay 19.0 58 lune16 Hay 41.7 48 lune4 Vetch 80.6 62 June 8 Hay 2.4.3 Parasitoid identifications
Collections of parasitized nymph and adult plant bugs in fields with higher parasitisrn rates were made at irreguiar intervals. Rearing of these samples produced adult wasps for species identification. In 1997, collections were made June 24, July 9,
July 14, July 17, August 26, August 28, Sept. 1, Sept. 9 and Sept. 15. In 1998, collections were made June 5, June 20, June 21, July 5, July 16, July 30, July 3 1 and August 14, In
1999, collections were made June 4, June 5, June 12, June 16, June 18, July 2, July 6 and
Jdy 30.
Approximately 3400 nymphs and adults of L. Zineoluris were collected for rearing and identification in 1997. Three species of native parasitoids, Peristemrs pallipes,
Leiophron lygiorus, and Leiophron sp. (near brevipetiolatus), were found and identified fiom the survey in 1997. Peristenus paZZ@es was collected from July 9 to July 17. One speciinen of L. Zygivorus emerged fiom the collection of TPB nyrnphs on July 14, while the other 24 specimens emerged fiom collections made fiom August 26 to September 26 on a weedy field containing Solidago spp. A unidentified species of Leiophron (near brevipetiolatus) was also collected, and 5 specimens emerged fiom collections made
Sept. 1 and Sept. 9.
Approximately 1800 mirids (mostly L. lineolaris and A. lineolatus) were collected in 1998 for rearing and identification. Three species of nymphal parasitoids were reared f?om collections made in 1998: P. pallipes, P. pseudopallipes and Leiophron solidaginis
Loan. PeristenuspaZZipes emerged from collections made between June 5 and July 5. P. pseudopallipes emerged fiom nymphs collected between July 16 and August 14, and two individuals of L. solidagins emerged fiom collections made June 5 and June 20. PeristenuspaZIIi,es can be distinguished fiom P. pseudopaZZ@es by the well defined punctures between the antennal socket and the median ocellus. Morphological differences within P. paZl@es were also observed depending on the host insect. A pale clypeus characterizes the Lygus type, whereas the Adelphocoris type has a dark clypeus.
More systematic work is necessary to get a complete picture of the P. pa1Zipe.s complex of species.
In 2999, more than 600 adults and nymphs of L. lineolaris, A, lineolatus and
Lepiopterna dolabrata were collected for rearing and parasitoid identification. Number of each mind species collected, date of collection, parasitoid species and emergence time are presented in Table 2.4. Emergence tirne was significantly different for P. pallipes reared fiom Lygus, AdeZphocoris, and Leptopterna hosts @ Micractonus sp. (Hymenoptera: Braconidae), parasites of the alfalfa weevil, emerged fkom the collected material in 1999, probably mistakenly included with the alfalfa plants used to feed L. lineolaris. Table 2.4 Parasitoid emergence (PeristenuspaZZ@es)fkom three mirid species collected in 1999 and held at 26°C fier diapausea. Mirid No. Dates No. No. Mean emergence time species collected collected cocoons ernerged (days * std) LYW 257 June18- 36 25 19.7 * 2.9 a lineolaris July 6 Adelphocoris 130 June 16 23 13 17.0 * 2.2 b Zineola t us Leptopterna 140 June 4 - 48 23 9.3 * 1.6~ dolabrata 12 a A few Microctonus sp. (Hym.: Braconidae) emerged fiom the collected material, and were mistakenly not immediately distinguished fiom the Peristenus paZlipes ernerged fiom Lygus, Adelphocoris and Leptopterna. Number of cocoons found fiom searching the pupation substrate (vermiculite). Average within the same column followed by the same letter are not significantly different (P > 0.05; Tukey's HSD multiple cornparisons test). 2.5 Discussion Five species of native parasitoids have been coliected in the Guelph area in southern Ontario: Peristenus pallipes (Curtis), Peristenus pseudopallipes (Loan), Leiophron Iygivoms (Loan), Leiophron solidaginis Loan, and Leiophron sp. near brevipetiolatus Loan. Further taxonomie studies may indicate that the spring P. pailipes complex in fact contains two or tbree different species, atîackùig different hosts (L. lineolaris vs. A. Iineolatus vs. L. doZabrata) (H. Goulet, pers. comm.). Differences in emergence time for P. pallipes collected fiom the three bost-species after diapause (Table 2.4) seem to support this assumption. Another species, Leiophron un@rmis (Gahan), was not collected in the Guelph xea, but is now known to be present in southern Ontario (Broadbent et al. 1999). The univolthe species P. paliipes was comrnon during the peak of first generation TPB nymphs. Peristenus pseudopallipes was observed parasitizing the second generation TPB nyrnphs at the end of July and the beginning of August. The multivoltine species, L. lygvorus, was more noticeable at the end of the period of the second generation nymphs, in late August/early September, sirnilar to the Leiophron sp. near brevipeliolatus, which is most likely also a multivoItine species. Overall parasitism rates were consistent for the three years studied (see Table 2. L), although greatly influenced by different host-plants and the sampling time. Fields with a mixture of weeds were more heavily parasitized than alfalfa fields, probably due to the continuous source of nectar fiom flowering plants available for the parasitoid adults. Fields with continuous pwthof plants (weedy fields mostly) usually sustained parasitoid populations for most of the spring/sumner, whereas cutting of alfalfa seemed to drastically reduce both host and parasitoid populations (see Figures 2.5A and 2.5B). We observed very little parasitism by P. pseudopallipes (end of JuLy/early August) in alfalfa fields versus weedy fields, which is in accordance with data fiom Shahajahan and Streams (1 973) and Lim and Stewart (1976b). Host-plant preference might explain, in part, this clifference, because P. pseudopaZIipes seems to be attracted to Erigeron spp. which would serve as a nectar source (Shahajahan and Streams 1973, Shahajahan 1974). As well, L. Zineolaris feeds on a wide variety of weeds (Snodgrass et al. 1984) and is often found in very high densities in weeds surrounding agricultural fields (Cleveland 1982). In contrast to P. pallipes, most of the larvae of P. pseudopallipes will emerge fiom the late instars of TPB nymphs rather than the adult (LM and Stewart 1976a). However, some parasitized adults in July/August were observed, which niggests the effect of another parasitoid acting in conjunction with P. pseudopallipes, probably L. lygivorus. However, very few L. Zygivorus were reared fiom collections at this time of the summer. Further refinement of rearing techniques or the development of molecular marker technologies would greatly enhance our capabilities of identimg parasitoids and understanding their host-relationships. Host-preferences of some parasitoids present in northeastern USA are known @ay 1999), but more studies on non-target species, parasitoid biology, and dso on host-range of L. Zygivorus are necessary. The hi& number of TPB and generally low percent parasitism in many of the fields sampled suggests a need for fürther studies on the potential introduction of exotic natural enernies of TPB into Ontario. The introduction and establishment of the parasitoid Peristenus digoneutis Loan in northeastem USA (Day 1996, Day et al. 1998), and the subsequent increase in percent parasitism of Lygus suggests that Merintroductions in areas where P. digoneutis is not present codd enhance control of Lygus bugs. We now possess baseline data on Noah American parasitoids of 3 important mirid species in southem Ontario, and the establishment of European parasitoids to control these pests should be considered. Chapter 3. Synchrony Between the Tarnished Plant Bug, Lygus lineoiark, and Two Exotic Braconid Parasites, Per&enus spp. 3.1 Abstract Poor synchrony in a parasite-host relationship may lead to low rates of parasitism, few offspring for the parasitoid and population extinction. The synchrony of emergence of the parasitoids Perisienus styginrs and P. digoneutis with kt-generation Tamished Plant Bug (TPB) nymphs was assessed by deteminhg day-degree development for TPB and day-degree requirements for emergence of the parasitoids afler diapause. The percent development per day for Lygus lineolaris was proportional to temperature, and permitted the determination of an egg laying threshold of 12.7"C, a nyrnphal development threshold of 7.g°C, and a generation threshold (fiom end of diapause to newly emerged adults in the spring) of 9.6"C- The emergence threshold for P. digoneutis afier 9 months in diapause was 0.3OC, and was 58°C and 55°C for P. stygicus after 6 months and 3 months in diapause, respectively. Accumulated degree-days in the field and predicted occurrence of parasitoids and various stages of TPB showed a gap of almost 30 days between parasite emergence and appearance of TPB nymphs. Ninety percent of parasitoid emergence would have occurred between May 24 and June 10, while ninety percent appearance of the second and third instars of plant bugs, which are the target stages for parasitism, would not be reached until July 7 and July 12, respectively. However, predictions for TPB appearance fiom laboratory studies do not seem to reflect peak coliections of nymphs in the field, and poor correlation may be due to temperatures monitored in field Locations and used for predictions. 3.2 Introduction The Tamished Plant Bug (TPB), Lygus Iineolaris (Miridae: Hemiptera), is an important agricuitural pest in North America. It attacks more than 300 different plant species (Young 1986) and in eastern Canada and the United States, it is the primary pest of lettuce, celery, peppers, strawbemes, apples, and various other crops (Cleveland 1982, Fleischer and Gaylor 1987). Despite occasional high rates of parasitism observed, this pest is not significantly controlled by native species of parasites (Clancy and Pierce 1966, Codson 1987, Day 1987, Day et al. 1990) or predators (Anioldi et ai. 1991). A complementary action by introducing exotic parasites would probably enhance control, however, a better understanding of parasite-host interactions is essential to ensure the success of a biological control program. Lack of synchrony is one important reason why natural enemieç fail to become established in a biological control program (Hagvar 199 1, Stiling 199*3). Poor synchrony in a parasite-host relationship will lead to low rates of parasitism, few offspring fiom the parasitoids, and possibly Iack of establishment of the released agent. This situation can often be indirectly attributed to climate, which may disrupt the synchrony of the parasite/host life-cycles. Studies attempting to correlate heat requirements for the development of hosts and their parasitoids arescarce, although essentiai (Miller 1983). Many s~dieshave been carried out to predict the appearance of the nymphal stages of TPB in various crops using day-degree models (Roberts 1982, Bostanian et al. 1990, Al-Ghamdi cet al. 1995), but there are none in the literature to predict the emergence of TPB parasiitoids. Exotic European parasitoids considered for release in North America are multivoltine or, at least, bivoltine. As such, they would be able to attack and reduce densities of the &st and second generations of the tamished plant bug. Peristenus digoneutis has been released success£üily against the tarnished plant bug in the United States (Day 1996, Day et al. 1990), however attempts to release and establish P. stygicus have failed (VanSteenwyk and Stem 1977, Craig and Loan 1984b, Coulson 1987). The purpose of this research is to determine emergence patterns of the exotic braconid parasitoids Peristenus stygicus (Loan) and P. digoneutis (Loan), as weil as the synchrony of emergence of the parasitoids with first-generation tarnished plant bug nymphs . 3.3 Material and Methods 3.3.1 Diapausing plan f bugs and parasitoids Tamished plant bugs were collected on goidenrod, Solidugo spp., and Canada fleabane, Erigeron canadensis, in Guelph, Ontario, in mid-October 1996. They were placed in outdoor plexiglass cages (60 cm x 50 cm x 50 cm) and supplied with keshly cut goldenrod and sprouting potatoes as food. The bottoms of the cages were filled with a mixture of comgated cardboard and felt pieces to serve as a substrate for ovenvintering adults. Diapausing adults were kept outdoors until the end of March of the following year to simulate conditions found in nature and then transferred to a 2 * 1OC cold room until the start of the experiment in early June 1997. Due to parasitoid quarantine and a required length of time in diapause, it was impossible to start the experiment before June. Overwintering parasitoids of the two Peristenus species were obtained by rearïng paraçitized TPB at short day photopenod (l2hL: 12hD) for a month. Afier that period, the temperature was decreased in 7 day intervals to 14"C, 7OC and then 2OC for the remainllig thne in diapause. Diapausing period was calculated to be the length of time at 2°C. 3.3.2 Assessment of synchrony Mer diapause periods of 3,6 or 9 months, parasites (diapausing cocoons) and plant bugs were placed in growth chambers at constant temperatures ranging fkom 15 to 33°C and at 16hL: 8hD. As well, two cabinets with fluctuating temperatures (26-20°C, avg. 24°C; 23-1 7"C, avg. 2 1OC; both l6hL: 8hD) were included in the experimental set up, to evaluate the effect of different day and night temperatures on development. Twenty-five addt plant bugs were placed in 2 separate containers (4 L buckets covered with mesh) at each temperature, with fiesh food (sprouting potatoes, romaine lemice and green beans) added every 3 days. Old food (serving as egg laying material) was then transferred to a new container. Those containers newly filled with egg laying material were monitored every 3 days, given fiesh food, and stages of the TPB recorded. Upon reacbg the adult stage, the plant bugs were removed fiom the container and developmentai time (nurnber of days) to adulthood was recorded, Emergence of parasitoids at each temperature was checked daily. Emergence thresholds for parasitoids as well as egg laying and nyrnphal development thresholds for the TPB were determined using developmental data collected fiom each temperature (% development/day at each temperature). Day-degree PD) accumulation above developmental thresholds (or emergence threshold) was calculated for parasitoids and plant bugs (see below for formula used). Comparisons of parasitoid emergence and TPB development at different temperatures were subsequently perfomed. Values in day-degrees above the threshold for IO%, 50% and 90% emergence of parasitoids as well as for appearance of each stage of Lygus nymphs were obtained. 3.3.3 Assessrnent offield synchrony To assess putative synchrony in real field conditions, mean soi1 (5 cm below surface) and grass temperatures recorded for the last three years at the Guelph Turfgras Institute, Ontario, were compiled. niresholds of emergence of parasitoids and development of TPB were used to determine degree-day accumulation curves for the spring using average temperatures of the three years. Degree-days were calculated as the difference between mean temperature and the lower threshold of development (or emergence) with the foliowing formula: degree-day = (Tmin + Tmax)/2 - Tb where Tmin is the minimum temperature observed during the day, Tmax is the maximum temperature, and Tb is the Iower developmental threshold. Although hourly data may calculate more accurate estimates of degree-day, we used this simple method to maximise practical use. Comparisons of IO%, 50% and 90% development above thresholds for the TPB, and emergence of parasitoids, were used to compare putative synchrony in the field. Plant bugs were also collected fiom the field during l997-l999, and number of day-degrees for peak appearance of nymphs and adults were compared with degree-day data collected fiom the laboratory experiment. 3.4 Results Developrnentd theof Lygus lineolaris after diapause and emergence of the parasitoids at various temperatures derdiapause are presented in Tables 3.1 and 3 -2. Resuits showed that the higher the temperature, the shorter the developmental time was for L. lineoiaris, although a constant temperature of 33OC appears to have a detrimental effect, slowing domthe development of TPB (Table 3 -1). A similar effect of temperature is observed for the emergence of the parasitoids, with temperahue above 30°C slowing down emergence, as well as decreasing sumival (Table 3-2). Longevity for P. ssgicus and P. digoneutis is presented in Table 3 -3. Peristenus sStgicus emerging after 3 months in diapause survived longer than after 6 months, at ail the temperatures observed. At 21°C, survivd time was 38.7 days for P. stygicus 3 months in diapause, 22.9 days for P. sîygicus 6 months in diapause, and 24.4 days for P. digoneutis 9 months in diapause (Table 3.3). The percent development per day for Lygus Zineolaris was proportional to the temperature and therefore permitted the determination of an egg laying threshold of 12.7"C, a nymph developmental threshold of 7.9"C, and a generation threshold (fi0111 end of diapause to newly emerged spring adults) of 9.6"C (Figure 3.1). For the parasitoids, slight variations in the threshold temperature were observed for different lengths of time in diapause (Figure 3.2). The emergence threshold for P. digoneutis after 9 months in Table 3.1 Cumulative number of days for development of Lygm Zineolaris at various temperature fier diapause. Egg Iaying Second instar Third instar Temperature N Average N Average * N Average (da~s) (days t std) - (days t std) 15 5 28.0 5 67.8 t 6.0 a 6 74.7 -t 6.0 Table 3.1 (con tinued) Fourth instar Fifth instar Adult Temperaîure N Average N Average N Average* (days f std) (days i std) (days & sîd) 82.8 k 7.4 90.0 16.9 97.8 t 4.6 a 64.1 I 1 t .9 71.8 + 13.1 82.2 + 142 b 43.4 i 10.6 48.2 I 10.4 55.5 t ro.5 c 43-1t- 12.9 47.4 2 13.1 54.1 3- 13.0 c 31.3 t 8.3 34.0 + 8.2 39.5 f 8.4 d 29.5 I4.9 32.9 + 4.9 38.1 f 4.7 d 25.8 t 5-4 28.4 t 5.3 33.7 I5.4 e 24.0 t 4-5 26.2 f 4.5 30.4 4 4.5 ef 33 315 24.1 I3.1 361 25.3 13.3 166 29.4 f 3.9 f * Means with the same letter are not significantly different (Waller-Duncan, P c 0.05). " Day and night temperature in parenthesis Average (days) = Average N2 - (Development Tirne NI + egg incubation tirne), where Development time N1 = 0.0547X2 - 2.934X + 42.695 Egg incubation time = 0.04~' - 2.69X + 53.50 (for temperature 20 to 30°C) Egg incubation time = 0.1 2~'- 6.987X +- 106.61 (for temperature 15, 18,33"C), fiorn Roberts (1982) X = temperature ("C) Table 3.2 Emergence of Perktemcs species folIowing 3,6 or 9 rnonths in diapause P. stygr'cus 3 months P-stygi~t~~ 6 months P. digoneutis 9 montfis Temperature N Average N Average N Average (days Igd) (days t std) (days 3- std) 15 11 37.27 t 4.6 a 28 25.8 t 2.2 a 12 29.2 3.5 a 18 13 32.7f 2.9 b 10 19.9 t 1.4 b 15 22.9 -t 2.1 b 2 1 20 19.9 12.4 cd 16 15.4I 1.5 c 20 18.3 f 2.2 cde 21 (23-17) a 12 20.7 k 2.6 c 19 14.5 I1.2 c 15 18.7 t 3.1 cd 24 19 18.4 i 1.9 de 11 11.6+1.1 d 18 17.0 t 1.5 def 24 (26-20) a 10 19.9 i 1.9 cd - - 17 16.5 + 1.7 ef 27 9 16.8 t 2.2 e 9 I1.7t0.7 d 12 15.8 k 2.9 f 30 O - 1O 12.1 + 1.3 d 1O I5.4f 12f 33 O - O - 3 20.0 f6.9 c Means with the same letter are not significantly- different (Waller-Duncan, P c 0.05). a Day and night temperature in pareibiesis Table 3.3 Longevity of Perktem sWctls at various temperature foIlowing 3,6 or 9 months in diapause. P. stY'gm 3 rnonths P. stygicus 6 months P. digoneais 9 months Temperature N Average N Average N Average (days + std) (days t std) (days f std) 15 11 57-7t 10.8 17 53.1 t 13.1 - - 18 13 46.8 f 13.7 23 30.6 f 7.6 - - 21 20 38.7t 8.7 18 22.9 t 14.6 19 24.4 + 1 1.5 21(23-17)a 12 29.3t8.7 20 20.9 t 7.5 - - 24 19 25.4213.6 23 12.2 + 8.5 5 16-6 f 8.9 27 9 17.6 f 7.2 10 14.6 f 7.7 - - 30 O - 13 5.4 + 3.9 - - 33 O - O - - - a Day and night temperature in parenthesis 16 - Egg Iaying y = 0.927~ 11-80 eA 12 - 'P R~= 0.89, threshold = 12.7OC a!z 10 8- -O y=O.l67x - 1.60 Q) 6- m5 ' 4i 2 - O 1 I I O 5 10 15 20 25 30 Temperature (OC) Figure 3.1 Temperature thresholds of developrnent for Lygus lineolaris ' 1 P. digoneutis 9 months 1 8 - . 1 A P. stygicus 3 months 7 - O 5 10 15 20 25 30 Temperature ("C) Figure 3.2 Thresholds of emergence for Peristenus stygicus and P. digoneutis derdiapause periods of 3,6 and 9 months diapause was 0.3OC, and was 58°C and 55°C for P. stygicus afler 6 months and 3 rnonths in diapause, respectively. The emergence temperature thresholds determined for the parasitoids were used subsequently for day-degree calculations, as well as the first generation temperature threshold for the TPB (9.6"C). Mean emergence times for Peristenus digoneutis and P. sîygicus as a function of the number of weeks in diapause reveal a plateau after 5 months for three shains of Peristenus spp. (Whistlecraft et al. 2000). We can thus assume that values obtained for P. digoneutis emergence after 9 months in diapause and P. sîygicus after 6 months in diapause (Table 3.2), would reflect emergence tirne of the species fiom 5 to 10 months after diapause. Direct cornparisons of the mean number of days until parasitoid emergence with the appearance period of 2nd and 3rd instar nymphs of the TPB at various temperatures appeared to show a lack of synchrony between the parasitoids and the host (Figure 3 -3). For most of the temperatures observed, mean days to emergence of the parasitoids was reached before any 2nd instar nymphs were observed. As the temperature increased, however, the gap between emergence of parasitoids and appearance of young nyrnphs seemed to decrease (Figure 3.3). Emergence of parasitoids and developmental stage of TPB should be compared by degree-day accumulation above the thresholds of development. Number of DD accumulation over their respective developmental threshold for parasitoids (see Figure 3.4) showed that IO%, 50% and 90% emergence occurred at about 204.1,234.0 and 263.8 DD above 5.8OC for P. st~@cusafter 6 months diapause, and at 284.9,335.2 and 405.4 DD above 5S°C for P. stygicus after 3 months in diapause (Table 3.4). Cumulative emergence - - L. lineolaris 2nd instar 0 L. lineolaris 3rd instar 0 P. stygicus 3 months I P. stygicus 6 months j 0 P. digoneutis 9 months O --- 10 15 20 25 30 35 Temperature ("C) Figure 3.3 Cornparison of laboratory ernergence of parasitoids (3,6 or 9 months in diapause) and appearance of early nymphs of Lygus lineolaris after diapause I en 6. z 401 A A .)* P. digo9 cumul. > 0.3 OC I P. sty6 cumul. > 58°C A P. sty3 cumul. > 5.S°C O L I l 1 1O0 200 300 400 500 600 DD > threshold Figure 3 -4 Cumulative emergence of Perisfenzis digoneufis and P. stygims above their developmental threshold, after a diapause period of 3, 6 or 9 months Table 3.4 Day-degree requirements for emergence of parasitoids and appearance of plant bug nymphs in the laboratory, and predicted dates using average field temperatures (1997-1 999) at the Guelph Turfgrass Institute Day-degree accumulation Predicted date* Predicted date* Predicted date* TO - Laboratory - Air temperature Soil temperature Grass temperature thresliold 10% 50% 90% 10% 50% 90% 10% 50% 90% 10% 50% 90% P. digoneutis 0.3 338.6 404.2 469.8 May May May May May May May May May 9 months diapause 16 22 28 15 20 24 12 17 22 P,sîygicus 5.8 204.1 234.0 263.8 May May June May May May May May May 6 months diapause 29 3 1 4 24 28 31 20 24 28 P. siygicus 5.5 284.9 335.2 405.4 June June June May June June May June June 3 months diapause 5 11 16 31 6 10 29 3 8 Lygw 2ndinstars 9.6 242.7 387.9 533.1 June July July June June July June June JUIY * 2 1 5 20 13 26 9 12 25 7 Lygus 3" instars 9.6 289.6 433.0 576.4 June July July June June Jul y June June July 4 c. 2 5 11 25 18 3O 13 17 28 12 Lygur 4' instars 9.6 323.5 476.2 629.0 June July July June July july June July July 28 15 30 2 1 4 16 20 2 16 9.6 377.5 523.4 669.2 July July August June July July June July July 4 19 3 26 8 19 24 6 18 9,6 444.4 601.7 759.0 July July ----a July July Jul y June July July 12 27 1 14 27 29 14 26 'Predicted date determined from average temperature of 1997-1999 at Guelph Turfgrass Institute, Dates in bold types are most probable given the habitat of parasitoid cocoons (soil) and plant bugs (plant canopy). of P. digoneutis showed that IO%, 50% and 90% emergence occurred at 338.6,404.2 and 469.8 DD above the threshold of O.3"C. For the tamished plant bug, the number of DD accurnulated above the threshold of 9.6OC (Figure 3.5) showed that IO%, 50% and 90% appearance of early nymphs, which are target stages for parasitism, were 242.7,387.9 and 533.1 DD for second instar, and 289.6,433.0 and 576.4 DD for third instars, respectively (Table 3.4). Degree-day accumulations are also presented for third, fourth and fia instars as well as adult TPB (Table 3 -4). Parasitoid cocoons will remain in the soil mtiI emergence, while plant bug ad&, eggs and nymphs are influenced by the temperature at the canopy level. As such, we compared parasitoid/host synchrony by using soil and gras temperature data fiom 1997- 1999 fiom the Guelph Turfgrass Institute, Guelph, Ontario. Accumulated degree-days in the field (Figure 3.6) and predicted occurrence of parasitoids and various stages of TPB (Table 3.4) showed a gap of almost 30 days between parasite emergence and appearance of TPB nymphs (Figure 3.6). Ninety percent parasitoid emergence would be reached between May 24 and June 10, although ninety percent appearance of the second and third instars of plant bugs would not be reached until July 7 and July 12, respectively (Table 3.4). Data fkom TPB field collections showed that the peak of late instar nymphs of TPB was observed on the week of June 15 for 1998 and 1999 (Figures 2.1A and 2.1 B, Chapter 2). This does not correspond well with predicted values of appearance of fourth and fifih instars of TPB (Jdy 2 and July 6, respectively) based on laboratory measurements of degree-day and corresponding values in the field (Table 3.4). ------1st instar 2nd instar A 3rd instar x 4th instar + 5th instar , Adults Figure 3.5 Cumulative development of Lygus ZineoZaris above developmentd threshold of 9.B°C 8 Q O DD > 5.8OC (Soil TO) Q O x DD > 5S°C (Soi1 TO) Q O Apd May May June J~Y 10 5 30 24 19 Date Figure 3 -6 Day-degree accumulation above thresholds, calculated fiom 1997- 1999 average temperature (soi1 or grass), and predicted dates of appearance (arrows) of L. lineolmis, P. stygicus and P. digonezttis fiom laboratory study 3.5 Discussion In the spring, &er the emergence of TPB fiom diapause, air temperatures higher than 12.7OC are needed for egg iaying. This is higher than the developmental threshold of 7.g°C observed for nymphal development. Because days of warmer temperatures are important for egg laying at the beginning of the spring, an unuçualIy cold spring could lead to delayed development of the first generation of the Tarnished Plant Bug. The food source used by TPB for the determination of the thresholds was a combination of beans, lettuce and sprouting potatoes. The addition of coddled lepidopteran larvae as food improved the development and survival of L. hespems (Knight) (Wheeler 1976), which rnight imply that the threshold values would have been slightly different with more protein-rich food. Fleishler and Gaylor (1988) also found a variation of the lower threshold of development for L Zineolaris depending on the food source, with a range of thresholds fiom 8.03 to 11.70°C on nine different food sources (8 weeds and beans). However, no significant ciifferences among hosts were detected. Other factors can affect developmental rates (Hilbert 1995), such as feeding behaviour, food availability and quality, and size at the end of the juvenile stage. These factors also Vary in time, which cm complicate and decrease precision of modelling of developmental rates. Compromises may have to be made between precision and practicability of developmentai threshold and day-degree prediction models (Pruess 1983). However, the developmental thresholds found for the tamished plant bug are in agreement with those in the literatue, which vary fiom 9.30~to 10.8OC fiom egg hatch to adulthood (Ridgway and Gyrisco 1960, Roberts 1982, Fleischler and Gaylor 1988). The emergence thresholds of O.3OC, 5S°C and 5.8"C observed for the two parasitoids seem to indicate that these species are adapted to cotder climates. Peristenus digoneutis threshold was the lowest of al1 at 0.3OC, but the long period of diapause (9 months) may explaùi in part the clifference between P. digoneutir and P. stygicus. Researchers have found P. digoneutis to be more abundant in northern parts of France, Germany, Switzerland and Poland, which could explain the lower threshold (Bilewicz- Pawinska l976a, D. Coutinot, pers. comm. 1999). The plateau of emergence obtained after 20 weeks in diapause (Whistlecraft et al. 2000) indicates that the time for emergence will not vary extensively, or decrease, after a period of this length at diapausing temperature. Comparisons of emergence of the parasitoids with the appearance of the TPB using degree-day data must consider the habitats and life cycles of the parasitoids and hosts. In a field environment, the cocoons of the parasitoid wasps will stay in the soil until emergence, therefore, accumulation of physiological time at temperature >threshold should be calculated fiom soil temperatmes. However, no studies have been published on diapausing habitats of these parasitoids. We assumed that soil temperatures at 5 cm would better reflect temperatures inifluencing the cocoons, since Bilewicz-Pawinska and Pankanin (1974) found the largest number of cocoons near the roots of the plants on which the hosts were reared. For more accuracy using microhabitat temperatures to determine and evaluate thresholds for the parasitoids, a thorough study of cocoon distribution in soil and leaf litter should be conducted. TPB adults will diapause in leaf litter and climb to surrounding vegetation as temperature increases. Therefore, temperature at gras level, which probably better reflects canopy temperatures, seems to be appropriate for degree-day caiculations for TPB, aithough again, variations of microhabitats might slightly affect these calculations. The predicted appearance of early nymphs of Tarnished Plant Bug with the day- degree mode1 used does not seem to reflect accurately the time of appearance of nymphs in the field. Field colIection of 2nd and 3rd instar nymphs (Chapter 2) was performed before the predicted appearance of nymphs at the end of June/early July. Boivin and Stewart (1983) found peak densities of various nymphal stages of Lygus in Quebec to be eariier than predicted values found here using degree-day data. However, large variations have been observed depending of the year or the host-plants sampled (Boivin and Stewart 1983) and direct calendar cornparison dates may even be more misleading for predictions. Khattat and Stewart (1980) found peak capture of 4th instar Lygus lineolaris occurring at the end of June in Quebec, which is in accordance with the predicted values obtained in this study. ALGhamdi et al. (1995) observed the peak of nymphs in early June in Quebec at 257 DD (calculated fiom air temperature) above a threshold of 8.0°C. Upon the use of a similar threshold to calculate degree-day from our field data (Guelph Turfgrass Institute), 257 DD fiom air temperatures would correspond to the appearance of the peak number of nymphs on June 14. The method used to measure stages of growth of TPB in rearing cabinets may have overestimated the number of degree-days necessw for emergence. Three or four days separating the observations might lead to the sarne instar of TPB being observed twice, moreover at lower temperatures. Space limitation did not permit the transfer of TPB instars to a new container after each moult, which would have avoided the recounting of specimens at the same stage. Using gras temperature and soi1 temperature (5 cm) compiled nom field data fiom the Guelph area, TPB and parasitoid thresholds showed that approximately one month would separate 50% parasite emergence and 50% appearance of 2ndinstars of L. lineolaris. Even though P. stygicm emergence seems to be more synchronous with the TPB than that of P. digoneutis with temperature field data fiom the Guelph area, neither appear to have adequate synchrony for control based on laboratory studies. Using another set of temperature data from a more Southern (and warmer) climate may reveal a more appropnate synchrony host-parasitoid. An alternate host such as the alfalfa plant bug, AdeZphocoris lineolatus (Goeze) may permit the suMva.1 and establishment of Peristenus spp. in the field since it overwinters in the egg stage and thus nymphs appear earlier in the spnng than do nymphs of the TPB. Chapter 4. Interspecific Cornpetition Between Exotic and Native Parasitoids of the Tarnished Plant Bug, Lygrcs lineolaris (Palisot de Beauvois) 4.1 Abstract European parasitoids considered for introduction to control the Tarnished Plant Bug in North Amenca have no previous association with the target pest, Lygus lineolaris (Palisot de Beauvois). In Europe, they attack primarily Lygus rugulipennis Poppius and L. pratensis (L.). Compatibility of these parasitoids and their hosts in a "new- association", the specificity of such biocontrol agents, and the potential cornpetition with native parasitoids could be issues for the prospect of using European species in biological control programs against L. lineolaris. In-host compatibility and competitiveness of the exotic multivoltine parasitoids, Peristenus slygicus and P. digoneutis, with the native parasitoids, Leiophron Zygivorus (rnultivoltine), P. pallipes and P. pseudopallipes (both univoltine) were assessed. Dissection of hosts indicated that over 92% of the parasitoid attacks on L. lineolaris nyrnphs resulted in oviposition and development of the larvae for the five species studied. In suitability tests, 84% of P. styginis adult wasps emerged fiom parasitized Tamished Plant Bugs, compared to 67% and 69% for P. digoneutis and L. wvorus, respectively. P. digoneutis was superior in the in-host competition with P. stygicus and L. lygivoms . Peristenus stygicicus was dominant in the in-host competition with the three North American parasitoids. The length of the dominant larva inside the host for P. stygiczu seven days after oviposition was iduenced by the number of larvae present. When only one larva was developing, the average size was 2.3 + 0.2 mm for the dominant larva, and the size decreased as the number of larvae increased. 4.2 Introduction The Tamished Plant Bug (TPB), Lygus lineolaris (Palisot de Beauvois), feeds on - over 300 spp. of plants (Young 1986), and is a major pest of alfalfa, cotton, strawbemes, apples, canola and several species of vegetable crops. In North Amenca, this Pest species is attacked by the native parasitoids Peristenus pall@es (Curtis), P. pseudopallipes (Loan), Leiophron uniformis (Gahan) and L. l'ygfvonrs (Loan). Unfomuiately, parasitism rates on Lygus lineolaris by the native parasitoids are usuaily too low to give effective control (ç20%), despite occasional records of 50-60% parasitism of single samples. Most previous attempts to release European parasitoids to control the TPB in Canada and the United States have failed (Day 1987, Craig and Loan 1987). Releases of P. digoneutis Loan, P. stygzgzcus(Loan), P. adelphocoridis Loan and P. rubr icollis (Thornpson) have been performed at different locations in western Canada between 1978 - 1981 (Craig and Loan 1987). Releases of the same four species were also made in California, Arizona, Delaware and New Jersey, USA, fiorn 1964 to 1984 (Day 1987). Reasons for the iack of establishment might include low numbers released, poor synchronization with the host, encapsulation of the parasitoid larva, or cornpetition with native parasitoids. However, despite some failures, one species, P. digoneutis, is now established in the northeastern United States @ay et al. 1990, Day 1996) and has decreased L. lineolaris populations there by up to 75% (Day 1996). It is now present in Québec, Canada (Broadbent et al. 1999) and will likely be released in Ontario in the near future. Another species, P. s~gicus,seems to possess desirable charactenstics for release such as facultative diapause, short developmental time, high ievel of parasitization and ease of mass rearing Proadbent 1976). European parasitoids considered for introduction have no previous association with the target Pest, Lygus Zineolaris, in North America. Compatibility of parasitoids and their hosts in such a "new-association" (Hokkanen and Pimente1 1984), as well as the specifïcity of such biocontrol agents (Goeden and Kok 1986) could be issues for the prospect of using this biological strategy (Waage and Greathead 1988). However, the new-association strategy represents an alternative to classical biological control and has been used successfully on many occasions (Hokkanen and Pimentel 1989). Some programs are still in progress to htroduce additional European parasitoids to North Amenca (Kuhlmann et al. 1998). Although searcbg ability of a parasitoid might be the most important feature to assess its competitiveness, in-host competition might in some cases result in displacement of one species by another (Ehler and Hall 1982). For most parasitoids, and as is the case here with euphorine parasitoids, only one species will survive in the host when involved in interspecific competition. The introduction of a new exotic species of parasitoid into a guild of native species will ceaainly have an effect on the dynamics of host-parasitoid relationships. In this study, two of the three most cornmon parasitoids of the European Lygus (Lywrugulipennis Poppius), P. digoneutis and P. s~ygicuî,were evaluated for their competitiveness with native North Amencan parasitoids. Three species of North Amencan nymphd parasitoids of TPB were available for cornparison: P. pallipes (emerged fiom L. Zineolaris), P. pseudopallipes and L lygivoms. The two North Amencan Peristenus species are univolthe, P. pa1Zipe.s attacking the ktgeneration nymphs in May-June and P. pseudopaZZ@es attackuig the second generation in July- August (Loan 1965, 1974b, 1980). Leiophron species are multivoltine, but the individuals of L. lygivorus, a poorly known species, studied here were coilected mostly in late summer in 1997 (August-September) (Lachance, Chapter 2). The objectives of this study were to determine the in-host compatibility and the competitiveness of the exotic multivoltine parasitoids, P-sWas and P-digoraeutis, with the native parasitoids, L. [ygivorus, P. pallipes and P-pseudopalliges. It is important, before release, to predict the potential outcome of interspecific competition between the introduced agents and the native parasitoids. Introduced parasitoids may fail to become estabiished because of competition with native ones, or may be established but displace natural enemies already present. 4.3 Materials and Methods Second and earIy third instar nymphs of L. lineolaris were used for parasitism. Parasitized nymphs were reared on lettuce and bean pods, and kept at 16hL: 8hD and 22 * I OC. Parasitoid adults were held at 14OC, 16hL: 8hD. Upon emergence, male and female wasps were held together, by species, in a 3.5 L glas Mason jar, and kept at 22°C for at least 90 minutes after emergence (usually during the fïrst 3 hours of the photophase) for mating. The jar was then returned to 14OC. Honey and water were provided. Female wasps used in the experiments were selected fkom 3 to 15 days mer emergence, to ensure they were properly mated. Each female was then put in contact with TPB nymphs in a srnall via1 (40 ml) for parasitism. Between 3 and 6 nyrmphs were presented at once, and nymphs attacked by the female were immediately withdrawn kom the via1 and set aside in a cylindncal plexiglass cage (15 cm ht x 9.5 cm diam) with beans and lettuce for food. Sets of about 10 parasitized nymphs were grouped together in the cage. Food was changed every 4 days, and vermicuiite was added in a container undemeath the cage to serve as a pupation site for the parasitoids. Each female parasitized between 3 and 5 nymphs per day, for approximately 5-7 days. Suitability of L Iineolaris as a host for each species of parasitoid was assessed by dissecting sorne parasitized nymphs about seven days after parasitism (parasitism success) and by rearing the balance of the parasitized hosts until emergence of adult wasps (emergence success). P. digoneutis eggs were reported to hatch about five days &er oviposition at 22 1°C (Carignan et al. 1995), so perfo&g dissections 7 days after attack fiom a wasp ensured that al1 eggs would have hatched. Non-emerged parasitoids fkom each of the 5 parasitoid species were dissected fiom their cocoons, and number of dead pupae counted. For the in-host cornpetition study, nyrnphs were parasitized by one parasitoid species at time zero and then parasitized again by anorher species 24 hours later. Most combinations of parasitoids were compared, but sample sizes were greatly dependent on parasitoid availability and emergence. Peristenus digoneutis was not tested against al1 native species. Pairings were made over an extended time period under identical temperature, photoperiod and rearing conditions to provide sufficient replications for each species combination. Date and species of parasitoid that subsequently emerged were recorded. After al1 emergence had occurred, cocoons remaining in the vermiculite were dissected and number of dead parasitoids recorded. The effect of supernumerary Iarvae inside the host on the size of the surviving larva was also assessed. Thirtj 2nd or early 3rd instar nymphs were introduced daily in a 3.5 L jar containhg about 15 females of P. sîygicus, and withdrawn after 60 minutes. The nyrnphs were reared as stated above, and dissected at 7 days &er parasitism. The length of the Live (dominant) larva was measured, and the number of larvae present was recorded. 4.4 Results Dissection of hosts indicated that 92-100% of attacks ty 1 a single parasitoid specie on plant bug nymphs in the laboratory resuited in oviposition and development of larvae for the five species studied (Table 4.1). No signs of encapsulation were observed for any parasitoid species, as almost al1 of the eggs had hatched and the larvae developed. Attacks by Peristenus stygicus, P. pallipes, P. pseudopdlipes and L. lygivorus occurred quickly, as the wasps responded to the proximity of TPB nymphs by intensive searching in somewhat scattered movexnents. Peristenur digoneutis was more sluggish, and did not always show signs of active searching behavior. Parasitization by P. digoneutis occurred almost exclusively when the host and the wasp collided together by chance in the vial. Percentage emergence of adult wasps of the dif5erent species when each species attacked hosts without cornpetition, was recorded (Table 4.2). Eighty-four percent of P. sfygicus adults emerged compared with 67.3% and 69.0% for P. digoneutis and L. mvonrr, respectively. Cocoons were occasionally found to contain a wasp that appeared alive but had not emerged fiom the pupation substrate (vermiculite), for these three species. In such cases, they were included 21 the number of emerged adults. Table 4.1 Parasitism rates of nyxnphs of Lygus lineolaris after a single attack by each of the five different species ofparasitoids. Parasitoid species No. nymphs No. nymphs % parasitism attacked parasitized * P. stygicus 79 78 98.7 P. digoneutis 40 40 100 L. lygrgrvorus 51 47 92.2 P. pallipes 40 39 97.5 P. pseudopallipes 16 16 100 * Dissection of nymphs was performed 7 days after parasitism. Table 4.2 Percent emergence of 5 species of adult parasitoids fiom the host Lyws Zineolarïs dera single attack by a female wasp. Parasitoids species No. No. emerging YO No. of parasitized (adults) a emergence dead pupae P. stygicus 183 153 83-6 4 P. pseudapallipes 35 1 2.9 14 a Cive parasitoids occasionaHy found during dissections were included in totds Peristenus pallipes and P. pseudopallipes (univoltine species) must go through diapause before emergence, and mortality during diapause was high. No emergence was recorded for P. pallwes, although 9 dead pupae were found; only one adult of P. pseudopaZlipes emerged, and 14 dead pupae were found (Table 4.2). Peristenus digoneutis was superior in in-host competition with P. srygim (Table 4.3). P. digoneutis offspring emerged 40% of the time if their parents parasitized a host initially parasitized by P. sîygicus. Offspring of P. digoneutis emerged 88% of the time if their parents parasitized the host £kt(Table 4.3). When P. digoneutis competed with L. mvoncr, similar results were observed. Forty-one percent of the offspring were P. digoneutis when it parasitized second, and 84% were P. digoneutis if it parasitized first. However, in pairings with P. digoneutis, more L. bgivorus (45.6%) thm P. stygicus (28.0%) offspring emerged if P. digoneutis parasitized second. Peristenus stygicus was dominant in the in-host competition with the Siree North Amencan parasitoids (Table 4.3). In more than 60% of the cases, a P. siygicus wasp emerged fiom a host parasitized by this species and L. Zygivorus or P. pallipes, even if P. swcus parasitized the host 24 hours &er these two latter species (Table 4.3). From 69.1 % to 79.7% of P. stygicus emerged if competing with L. Zygivorus, and 60.0% to 68.4% of P. stygicus emerged if competing with P. paZZipes. Fewer P. stygicus emerged if matched against P. pseudopallipes (3 8-5 to 45 9% emergence) (Table 4.3). Peristenus palZ@es and P. pseudopallipes seemed to be better in-host cornpetitors than L. lygivorus, as the competition between them and L. Zygivonrs yielded ~6.4%of L. Zygivorus wasps (Table 4.3). However, mortality of the univoltine parasitoids P. pallipes and P. pseudopallipes during diapause was high. The sequence of parasitism only Table 4.3 Emergence and mortality of parasitoids following parasitism of a Lygus lineolaris nymph with 2 different species at 24 hours interval. Species attacking No. of Emergence of parasitoids Number of dead pupae Total number First (t=O) Second (t=24h) attacks First sp. Second sp. First sp. Second sp. Unknown sp, First sp, (%) Second sp, (%) P. stygicus P. digone utis 25 7 1O O O O 7 (28.0%) 1 O (40.0%) P. digoneutis P. stygicus 5 O 44 3 O O O 44 (88.0%) 3 (6,0%) ,llll-l---ll-----l------. "C---..---.-..------.---.----.---.------..**~*.--.-....-.....*-.....----.----.--.------.-*------.---*-".-----..----. L, lygivorus P. digone ut is 46 2 1 19 O O 1 21(45,6%) 19(41.3%) P. digoneufis L. iygivortrs 44 37 3 O O O 37 (84,1%) 3 (6.8%) .------ll------".-~------*------*.-.------*------*-.--.-.*------.-...-.---..--..-.-*------.-*--.----.----.----.------..------*---* P. stygicus L. iygivorus 79 63 1 O 2 2 63(79,7%) 3(3.8%) P. stygictrs P, pullipes 10 5 0 O 1 O 6 (60.0%) 1 (10.0%) P. pseudopullipes P. stygictrs 37 2 17 1 O O 3(8,1%) 17(45.9%) .-1-..11-----1.1-----"*-..-*--.------.--.-.-----.----..------~..--.------*------"---.-".-*--.-----*------.--.------*----*-----.------*.. L. lygivorus P. pallipes 39 1 2 O 20 O 1 (2,6%) 22 (56.4%) P,pallipes L, lygivorus 47 5 3 15 O O 20 (42.5%) 3 (6.4%) L. lygivorus P. pseudopallipes 20 1 4 O 8 O 1 (5.0%) 12 (60,0%) P. pseudopallipes L. lygivorus 11 O O 8 O O 8 (72.7%) O (0%) " Live parasitoids occasionally found during dissections were included slightly affected emergence for a particular species of parasitoid. Only when competing with P. digoneutis did the sequence of parasitism af5ect the outcome of adult emergence. The size of the dominant larva inside the host for P. siygicus was influenced by the number of larvae present (Table 4.4). When only one larva was developing, the average length was 2.3 I 0.2 mm for the dominmt lama, and the size decreased as the number of larvae increased (Table 4.4). Up to 12 larvae were found in one host, but in al1 superparasitisrn cases only one larva per host appeared dive at the time of dissection. Table 4.4 Length of survïving larva of Peristenus slygczis in the host Lygus Zineolaris seven days derparasitisrn No. of larvae in host N Lena (mm) STD 1 5 2.32 a 0.19 2 11 1.68 0.23 3 9 1.54 bC 0.24 24 12 1.38 O. 14 Means with the same letter are not significantly different (Waller-Duncan, P < 0.05). 4.5 Discussion Based on the dissection results, the five parasitoids studied almost dways laid an egg with every attack. This result differs fiom the flndings fiom Drea et al, (1973), where only 11 out of 18 L. rugulipennis nymphs contained a larva after an attack by P. stygzgzcus. In this case, it rnight be possible that L. ruguZïpennis, being the most cornmon native host of P. sîygicus in Europe, has developed an immune defense by encapsulating and killing the parasitoid egg. Although the three species tested in this study (P. pallipes, L. Mvorus and P. pseudopallipes) are Nearctic [there is still some uncertainty about P. paZZ@es, @ay 1987)] and have CO-evolvedwith L. lineolaris, no encapsulation was observed. However, P. pallipes might have a different host preference than L. lineolaris @ay 1999, P. Mason, pers. comm.). Another North American species, L. unformis, does not seem to utilize L. lineolaris as a preferred host, as only 6.7% of the nymphs attacked yielded a cocoon (Debolt 1989a). This is not the case with L. lygivorus, where Iarval development, pupation, and adult emergence occur without any complications. Although more than 92% of attacks resulted in egg-laying for the three multivoltine parasitoid species, only 67.3 to 83.6%adult wasps emerged, depending on the species (Tables 4.1 and 4.2). Mortality therefore occurred mostly during the deveiopment of wasp larvae inside its host or before pupation, as most of the cocoons found in the pupation substrate were empty indicating ernergence (aithough occasionally not al1 the cocoons could be found in the substrate). Natural mortaiity may have resulted fiom stinging trauma (Day 1994), cannibalism of nymphs, encapsulation (not likely here), and pupation failure by parasitoid larvae. A few parasitoids found in their cocoons were fully formed and appeared alive. These wasps may have entered diapause, although the 92 photophase was 16 hours. This situation has been obsemed more fiequently with the bivoltine P. digoneutis (Lachance Chapter 5, J. Whistlecraft pers. comm.). A long diapause period seems to act as a major mortality factor for the univolthe parasitoids P. pallipes and P. pseudopallipes, as emergence rates were often low, and many dead pupae were found. The amount of moishire was likely an important factor. The fist lama to hatch inside the host-nymph most likely killed the other species present by mechanical means using its mandibles (Carignan et al. 1995). Development of P. stygicus at 22OC resulted in hatching of the egg almost invariably at 5 days after parasitism (Lachance, Chapter 5). The same has been observed for P. diganeutis at 2 1°C (Carignan et al. 2 995). Although the fist larva to emerge is likely to survive, the presence of other eggs or larvae in the same host had an influence on its development. The more larvae that were inside the host, the srnaller the remauiing live larva was at 7 days after parasitism (see Table 4.4). Whether surviving larvae would recover fiom this growth inhibition by the pupation stage is unknown. A thorough study of the developmental stages of al1 these parasitoids would shed valuable light on the outcome of cornpetition. A difference of only one day between parasitisrn of the host by two species of adult wasps as performed in this study might not have been enough to account for the variation in egg hatch among all species. A large variation in egg hatch might explain the slight difference in wasp emergence due to the sequence of parasitism, although for P. stygcuî and P. digoneutis this was not the case. An interval of 2 days would most likely favor the first attacker, Surprisingly, although P. digoneutis was a better cornpetitor than P. stygicus when developing together in the same host, when they were competing with L. lygivorus, P. stygicus appeared to have a slight advantage. Some factor, other than mechanical damage, must therefore be involved in the competition, or alternatively, the second parasitoid to attack might not have laid an egg. A parasitoid that encounters a host already parasitized might accept the host and lay an egg, or reject the host. Most parasitoids are not able to recognize a host previously parasitized by a dif%erentspecies and wiiI therefore multiparasitize the host upon encounter (Hagvar 1989). It would be unlikely that a rejection fkom the second wasp occurred here, due to the 24 hours interval between parasitism, and the usually aggressive attack of the second species of parasite used for competition. Moreover, it is usually accepted that markers used by parasitoids are species-specinc (Bauer 1985, Laing and Corrigan 1987) and that multiparasitism is a better strategy in many situations (Hagvar 1989). Host-preferences, habitat (host plants) and tirne of activity of the various parasitoids will have a decisive impact on parasitoid population dynamics in the field. The parasitoid L. uniformis (not tested here), a common species in the United States, is thought to utilize the mirid Halticus bractatus (Say) as a preferred host (Day et Saunders 1990) and therefore should not be an important competitor with introduced parasitoid species for the host L. lineolaris. Peristenus pallipes, although found parasitizing L. ZineoZaris, also has other preferred hosts such as Leptopterna dolobrata L., Trigonotylus coelestialium (Kïrkaldy) and possibly Adelphocoris lineolufus (Goeze) (Day 1999, P. Mason, pers. comrn.). The two parasitoid species that might interact significantly with an introduced species would be L. lygiorus with first and second generation TPB and P. pseudopallipes with second generation TPB ody. However, the habitat preference of L. lygvorus seems to be weedy areas characterized by the presence of Erigeron and Solidago spp. (Loan 1980, Lachance Chapter 2), which would reduce the iikelihood of cornpetitive in-host interaction with an introduced parasitoid species. The European parasitoids, P. S@~CUSand P. digoneutis, seem to be more adapted to search in alfia than the North American species, which are found commonly in weedy areas @ay 1987). In northeastern United States, P. digoneutis seems to parasitize TPB slightly after P. paZZipes @ay, pers. comm, Lachance, pers. observations), which might confirm the differences in host preference and penod of emergence. P. pseudopallipes would not directly compete with other parasitoids, or at least its influence would be lirnited because of its late summer appearance. Although the European P-digoneuris and P. sStgicus wodd be better in-host cornpetitors than the native species tested, they would most likely not exclude any other parasitoid fiom the current guild. Their establishment would also not be at risk of failure because of competition wiîh native North American species. A direct interaction that might occur between parasitoids would be in the case of the release of P. stygicus in areas where P. digoneutis is now established (northeastem United States). The outcome of such competition in the field is difficult to predict, although in the laboratory study P. digoneuris appears to be a slightly better in-host cornpetitor than P. stygicus. Successful establishment and control of plant bug popdations have been achieved after the release of P. digoneutis, with a decrease of up to 75% of Lygus populations in alfalfa @ay 1996). Successful establishment of P. stygicus might be difficult in areas occupied by P. digoneutis due to the in-host advantage of P. digoneutis, and the apparent limitation of hosts in alfdfa due to the reduction in Lygus populations by P. digoneulis @ay 1996). Peristenus stygicus, given its aggressiveness and cornpetitiveness, might still be a good candidate for release and establishment in North America. However, previous releases of that species have not been successflll (Van Steenwyk and Stem 1977, Cmig and Loan 1987), and have been explained by the poor dispersal of the adult wasps çVan Steenwyk and Stem 1977). A parasitoid chosen for release needs to fit into the existing community of natural enemies, with minimal cornpetitive interference (Mïller 1983). Although the European P. digoneutis and P. styginrs seem to be better in-host cornpetitors than native species when tested in the laboratory, it appears, fiom the biological characteristics of native and exotic parasitoids, that if the two European parasitoids were to be released in Canada, they would probably cause no major distubance in the population dynarnics of native North American species. Chapter 5. Diapause Induction in the Parasitoids Peristenus stygicus and Leiophron @gnlorus(Hymenoptera: Braconidae: Euphorinae) 5.1 Abstract Knowledge of diapause requirements is an important part of a rearing program for biocontrol agents. In order to "stockpile" large number of parasitoids for mas-release, one should be able to induce and terminate diapause, as well as to successfully store the diapausing stage. Induction of diapause for Peristenus stygicus Loan and L. Zygïvorus Coan) both multivoltine parasitoids of the tamished plant bug, Lygus lineolaris (Palisot de Beauvois) was studied. Parasitized nymphs of L. Zineoluris were transferred to short day photopenod (12hD: 12hL) at intervals of O to 11 days afler parasitism, in order to induce diapause of the parasitoids. Fi* percent of P. stygicus were induced into diapause when the transfer occurred at or before 6.5 days. More than 75% of P. stygïcus emerged without diapause if the transfer occurred > 8 days after parasitisrn. Although a slight increase in the percent of individuals emerging without diapause was observed if the transfer of the parasitized nymphs occurred later in the life cycle of L. lygvorus, no significant difference in diapause induction due to a transfer to short photopenod was recorded. At 22*C, eggs of P. srygicus hatched inside the host 5 days after parasitism, therefore the induction of diapause occuned when the parasitoids were in the egg stage or early 1" instar. 5.2 Introduction The Tamished Plant Bug (TPB), Lygus lineoluris (Palisot de Beauvois), is a polyphagous pest feeding on more than 300 host-plants in North Amenca (Young 1986). A few native natural enemies exist, but do not regulate the pest population successfully (Clancy and Pierce 1966, Day 1987). In North America, four nyrnphal parasitoids (2 Peristenus spp. and 2 Leiophron spp.), known to be Nearctic, attack L. lineolaris; a fifth [(PeristenuspaZZipes (Curtis)] is believed to be Holarctic. Five parasitoids (dl Peristenus spp.) are also present in Europe, attacking two close relatives of L. lineolaris, L. mgdipennis (Poppius) and L. pratensis (L.). Synchrony of life cycles with those of their hosts through diapause is a prerequisite for survïval of parasitoids. Several nymphal braconid parasitoids of TPB are univoltine, and undergo a diapause period of 8-10 months. Others are multivoltine and will have two or sometimes three generations each summer before entering a 7-8 month diapause. Study of the biology or any other aspect of the life history of univoltine parasitoids is hampered by their obligatory diapause. Fortunately, diapause can be reduced in the laboratory to a minimum period of about 3.5 months (Bilewicz-Pawinska and Varis 1990) prior to successful emergence of adults. Multivoltine parasitoids are preferred for biocontrol of Lygus pests, because of their potential to attack the 2-3 generations of plant bugs occurring during the summer months (Day 1987). Peristenus digoneutis Loan and P. sîygicus Loan are two Palaearctic multivoltine parasitoids considered for release against the TPB in North Amerka. Peristenus digoneutis was reieased in New Jersey, USA, at the beginning of the 19801s, and has become established @ay 1996, Day et ai. 1990, L998). Peristenus sslgicus, on 98 the other hand, was released in California, USA, and in Saskatchewan, Canada, but never established, possibly due to low numbers released, low female: male do,poor dispersal and/or poor density-dependent response (VanSteenwyk and Stem 1977, Craig and Loan l984b, Coulson 1987). However, previous and curent laboratory studies on P. s~cus indicate that this parasitoid possesses several desirable qualities for release, such as facultative diapause, short developmental fime, hi& levels of parasitism and ease of mass rearing (Broadbent 1976). Other multivoltine North American parasitoids are Leiophron lygivorus (Loan), L. uniformis Gahan and Peristenus howardi Shaw. These parasitoids could be introduced in other parts of North Amerka where they are not present and their potential assessed. Knowledge of diapause requirements should be an important part of a rearing program for biocontrol agents. In order to "stockpile" large numbers of parasitoids for rnass-release, one should be able to induce and terminate diapause, as well as to successfully store the diapausing stage. Studies on diapause in Peristenus spp. have shown that the minimum time in diapause before interruption was 2.5 to 3.5 months, and that the longer the diapause, the shorter the emergence tirne (Bilewicz-Pawinska 1978, Bilewicz-Pawinska and Varis 1990). Sdval&er diapause for four species of European Peristenus was between 50-80% (Bilewicz-Pawinska 1977d), and P. digoneuris seems to emerge earlier (2-22 days) than P. ssrgicus (1 1-34 days) after diapause (Bilewicz- Pawinska 1978). For nearly all P. stygcus, diapause was induced when the parasitized nymphs were reared at 42.75 h of light, and a very low incidence of diapause was observed for parasitized nymphs reared at >14 h of light (VanSteenwyk and Stem 1976). However, the life stage of the parasite when diapause was induced was not determined. Induction of diapause for P. stygicus and L. [ygivoncî, both mdtivoltine parasitoids of L. Zineolaris, was investigated. The objectives were to determine the period when the induction of the diapause would occur for P. stygiinrs and L. lygvonrs, and the stage of the parasitoid at which induction occurs. 5.3 Materiais and Methods 5.3.1 Diapause induction Second and early third instar nymphs of Lyps lineolaris were placed in plastic vials (40 ml) in groups of 10 or 15. A single female wasp of either P. ssrgicus or L. lygivonrs was introduced for a period of 2 hours. About 100 - 120 nymphs were parasitized for each replicate. After parasitism, nymphs were intemiixed (separate parasitoid species), randornly selected in groups of 10 or 15 and placed in plexiglass cages (9.5 cm diam. x 15 cm ht.) with vermiculite at the bottom for a pupation substrate. Lettuce and bean pods were added to the cages for food, and were changed every 4 days. Cages were placed in a growth chamber at long day conditions 16hL: 8hD and 22OC. Prelirninary observations suggested that induction of diapause occurred during the first 7-8 days after parasitism by P. swcus (A. B. Broadbent, pers. comm.). To determine the effect of timing of transfer to short photoperiod on diapause induction, a cage was transferred every day or every second day for a period of approximately 11 days, hmthe long day photoperiod charnber to another at the same temperature but shorter photoperiod (12hL: 12hD). Twenty days after the start of the experirnent, al1 cages were brought back to 16hL: 8hD and 22OC. Thereafter, cages were checked daily for emergence of adult parasitoids up to 40 days after parasitism. Four replicates of nymphs parasitized by L. lygnoms, and 5 replicates for P. stygicus7 were carried out. If no Meremergence of wasps occurred beyond forty days after parasitism, diapause was assumed to have occurred. Any non-parasitized hosts (usually an addt TPB) was removed fiom the cages and excluded fiom the counts, while other nymphs were assumed to be parasitized. Cocoons remainiog in the verrniculite were transferred at 7-10 day intervals from 14°C to 7"C, and then held at 2OC for a 5-6 month period of diapause. After this time, dl cocoons were placed at 22"C, 16hL: XIiD, where emergence was checked daily until al1 wasps had emerged, Remaining cocoons were dissected and recorded as dead adult, dead larva, or empty cocoon (emerged wasps). 5.3.2 Stage of diapause induction in Peristenus stygicus About 35 second and early third instar L. lineolaris were initially placed in contact with approximately 15 P. sStgicus females for about an hour in a 3.5 L glasjar. Newly parasitized nymphs (some may not have been parasitized) were placed in a growth cabinet at 22°C and 16hL: 8hD. To determine the time of egg hatch inside the host, as well as the stage of parasitoid development, parasitized nyrnphs were dissected daily fiom 4 to 9 days derparasitism. Dissected nymphs were recorded as containing eggs or larvae, and length of eggs and larvae measured. These data were then used to infer at what developmental stage of this parasitoid diapause was induced in the previous set of experiments. 5.4 ResuIts 5.4.1 Parasitoid survival Adult wasps emerged fkom 41% of the nymphs parasitized by P. stygicus, compared with 55.3% of those parasitized by L. Zygivoms (Table 5.1). Sumival rates were similar before or after diapause for development and emergence of parasitoids; a total of 2 1.8% of P. srygicus emerged without diapause, and 24.0% after diapause (Table 5.1). Similarly for L. lygivorus, 34.2% emerged without diapause and 32.0% after diapause. For P. stygicus, 96.5% of the empty cocoons (wasp had emerged) were found in the vermidite, although only 2 replicates of the experiment were searched for cocoons in the vemiiculite. Twenty-two cocoons contahhg a dead adult wasp were found (17.6% of total cocoons), and 17 containing a dead larvae (13.6% of total cocoons). Therefore, the addition of dead cocoons and empty ones surnrned up to 125 cocoons formed, although 203 nymphs were put in contact with adult wasps (counts for 2 of the 5 replicates). For L. mvonrs, although the emergence rate was higher, only 66.7% of the empty cocoons were retrieved in the vermiculite after emergence of the adults. Eight cocoons contained a dead adult wasp (S. 1% of total cocoons) and 2 cocoons with a dry larva (1.3% of total cocoons) were also found afler dissection of the cocoons remaining in the vermiculite. Therefore, at least 157 larvae of L. Zygivorus pupated in the vermiculite, based on the number of emerged adults and cocoons found, although a total of 266 nymphs were potentially parasitized. Table 5.1 Percent emergence of non-diapausing and diapausing individuals of the parasitoids Peristenus stygicus and Leiophron lygivorus from nymphs of Lygus lineolaris. Parasitoid No. nymphs No. adult wasps emerged Percent emergence species Parasitized a Total Non-diapaused Diapaused Total Non-diapaused Diapaused P. stygicus 367 149 80 69 40.6 21.8 24.0 L. lygivorus 266 147 91 56 55.3 34.2 32.0 a Non-parasitized hosts were excluded from the counts (see Materials and Methods) No. diapaused I (Total No. parasitized - No. non-diapaused) x 100 5.4.2 Diapause induction When transfer to short day occurred S 7 days after parasitism, more wasps of P. st~~gicuswere induced into diapause than not. When the transfer occurred 8 days or more afler parasitism, more than 75% of the wasps did not enter diapause (Table 5.2A). For L. lygivonrs, a slightincrease occurred in the proportion of wasps that were not induced into diapause when parasitized nymphs were transferred to shoa day conditions at 4 days or more (Table 5.2B). By plottuig the day of transfer to short photopenod and the percent emergence without diapause, a threshold was deterxnined at which more than 50% of the individuals were induced into diapause. For P. smcus, the emergence before diapause was fitted with a Linear regression (P=0.002,del 1, ~~=0.64)(Figure 5.1). Fiepercent of P. ssrgicus were induced into diapause at 6.5 days. Although an increase in the proportion of individuals that were not induced into diapause was observed if the transfer of the parasitized nymphs occurred at 4 days or more in the life cycle of L. Zygivorus, the R~ value of the linear regression was low (~~=0.42),but significant (P=0.042,del 1) (Figure 5.2). 5.4-3Stage of diapause inductionfor Peristenus stygicus At 22OC and long day photoperiod, eggs of Peristenus stygicus started to hatch 4 days &er parasitism, although most of them hatched 5 days deroviposition (Table 5.3). Before day 5, only one hatched egg was found out of 15 nymphs dissected, and at day 6, Table 5.2A Percent emergence of Peristenus sfygicus from nymphs of Lygus lineolaris after transfer from long day photoperiod (16hL: 8hD) to short day photoperiod (12hL: 12hD) at daily intervals following parasitism. Day of transfer No. nyrnphs No. adult wasps ernerged Percent emergence to 12hL: 12hD parasitized a Total Non-diapaused Diapaused Total Non-diapaused Diapaused O 23 7 O 7 30.4 O 1 O0 1 28 12 6 6 42.9 50.0 50.0 2 19 9 O 9 47.4 O 1O0 3 29 3 O 3 10.3 O 1 O0 4 24 10 O 10 41.7 O 1 O0 5 5 1 11 2 9 21.6 18.2 81.8 6 24 Il O II 45.8 O 1O0 7 50 19 9 10 38.0 47.4 52.6 8 22 12 9 3 54.5 75 ,O 25 9 24 13 12 1 54.2 92.3 7.7 10 48 25 25 O 52.1 1 O0 O 11 25 17 17 O 68,O 1 O0 O a Non-parasitized hosts were excluded from the counts (see Materials and Methods) Table 5.2B Percent emergence of Leiophron lygivorus from nymphs of Lygus lineolaris after transfer from long day photoperiod (1 6hL: 8hD) to short day photoperiod (12hL: 12hD) at daily intervals following parasitism. ------Day of transfer No. nymphs No. adult wasps emerged Percent emergence to 12hL: 12hD parasitized a Total Non-diapaused Diapaused Total Non-diapaused Diapaused 11 O - " - - - 'Non-parasitized hosts were excluded from the counts (see Materials and Methods) O 2 4 6 8 10 12 Day of transfer to short photoperiod Figure 5.1 Emergence of non-diapaused Perisfenus sfygicus after transfer to short day photoperiod (12hL: 12hD) following parasitism Day of transfer to short photoperiod Figure 5.2 Emergence of non-diapaused Leiophron Zygivors fier tramsfer to short day photoperiod (l2hD: 12h.L) following parasitisrn no eggs were found inside the host Length of eggs and larval stages of P. smms are presented in Table 5.3. The number of fernale P. stygims was high during parasitism of nymphs, and the average number of larvae found inside individual hosts was between 1.5 and 3.5. Up to 12 larvae were found inside one host, although only one appeared to be dive in alI cases of superparasitism. Table 5.3 Stage and length of parasitoid larvae of Peristenus sîygicus developing in nymphs of Lygus lineolaris at daily intervals following parasitism. ' Day after No. nymphs No. Avg. length Avg. no. of No. nymphs Avg. length Avg. No. of parasitism dissected nymphs of live larvae larvae in with eggs of eggs eggs in host with larvae (mm k std) host (h std) (mm std) (h std) 4 15 1 0.47 1 14 0.28 st 0.03 1.5 i 0.8 5 18 12 0.61 *O.Il 1.5I 1.2 13 0.38 & 0.06 1.5 A 1 .O 6 15 15 1.33rt0.19 2.8k2.0 O - - 7 37 37 1.63 3t 0.36 3.5 * 2.4 O - - 8 15 15 1.86 f 0.38 3.3 f 2.2 O - - 9 16 16 2.59 * 0.38 2.7 I2.1 O - - a Rearing conditions were 22OC and 16hL: 8hD photoperiod. No. containing eggs or larvae. The non-parasitized individuals were not included. 5.5 Discussion Short photoperiod has a major effect on incidence of diapause for P. stygicus, and this effect is induced within the fist 7 days after parasitism at 22°C. Results are Iess conclusive for L. lygz%orus, as very few additional diapausing wasps were recorded if exposed to shoa photopenod early in the developrnent of the lama inside its host. It is possible that the induction arises later in the parasitoid development for L. lygivorus, or that other factors may be involved. For P. sStgicus, it has been well-documented that rearing parasitized host at -42.75 hours photopenod induced diapause (VanSteenwyk and Stem 1976). It is obvious that the fkst 7 days are the most cntical for inducing diapause and that the stage involved is most likely the egg or the early first instar larva. Carignan and BoiWi (1995), studying P. digoneutis development at 21°C, found eggs in the host up to 5 days after parasitism, and 1st instar Iarvae fiom the 5th to the 15th day after parasitism. Development at 22°C rnight be a little faster for P. stygicus, but othenvise the two species show a very similar life cycle, as eggs started to hatch at the same time as observed with P. digoneutis (Carignan and Boivui 1995). Dissection of hosts derparasitism showed that the eggs laid had al1 hatched and first instar larvae were present after 6 days at 22OC. After dlemergence had occurred, the vermiculite was searched for any dead parasitoids. Cocoons of P. stygicus were more easily found than those of L. lygivorus, as they are slightly bigger, are fiequently formed on the walls of the containers, and more vermiculite pieces are stuck to them. It appears that mortality rates of the parasitoids experiencing a diapause period in the laboratory are similar to those not induced into diapause (Table 5.1). Mortality rates in the field may be higher, as more factors will infiuence survival rates. Many studies have demonstrated that more than one abiotic factor is ofien involved in diapause induction (Brodeur and McNeil 1989), although photoperiod is the most reliable index of seasonal change of temperate insects (Beck 1980, Tauber et al. 2 983). For L. Zygivoms, a short-day photoperiod of 12hL: 12hD alone does not seem to be enough to induce diapause, although a slight increase in diapause induction was observed with a longer time at short photoperiod. Other factors inducing diapause may be environmental cues, host's physiological state, or a combination of both (Maslennikova, in Tauber et al, 1983, 1986). Matemal age is also known to influence diapausing progeny, as older parasitic Hymenoptera will usually produce more diapausing offspring (Saunders 1962, McNeil and Rabb 1973, Parrish and Davis 1978). In this study, mated females between 3 and 15 days old were used, and it is therefore possible that some of the outliers in the data are a result of the 12 day age range of our females. However, longevity of P. srygicus at 20°C and 25°C is 28.1 and 17.4 days, respectively (VanSteenwyk and Stem 1976), much longer than the age of the females used here, and therefore the materna1 age effect should be slight in this study. In this laboratory study, the mortality of P. stygicus seems to be higher than L. wvorus (41% vs 55% emergence, respectively). However, this rnay be an artifact, as we cannot assume that al1 hosts were parasitized afier being in contact with female wasps. Although any non-parasitized adult host was removed fiom the containers, it is possible that the proportion of nyrnphs parasitized by L. lygivom was higher than by P. stygcus. Cannibalism of hosts, naturai beaths of hosts and parasitoids, and other mortality factors may aiso have occurred. Bilewicz-Pawinska (1 977) observed a higher percentage emergence after diapause (50-80%) for four species of parasitoids, including P. siygicus. In her study, the percent emergence was calculated fiom the ratio of number of cocoons used for the experiment to the number of emerged addt parasites. This is different than in our study, where the percent emergence was determined based on the number of nyrnphs parasitized (or believed to be parasitized), which is a higher number than the actuai number of larvae suMving up to pupation. In our study, a total of 125 cocoons were found in the vermiculite for P. sîygicus, with 83 being empty. Therefore, the survival frorn cocoon spinning to addt emergence was 66.4% (including both without diapause emergence and after diapause emergence). The emergence rate fiom cocoon to addt emergence for L. lygivorus was 93.6%. Producing and stockpihg diapausing cocoons cm easily be performed for P. stygcus under laboratory conditions. However, although some individuals of L. Zygivorus will go through diapause under short photoperiod, a rather large percent still emerged without diapause induction. A combination of short photoperiod and low rearing temperature may help in inducing diapause in L. lygvonrs. The fact that we have been collecting L. lygivorus in the field at the end of August/early September (Chapter 2) suggests that photopenod may not be the only factor inducing diapause. Chapter 6. General ConcIusion Integrated pest management programs consider implementing al1 techniques available to suppress a pest population below its economic injury level. Classical biological control, the control of pest species by introduced natural enemies (van den Bosh et al. 1982), is a powerfùl tool in the array of control methods, and moreover is permanent, cost-effective, selective, and requires minimal input once the natural enemy is established (Beirne 1984). It is also emerging as a key technology for biodiversity conservation (Waage 1996). With goals such as a reduction in pesticide use, and because the natural enemy - victim interaction is strong and persistent and is unlikely to result in resistance development (Holt and Hochberg 1997), biological control should be a very important tactic in fbture control programs. Moreover, as world trade increases the likelihood of introducing exotic pests (Waage 1996), classical biological control will likely be more and more important for control of these organisms. Most introductions of exotic pests are the result of plant introductions by humans (as ornamentals or economic plants) or accidental movement of goods (food, ballast water of ships, flowers, etc.) (VanIlriesche and Hoddle 1997). About one third of the arthropod pest problems in the United States are caused by exotic pests (Knutson et ai. 1990). The biological control approach used to suppress Lygus lineolaris in North Amenca is referred to as a "new-associationy' (Hokkanen and Pimentel 1984) because the exotic Euphorine parasitoids to be introduced are targeting a native species they have never been in contact with. "Old-associations" refers to control of an exotic pest by a nafmal enemy attacking the pest in its comtry of origin. Regardless of which approach one prefers, or uses, both old-associations and new-associations have been successfûl (Hokkanen and Pimentel 1984, 1989, Stiling 1990) and represent great tools for insect pest control (Wiedenman and Smith 1997). It is estimated that orchard and horticultural crop losses in Ontario due to Lygus spp., mostly Lygur lineolaris, amount to more than $12 million mually (Broadbent et ai. In Preparation). Few alternatives to insecticides exist at present to control the Tanrished Plant Bug, and a reduction of pesticide use would result fkom a successful introduction and establishment of effective parasitoids of these econornically important plant bugs in Ontario. Introducing exotic natural enemies to an ecosystem could have some potentially detrimental effects on various biotic entities present (Howarth 1991, Louda et al. 1997, Strong 1997). It is therefore important to select a biocontrol agent that has a narrow host- range (Wiedenmann and Smith 1997). It is also important to assess other potential environmental effects, such as displacement of native naturd enemies, Sefore the biological control agent is released, and to establish baseline data on naturaIIy occurring native biocontrol agents and their parasitism rates. Such information is necessary to assess environmental effects and effectiveness of the released agents. When the "new- association" approach is used, it is also important to test for compatibility of the target host and the biocontrol agents to be introduced (Wiedenmann and Smith 1997). In this study, five species of North American Euphorine parasitoids .were recorded fiom the mirids Lygus lineolaris, Adelphocoris lineolatus and Leptopterna dolabrata in Ontario, however overall rates of parasitisrn were found to be usually low. Annual pamsitism rates were below 8% for both L. lineolaris and A. lineolatus, with peaks of parasitisrn in June being lower than 35%. Only Leptopterna dolabrata indicated a high rate of parasitism with a peak of about 75% in early June, 1999. This plant bug species is a minor pest of agricultural crops in Canada, attacking mostly grasses in hay fields. However, it may serve as an altemate host for parasitoids attacking economically important rnirid pests. The parasitoids collected, in decreasing order of abundance, were: Peristenus pallipes, P. pseudopallipes, Leiophron lygivorus, Leiophon sp. near brevipetiolatus, and L. solidaginis. New species attacking economically important mirids, as well as species attacking other rnirids, will likely be described in the near future. P. pallipes is probabIy a complex of 3 different species (H. Goulet, pers. comrn.). Although the three types of P. pallipes are very similar morphologically, each of them seems to emerge at a different tirne after diapause, and each was found to be more common on one specific host (Lygus lineolaris, Adelphocoris lineolatus and Leptopterna dolabrata, respectively) (see Table 2.4). Advances in molecular marker techniques in Canada and the United States will provide a useful tool (Hoy 1994) for future survey work on Miridae and their parasitoids, both to fûrther resolve taxonomic problems and to study diversity in the Peristenus parasitoids (Foottit 2000). Low rates of parasitism by native species highlight the importance and the potential emphasis placed on the introduction of exotic parasitoids for control of these mirid pests, especially L. lineolaris, in Ontario. Lygus Iineolaris is the main target for biologicai control in northeastem North America because of the extensive darnage it causes to a large number of cornmodities. Although found in this study to be more cornpetitive (intrinsic competition) than native parasitoids, the European parasitoids P. stygicus and P. digoneutis would probably not have ecological impacts on the native parasitoids. Collectively, in-host competition, host-preference, habitat (host-plants) and time of activity of the various parasitoids (native and exotic) will all have an impact on parasitoid population dynamics, population structure, and outcome of competition. Therefore, biological characteristics of each parasitoid and the expected interaction between each other are important to consider. Leiophron uniformis (not tested here), a common species in the United States, is thought to prefer the mirid Halticus bractatus (Say) (Day and Saunders 1990) and therefore would not enter directly into competition with an introduced parasitoid species and its host L. lineolaris. PeristenuspalZïpes, although found commonly parasitizing L. lineolaris, aiso has other preferred hosts such as Leptopferna dolabrata L., Trigonotylus coelestialium (Kirkaldy) and Adelphocoris lineolatus (Goeze) @ay 1999, P. Mason, pers. comm.). Parasitism rates by P. pallees on L. lineolaris usually were found to be Iow. The two native parasitoids that might interact directly with an introduced species targeted to control L. lineoloris would therefore be L. mvorus with estand second generation TPB and P. pseudopallipes with second generation TPB only. However, the habitat preference of L. lygivorus seems to be weedy areas characterized by the presence of Erigeron and Solidago spp. (Loan 1980, Lachance Chapter 2), which would potentially exclude the likelihood of large scale competition with an introduced parasitoid species, which are commonly found in alfdfa. Peristenus pseudopallipes has al so been reported to be more abundant in fields containhg the weed Erigeron spp. (Shahajahan 1974, SiIIings and Broersma 1974, as P. pallipes). European parasitoids, P. sîygicus and P. digoneutis, seem to be better adapted to search in alfdfa @ay 1987) than the Noah Amencan species, which are found more in weedy areas. In northeastem United States, P. digoneutis attacks TPB slightly after P. pallipes (W. Day, pers. comm, Lachance, pers. observations), which might confirrn these 117 differences in host preference and periods of emergence. P. pseudopal2ïpes would not likely directly compete with other parasitoids, or at least its influence would be limited, because of its late summer appearance. A direct interaction that might occur between parasitoids would be in the case of the release of P. stygicus in areas where the European parasitoid P. digoneutis is now established (northeastern United States). The outcome of such cornpetition in the field is dificult to predict in a new ecosystem, although P. digoneutis appears to be a slightly better in-host competitor than P. stygicus. Based on studies conducted here (Chapter 4) and in studies by others, both parasitoids appear to be potentially acceptable-forrelease in Ontario. Given the low rates of parasitism by native species, and the biological characteristics of the parasitoids discussed above, we do not foresee any dramatic reduction of native species of parasitoids following introduction of these exotic species into northeastern North America. However, host-range evaluation (presently undenvay in Ontario) should be completed pnor to release to minimize the probability of detrimental effects on populations of non-target mirids. Decisions to introduce a naturd enemy should be based on an estimate of the host-range of the selected agent, on the value we place on these non-target species, and on the damage resulting fiom no action, or alternative actions, taken to suppress the invasive pest (VanIlriesche and Hoddle 1997). Merselection of an exotic parasitoid for release, it must be confirmed that the Tamished Plant Bug host will be abundant and at the proper stage when the parasite is emerging fiom its ovenvintering state. Even the best candidate for release, given poor synchrony with its host, would not reproduce effectively and may not establish. It is criticaily important to study synchrony when the biological controi agent and the target pest are not fiom sirnilar climatic regions, and also when the parasitoidmost association is a new one (Hokkanen and Pimentel 1984). The two exotic species, P. stygicus and P-dïgoneutis, do not seem to possess optimal synchrony with their host based on laboratory experiments. However, caution should be taken regarding the validity of results of laboratory experiments extrapolated to field conditions. Predictions obtained fiom laboratory rearing in this study on the appearance of first generation Tamished Plant Bug nymphs, compared with field collected nymphs, do not seem to be realistic. The lower temperature threshold of development (9.6"C) determined for L. lineoluris, however, is consistent with the literature (Ridgway and Gyrisco 1960, Roberts 1982, Fleishler and Gaylor 1988). Even though temperature is the main factor innuencing the development of poikiloterms, factors such as humïdity, photoperiod and the availability of food may account for the difference in predictions f?om laboratory experiments and patterns of plant bugs collected in the field. Upon entering diapause, the parasitoid adult is Mly fonned inside the cocoon (Loan 1980, Lachance, personal observation), although it requires a period of cold temperature before emerging. Relative humidity within the growth chambers used in this study was maintained at about 60% and the substrate containing the cocoons was sprayed with water every four days to keep it moist. Ofien parasitoids emerged one or two days after adding water to the substrate, as if moisture triggered an emergence response in the wasp. Temperature of the pupating substrate (soi1 or 1eafIitter) and humidity are most likely the only two major detemiinants of the emergence patterns of parasitoids after diapause. Predictions for emergence of parasitoids, therefore, can be regarded as more robust, because fewer environmental factors play a role in the induction of emergence. Predicted 119 emergence dates of parasitoid adults fiom laboratory experiments (mid-May to early June) corresponded to observations and coiiections of parasitized TPB nymphs in the field for the last 3 years in Ontario. Once diapause is induced, which was found to be mostly within the filst 7 days after parasitism (Chapter 5), a period of at lest 3.5 months at low temperature (<5-6OC) is necessary for subsequent emergence of the adult wasps milewicz-Pawinska and Varis 1990). Mortality of parasitoids, depending on the species, can be high during diapause. Mortality rates for the uoivoltine native parasitoids P. pallipes and P. pseudopallipes were very hi& in laboratory rearing (Chapter 4). The reasons are unlmown, as the conditions were the same for other multivoltine parasitoids, but humidity may be proposed as an important factor. High mortality and obligate diapause of univoltine species is a challenge for studying the biology of these parasitoids, as well as for rearing them prior to release. Surveys of parasitisrn rates before the introduction of a biological control agent are necessary to assess the real impact of an introduced and established agent on the target Pest and on non-target organisms. We now possess this baseline information, together with some other possible eEects of these biocontrol agents, and cm expect to introduce one or more of these parasitoids into Ontario for the control of the Tarnished Plant Bug. The first of these European parasitoids to be released in Ontario, Peristenus digoneutis, has been collected in southem Québec (Broadbent et al. 1999), after being introduced in New Jersey @ay et al. 1990) and will likely make its way to Ontario. A release in Ontario wodd likely gain a few years of reduced populations of the Tarnished Plant Bug. Post-release evaluations are necessary to evaluate the effectiveness of control, the dispersal of the agents, the interaction with plants and insects, and species-, cornrnunity- or ecosystem-level effects (Thomas and Willis 1998). The parasitoidmostmabitat system we have been studying and future work on post-release evaluations would allow US to gain knowledge of the important aspects of a responsible biological control program. Such work could serve as a model of effective planning and execution of biological control programs. If pesticide use is to be reduced in agriculture, biological control will become an increasingly important aspect of any integrated pest management program. Havùig a detailed model of a biologicd control program such as this one with Lygus spp., will benefit fürther efforts in implementing effective biological control agents in Canada. 7. References Al-Ghamdi, K. M., Stewart, R K. and Boivin, G. 1995. 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P. 1986. Host plants of the tamished plant bug, Lygus lineolaris (Heteroptera: Miridae). Am. Entornol. Soc. Am. 79: 747-762. 8. Appendix Parasitism of Lygus llineoluris, Adelphocoris lineolatus and Leptopterna dolabrata and mean nurnber of these species per 50 sweeps, collected in various fields in 1998 and 1999. June June June J~Y July Aug. Aug. Sept. 1 15 29 13 27 10 24 7 Date Appendix 1.1 Parasitism of Lygus Zineolaris , Alfalfa-Hay field, Concession 2, Guelph, 1998 5 - - 30 : % par. adults / % par. nymphs - 25 & - +L~~adults E 1 i-+-Lygusnymphs - 20 g V1 .d 3 - 0 +-, v5 1 - 15 2 2 - 10 5 0 -5 z 0- C -- - 0 May May June June June July Juiy Aug. Aug. Sept. 4 18 1 15 29 13 27 10 24 7 Date Appendix 1.2 Parasitism of Lygus Zineolaris ,Alfalfa, Wellington Rd.29, Guelph, 1998 - i I% par. adults j - ) % par. nymphs 1 - !-Eygus adults , i *Lygus nyrnphs j -! j !\1I a T W,V~*,A L -\* I11 May May June June June July July Aug. Aug. Sept. 4 18 1 15 29 13 27 10 24 7 Date Appendix 1.3 Parasitism of Lyps ZineoZmis ,Alfalfa, Jones Baseline, Guelph, 1998 1 % par. adults 1 EEd-% par. nymphs !-Lygus aduIts j-lygusi nymphs June June June J~Y Juiy Aug. Aug. Sept. 1 15 29 13 27 10 24 7 Date Appendix 1-4 Parasitism of Lygus lineo2uri.s ,Aifdfa-Hay field, Wellington Rd. 29, Guelph, 1998 ! % par. adults : i--.1i % par. nymphs ! -Lygus aduIts i 1-Lyp nymphs May May June June J~Y My Aug. Aug. 11 25 8 22 6 20 3 17 Date Appendix 1.5 Parasitism of Lygus lineolaris, Vetch, Stone Rd., Guelph, 1998 - 50 1 EEEi % par. nyrnphs i : *Lygus*Lygus nymphs i -dE 10- cn fa 8 - a 6 - 2 - May May June June June July July Aug. Aug. Sept. 4 18 1 15 29 13 27 10 24 7 Date Appendix 1.6 Parasitism of Lygus lineolaris ,Weeds, Student Housing, Guelph, 1998 May May June June July July Aug. Aug. Aug. Sept. Il 25 8 22 6 20 3 17 31 14 Date Appendix 1.7 Parasitism of Lygus lineolaris, Clover and weeds, Stone Rd., Guelph, 1998 May May June June July July Aug. Aug. Aug. Sept. 1I 25 8 22 6 20 3 17 3 1 14 Date Appendix 1.8 Parasitism ofLygus lineolaris , Echiurn sp. and weeds, Jones Baseline, Guelph 1998 j EEi % par. nymphs - 35 : !+LygusnymphsJ E 25- May June June Jme July My Aug Aug Sept. 18 1 15 29 13 27 10 24 7 Date Appendix 1.9 Parasitism of Lygus Zineolaris ,Malfa-Hay field, Concession 2, Guelph, 1999 16 - 1 % par. adula l4 Ï Im%par.nymphs 12 4 / us us adula 10; , ! Lygus nymphs .dEi C, - .d I May June June June July July Aug Aug Sept. 18 1 15 29 13 27 10 24 7 Date Appendix 1.10 Parasitism of Lygus lineolaris , Alfalfa, Wellington Rd. 29, Guelph, 1999 .- % par. adults ~n .- 4u % par. nymphs ~1 4- Lygus aduIts -35 E?0, -++ Lygus nymphs -30 2 O - 25 \ May June June June July July Aug Aug Sept. 18 1 15 29 13 27 10 24 7 Date Appendix 1.1 1 Parasitism of Lygics lineolaris .ALfaIfa, Jones Baseline, Guelph, 1999 1 - - 10 % par. adults - 9 . i--wi % par. nymphs 0-8 - -IIœ Lygus adults -8 -7 $ E Lygus nyrnphs rn -5 0.6 - - -6 -- \ % - E 5 a 0.4 + -4 ii i 3 s 1 1 -3 5 0.2 -2 - 1 O O May June June June July July Aug Aug Sept. 18 1 15 29 13 27 10 24 7 Date Appendix 1.12 Parasitism of Lygus lineolaris ,Vetch, Stone Rd., Guelph, 1999 1 - ! / % par. adults 0.8 - j i.csuzi % par. nymphs ~ i Lygus adufts 1 E l 1- Lygus nymphs -=C, 0.6 .d Vi 1, May June June June July Jdy Aug Aug. Sept. 18 1 15 29 13 27 10 24 7 Date Appendix 1.13 Parasitism of Lygus lineolaris , Weeds, Student Housing, Guelph 1999 . % par. aduits / EZSI% par. nymp hs A ' +Lygus adul ts -Lygus nymphs May June June June Jdy July Aug Aug. Sept. 18 1 15 29 13 27 10 24 7 Date Appendix 1.14 Parasitism of Lygur Zineolaris , Weeds, Bovey Bldg., Guelph, 1999 - 30 1 % par. nymphs - 25 aJ2i - 20 $! CA O - 15 ? f - 10 2 - 5 Z ------O Jme June June J~Y July Aug. Aug . Sept. 1 15 29 13 27 10 24 7 Date Appendix 1.1 5 Parasitism ofAdelphocoris lineolatus ,Alfalfa-Hay field, Concession 2, Guelph, 1998 - 30 t RiRSR % par. nymphs May May June June June Jdy July Aug. Aug. Sept. 4 18 1 15 29 13 27 10 24 7 Date Appendix 1.16 Parasitism of Adelphocoris ZineoZatus ,Alfalfa, Wellington Rd. 29, Guelph, 1998 5 - - 40 / % par. nymphs ; i+No.nymphs + - 35 i 4 - VI - 30 ,P. E V1 zrn .d 3 - - 25 .dC, O V1 - 20 T 2 i a2- - 15 g s s - 10 1 - p - 5 1 O - O May May June June June July July Aug. Aug. Sept. 4 18 1 15 29 13 27 10 24 7 Date Appendix 1.1 7 Parasitism of AdeZphocoris lineolafus, Alfalfa, Jones Baseline, Guelph, 1998 ( % par. nyrnphs / -+--No. nymphs i June June June July J~Y Aug. Aug. Sept. 1 15 29 13 27 10 24 7 Date Appendix 1.18 Parasitism of Adelphocoris Zheolatus ,Alfalfa-Hay field, Wellington Rd. 29, Guelph, 1998 35 - ' % pznyrnphs - 12 ! I No. nymphs 30 - + -- - 10 1 25 - E - 8 VJ :3 20 - sVi -- 6 15 - s 10 - I 5 - May May June June June July July Aug. Aug. Sept. 4 18 1 15 29 13 27 10 24 7 Date Appendk 1.19 Parasitism ofAdelphocoris lineolalus .Weedy hay field, Stone Rd., Guelph 1998 - 18 / B!?El%par. nymp hs May May June June July July Aug. Aug. Aug. 11 25 8 22 6 20 3 17 3 1 Date Appendix 1.20 Parasitism of Adelphocoris lineolutus ,Vetch, Stone Rd., Guelph, 1998 May May June June June July July Aug. Aug. Sept. 4 18 1 15 29 13 27 10 24 7 Date Appendix 1.2 1 Parasitism of Adelphocoris lineolatus ,Weeds, Student Housing, Guelph, 1998 14 - 1 % par. nymphs - 14 - 12 m P1 - 10 g 5 -8 O 'n \ -6 z Q -4 E - 2 ----A. O May May June June July Juiy Aug. Aug. Aug. Sept. 1 I 25 8 22 6 20 3 17 3 1 14 Date Appendix 1.22 Parasitism of Adelphocoris Zineolatus ,Clover, Stone Rd., Guelph 1998 - ; % par. nymphs May May June June July Jdy Aug. Aug. Aug. Sept. 11 25 8 22 6 20 3 17 3 1 14 Date Appendix 1.23 Parasitism of Adelphocoris lineolatus ,Echium sp. and weeds, Jones Baseline, Guelph 1998 'i % par- nymphs i -t- No. nymphs May June June June July Jdy Aug Aug Sept. 18 1 15 29 13 27 IO 24 7 Date Appendix 1.24 Parasitism of Adelphocoris Zineolatus ,Alfalfa-Hay field, Concession 2, GueIph, 1999 l I 1 % par. nympbs 1 l+No.nymphs 1 May June June June July July Aug Aug Sept. 18 1 15 29 13 27 10 24 7 Date Appendix 1 .X Parasitism of Adelphocoris lineolatus , Malfa, Wellington Rd. 29, Guelph, 1999 May June he June Jdy My Aug Aug Sept. 18 1 15 29 13 27 10 24 7 Date Appendix 1.26 Parasitism of Adelphocoris lineolatus , Alfalfa, Jones Baseline, Guelph, 1999 - 35 / % par, nymphs May June June June July Jdy Aug Aug Sep- 18 1 15 29 13 27 10 24 07 Date Appendix 1.27 Parasitism of Adelphocoris Iineolatus ,Weedy hay field, Stone Rd., Guelph, 1999 45 - Ui4il%par. nyrnphs - 90 40 - +NO. nyrnpns - 80 35 Ï -70 5 cnE 30- -60 .CI cn .z 25 ' - 50 a '.n td 20 40 I P, - - 15- - 30 25 10 + - 20 5 l 5 - - 10 O- O May June June June July Juiy Aug Aug Sept. 18 1 15 29 13 27 10 24 7 Date Appendix 1.28 Parasitism of Adelphocaris lineolatus , Vetch, Stone Rd., Guelph 1999 - ; % par. nyrnp hs May June June June Juiy July Aug Aug. Sept. 18 1 15 29 13 27 10 24 7 Date Appendix 1.29 Parasitism of Adelphocoris lineolatu ,Weeds, Student Housing, Guelph, 1999 May June June June July July Aug Aug. Sept. 18 1 15 29 13 27 10 24 7 Date Appendix 1-30 Parasitism of Adelphocoris linedatus , Weeds, Bovey Bldg., Guelph, 1999. j % par. nymphs ! +No. nymphs May June June June Jdy Jdy Aug Aug Sept. 18 1 15 29 13 27 10 24 7 Date Appendix 1.3 1 Parasitism of Leptopterna dolnbrata, Aifalfa-Hay field, Concession 2, Guelph, 1999 1 PBPI % par. nyrnphs 1 +No. nyrnphs May June June June July July Aug Aug Sep- 18 1 15 29 13 27 10 24 07 Date Appendix 1-32Parasitism of Leptopterna dolabruta, Weedy hay field, Stone Rd., Guelph 1999 1 % par. nymphs I 1 tNo. nymphs May June June June July July Aug Aug Sept. 18 1 15 29 13 27 10 24 7 Date Appendix 1.33 Parasitisrn of Leptopterna dolabram , Vetch, Stone Rd., Guelph 1999 (aai% par. nymphs I 1 i+No. nvm~hs ! May June June June Juiy JuIy Aug Aug. Sept. 18 1 15 29 13 27 10 24 7 Date Appendix 1.34 Parasitism of Leptopterna dolabruta, Weeds, Student Housing, Guelph, 1999 May June June June July My Aug Aug. Sept. 18 1 15 29 13 27 10 24 7 Date Appendix 1.35 Parasitisrn of Leptopternu dolabrata, Weeds, Bovey Bldg, Guelph, 1999