96: Rogers 285 DR. HARRY R. GROSS, JR.: CONTRIBUTIONS TO

Armyworm Symposium ’96: Rogers 285

DR. HARRY R. GROSS, JR.: CONTRIBUTIONS TO ARMYWORM RESEARCH

C. E. ROGERS USDA, Agricultural Research Service Insect Biology and Population Management Research Laboratory Tifton, GA 31793-0748

ABSTRACT

Dr. Harry R. Gross, Jr., USDA, ARS, IBPMRL (deceased) developed techniques and methodology for rearing and augmenting biological control agents to assist in the control of the fall armyworm, Spodoptera frugiperda (J. E. Smith), and the corn earworm, Helicoverpa zea (Boddie), in the southeastern USA. Dr. Gross’ career with the USDA spanned 27 years, during which he published 75 scientific papers and presented 41 oral papers on the results of his research. Dr. Gross conducted pioneering research on white fringed beetles, kairomones, and semiochemicals of beneficial insects, and patented a hive-mounted device through which exiting honey bees autodis- seminate Heliothis nuclear polyhedrosis virus to flowering plants for control of H. zea larvae. However, Dr. Gross’ greatest contribution to entomology was his development of rearing and augmentation technology to enhance the use of beneficial insects for controlling H. zea and S. frugiperda. Dr. Gross firmly believed in, and researched innovative ways to, use biological control for managing armyworms and other pests.

Key Words: Population management, mass rearing, augmentation, biological control

RESUMEN

El doctor Harry R. Gross, Jr., USDA, ARS, IBPMRL (deceso) desarrolló técnicas y metodologías de cría de agentes de control biológico para favorecer el control de Spo- doptera frugiperda (J. E. Smith) y Helicoverpa zea (Bodie) en el sureste de los Estados Unidos. La carrera del Dr. Gross en el USDA duró 27 años, durante los cuales publicó 75 artículos científicos y presentó 41 ponencias sobre los resultados de su investiga- ción. El Dr. Gross condujo investigaciones pioneras sobre escarabajos, kairomonas y semioquímicos de insectos benéficos, y patentó un equipo para las colmenas que posi- bilitaba a las abejas diseminar el virus de la polihedrosis de Heliotis a las plantas con flores para el control de larvas de H. zea. Sin embargo, la mayor contribución del Dr. Gross a la entomología fue su desarrollo de la tecnología de cría para aumentar el uso de insectos benéficos para el control de H. zea y S. frugiperda. El Dr. Gross firmemente creyó en el control biológico e investigó novedosas maneras de usarlo en el manejo de los gusanos trozadores y otras plagas.

The Armyworm Symposium of the 70th Annual Meeting of the Southeastern Branch of the Entomological Society of America (ESA) is dedicated to the memory of Dr. Harry Gross, Jr. and his contributions to armyworm research. Dr. Gross was Su- pervisory Research Entomologist, USDA, Agricultural Research Service (ARS), Tif- ton, Georgia at the time of his death; his entire entomological career of 27 years was spent within the geographical boundary of the Southeastern Branch, ESA. Dr. Gross was born in New Orleans, Louisiana, on 16 March, 1939, and died in At- lanta, Georgia, on 3 May, 1994, following a short illness. Dr. Gross obtained a passion

286 Florida Entomologist 79(3) September, 1996

Fig. 1. Harry R. Gross, Jr., Supervisory Research Entomologist (1939-1994) for entomology at an early age, as his father was a Pest Control Operator in the New Orleans area. After graduating from high school, Dr. Gross enrolled at Louisiana State University where he obtained the Bachelor of Science Degree (1960), Master of Science Degree (1964), and Doctor of Philosophy Degree in Entomology (1967).

Armyworm Symposium ’96: Rogers 287

Dr. Gross’ graduate student research dealt with the effects of water emulsions on pesticidal control of wood-boring beetles; the role of the American cockroach, Periplan- eta americana (L), and striped earwig, Labidura riparia (Pallas) in the degradation of household fabrics; flight habits of L. riparia; and the responses of L. riparia to hep- tachlor and mirex applied for controlling fire ants, Solenopses invicta (Buren) and S. molesta (Say). His research with L. riparia was used by the Louisiana Pest Control Association to develop management recommendations for households in the early 1970s. Dr. Gross’ entire professional career was spent with the USDA, ARS. Dr. Gross joined ARS in Gulf Port, Mississippi, in 1967 and for the following four years concentrated research on developing systems for collecting, rearing, storing and controlling white fringed beetles, Graphognathus spp., which are devastating pests of seeds and seedling-stage corn, cotton, peanut, soybean, and other crops in the Southeast. Dr. Gross retained interest in white fringed beetles throughout his career, and served as an expert witness in hearings by the Environmental Protection Agency in 1977 and the State of California Department of Food and Agriculture Science Advisory Panel in 1988. Dr. Gross transferred to the Insect Biology and Population Management Research Laboratory (IBPMRL) (formerly Southern Grain Insects Research Laboratory) in Tif- ton, Georgia, in 1971. At the IBPMRL, Dr. Gross pursued several research areas., e.g., double-cropping and host resistance in managing populations of the fall armyworm, Spodoptera frugiperda (J. E. Smith); overwintering and spring emergence chronology of the corn earworm, Helicoverpa zea (Boddie), and tobacco budworm, Heliothis virescens (Fabricius); methods for separating individual eggs of S. frugiperda from egg masses; developing an oviposition chamber for mass production of H. zea; devising flo- tation techniques for separating mature and immature pupae of H. zea and H. virescens for irradiation in large-scale sterility programs; assessing the importance of visual stimuli in enhancing trapping efficiency of H. zea; the effects of homogenates of H. zea and S. frugiperda larvae on inhibiting their oviposition in corn and aggregating the predators Coleomagilla maculata Timberlake and Geocoris punctipes Say; and identifying and studying the bionomics of the first known pupal parasitoid of S. frugiperda [Dipetimorpha intreota (Cresson)] and of H. virescens [Cryptus albitarsas (Cresson)] that he discovered. Other researchers are now actively pursuing research on the biology and efficacy of the pupal parasitoids as an addendum to biological control organisms which may be used to reduce field populations of noctuid pests of southeastern agriculture. Dr. Gross was one of the pioneer researchers demonstrating the role of kairomones in enhancing foraging behavior of parasitoids. He formulated the concept and demonstrated that pre-release exposure of Trichogramma pretiosum (Riley) to host kairomones and parasitization experiences increased its efficacy against H. zea eggs in the field. In spite of the progress that Dr. Gross and his colleagues made in this area of research in the early to mid-1970s, he became convinced that its use as an economical pest management strategy was not likely in the near future. Hence, Dr. Gross re- verted to researching a more conventional approach for biological control of H. zea and S. frugiperda. During the last 10 years of his career, Dr. Gross concentrated his research efforts on mass rearing and augmentation techniques for field evaluation of the tachinid Ar- chytas marmoratus (Townsend) as a biological control agent for H. zea and S. frugiperda. Dr. Gross developed an efficient mechanical extraction technique for removing A. marmoratus larvae from gravid females and an efficient method for applying them to whorl-stage corn. He also developed an efficient and economical mass- rearing procedure for A. marmoratus using larvae of the greater wax moth, Galleria

288 Florida Entomologist 79(3) September, 1996 mellonella (L.), as a factitious host, and demonstrated that laboratory-reared flies ef- ficiently seek and parasitize H. zea larvae in the field independent of host density. At the time of his death, Dr. Gross was in the second year of a large 3-year pilot program evaluating field augmentation of A. marmoratus to control H. zea larvae in whorl- stage corn. An innovative accomplishment by Dr. Gross shortly before his death was the de- signing and testing of a hive-mounted device by which the honey bee, Apis mellifera (L.), automatically contaminates itself with a talc formulation of Heliothis nuclear polyhedrosis virus (HNPV). As contaminated bees forage among flowers for pollen and honey, they distribute the HNPV. Helicoverpa zea larvae feeding on crimson clover, Trifolium incarnatum (L.), exposed to HNPV-contaminated honey bees experienced a significantly higher mortality due to virus than larvae feeding on non- contaminated clover. This development resulted in a patent and has generated considerable international interest and further current testing. Dr. Gross published his research results in 75 scientific papers and orally presented papers at 41 scientific meetings. In addition to his scientific accomplishments, Dr. Gross assumed several leadership roles in ARS as well as in professional societies. At the time of his death, Dr. Gross was Research Leader of the Insect Biology/Man- agement Systems Research unit at Tifton, GA, and had served as a Laboratory Direc- tor and Assistant to the Southeast Area Director (ARS). Dr. Gross served on several committees of the Entomological Society of America and the Georgia Entomological Society and served as Chair of this Symposium in 1982. Dr. Gross was President of the Southeastern Biological Control Working Group in 1990. In 1980, Dr. Gross represented the USDA in Argentina, Brazil, Paraguay, and Uruguay on an exploration trip to collect biological control agents for S. frugiperda and other pest species. Dr. Gross worked with a core group in 1987 to develop the ARS National Biological Control Pro- gram. In addition to his active participation in the Entomological Society of America, Dr. Gross also was active in the Georgia Entomological Society, Florida Entomological Society, Sigma Xi, American Registry of Professional Entomologists, Southeastern Bi- ological Control Working Group, and Southern Regional Projects. The void left by the sudden passing of Dr. Gross in May 1994 remains in the Insect Biology and Population Management Research Laboratory, the University of Georgia Coastal Plain Experiment Station, and the Tifton community. Dr. Gross’ dedication to his profession, his great empathy, his interpersonal communication skills, his willing- ness to assist subordinates, peers, and superiors in solving problems, and his positive outlook and jovial countenance are just a few of the characteristics of Dr. Gross that will not be forgotten and will be long-appreciated by his many associates and friends. Dr. Gross is survived by his wife, Marlene, Tifton, GA, and their daughter, Lisa, At- lanta, GA.

Armyworm Symposium ’96: Carpenter et al. 289

COMPATIBILITY OF F1 STERILITY AND A PARASITOID, COTESIA MARGINIVENTRIS (HYMENOPTERA: BRACONIDAE), FOR MANAGING SPODOPTERA EXIGUA (LEPIDOPTERA: NOCTUIDAE): ACCEPTABILITY AND SUITABILITY OF HOSTS

J. E. CARPENTER, HIDRAYANI1 AND W. SHEEHAN2 Insect Biology and Population Management Research Laboratory Agricultural Research Service, U. S. Department of Agriculture, Tifton, GA 31793-0748

1Andalas University, Padang, Indonesia

2E&C Consulting Engineers, Inc., 2175 Highpoint Road, Snellville, GA 30278

ABSTRACT

The potential for combining two alternative pest management tactics, F1 sterility and a parasitoid, was examined in the laboratory and in the greenhouse. Studies compared the acceptability and suitability of progeny from irradiated (100 Gy) and nonirradiated beet armyworm, Spodoptera exigua (Hübner), males as hosts for Cotesia marginiventris (Cresson). Results from these studies revealed that progeny of irradiated S. exigua males and nonirradiated S. exigua females are acceptable and suitable hosts for C. marginiventris development. Cotesia marginiventris females showed no oviposition preference for S. exigua progeny from females paired with either irradiated or nonirradiated males. Cotesia marginiventris and F1 sterility appear to be compatible tactics that potentially could be integrated into a preventative pest management program for S. exigua.

Key Words: Spodoptera exigua, Cotesia marginiventris, F1 sterility, inherited sterility, biological control

RESUMEN

Fue examinado el potencial de la combinación de dos alternativas de manejo de plagas, esterilidad de la F1 y un parasitoide, en el laboratorio y en un invernadero. Los estudios compararon la aceptación de la progenie del gusano de la remolacha, Spodop- tera exigua (Hübner), irradiado y no irradiado, como hospedante de Cotesia marginiventris (Cresson). Los resultados revelaron que la progenie de machos irradiados y de hembras no irradiadas de S. exigua constituye un hospedante aceptable y adecuado de C. marginiventris. Las hembras de C. marginiventris no mostraron preferencia ovopo- sicional por la progenie de S. exigua proveniente de hembras pareadas con machos irradiados o no irradiados. Cotesia marginiventris y la esterilidad de la F1 parecen ser tácticas compatibles que potencialmente podrían ser integradas en un programa pre- ventivo de manejo de plagas para S. exigua.

The beet armyworm, Spodoptera exigua (Hübner), is a serious pest in cotton in the southeastern United States, especially during outbreak conditions (Smith & Freeman

290 Florida Entomologist 79(3) September, 1996

1994). Although many factors have contributed to the outbreaks of S. exigua in cotton, an unusually high level of resistance to some pesticides is implicated (Sprenkel & Austin 1994). Alternative management strategies, such as conservation of natural enemies (Ruberson et al. 1994), mating disruption with synthetic pheromone (Waka- mura & Takai 1992), and inherited sterility, are being studied for their potential role in an integrated pest management program for S. exigua.

The potential for using F1 sterility as a component of regional management of lepidopteran pests has been suggested by Knipling (1970) and LaChance (1985), and numerous laboratory and cage studies on pests around the world have supported these ideas (LaChance 1985, Anonymous 1993). The successful application of the F1 sterility principle to a wild population of Helicoverpa zea (Boddie) during a recent pilot test en- couraged further development of this pest control strategy (Carpenter & Gross 1993). However, the high cost of rearing lepidopterans, relative to the cost of rearing dipter- ans, has moderated researchers’ enthusiasm concerning the use of F1 sterility for the control of lepidopteran pests. Nevertheless, Carpenter & Gross (1993) revealed that even a low irradiated:wild insect ratio could significantly reduce the seasonal increase of H. zea. In addition, population models (Knipling 1992, Carpenter 1993) have suggested that F1 sterility would be more efficient if combined with other pest control strategies. Therefore, recent studies have investigated the potential of integrating F1 sterility and parasitoids for increased efficiency in suppression of pest populations.

Mannion et al. (1994 & 1995) studied the compatibility of F1 sterility in H. zea and the tachinid parasitoid, Archytas marmoratus (Townsend). They found these two control strategies to be compatible and suggested that combining the two strategies may be useful for managing early season populations of H. zea. However, caution should be exercised in the extrapolation of these results to other lepidopteran pests, such as S. exigua. Parasite/host relationships are highly variable as a result of the various reproductive strategies of both host and parasite species. For example, H. zea females lay individual eggs spaced some distance apart which reduces mortality from larval cannibalism. Alternatively, S. exigua females lay eggs in masses, and larvae feed gre- gariously, in a patch, during their ﬁrst two instars. Also, A. marmoratus larviposits many maggots in the vicinity of late instar H. zea, whereas a common parasitoid for S. exigua, Cotesia marginiventris (Cresson), stings individual, early instars. The objectives of the present laboratory and greenhouse studies were: (1) to compare the acceptability and suitability of progeny from irradiated (100 Gy) and nonirradiated S. exigua males mated with nonirradiated females as hosts for C. marginiventris, and

(2) to relate these ﬁndings to the potential of combining the F1 sterility technique with resident or released C. marginiventris for managing populations of S. exigua.

MATERIALS AND METHODS

Insects Cotesia marginiventris and S. exigua were obtained from laboratory colonies at the Insect Biology and Population Management Research Laboratory, Tifton, GA. All S. exigua larvae were reared in plastic cups (30 ml) containing a meridic diet (Burton 1969) at 27 ± 1°C with a photoperiod of 14:10 (L:D) h. Cotesia marginiventris was reared on S. exigua larvae at the same temperature and photoperiod regime.

F1 Sterility Technique The “irradiation treatment” (I) larvae were those S. exigua larvae resulting from the mating of a nonirradiated female moth with an irradiated male moth. The “nor-

Armyworm Symposium ’96: Carpenter et al. 291 mal treatment” (N) larvae originated from crosses between nonirradiated moths. Lar- vae from both treatments were reared as above. Adult males undergoing irradiation were <24 h old and were irradiated (100 Gy) at about 20°C with a well-type 60Co source (Gammarad Irradiator, Model GR-12, U.S. Nuclear Division, Irvine, CA) delivering about 65 Gy per min. Dose calibration with an X-ray monitor and probe indicated a dose error of approximately ±5%.

Suitability of Progeny from Irradiated Males as Hosts

Spodoptera exigua larvae from both treatments (I & N) were stung by C. marginiventris. After a single sting, the host larva was transferred to a one-ounce, clear cup containing diet. A cardboard lid was placed on the cup and the host was allowed to complete its development. Hosts were scored as producing either a pupa or a parasitoid cocoon. Comparisons between larval treatments were made when host larvae were early 1st instars, late 2nd instars, early 3rd instars, and mid 3rd instars. Percent parasitism was calculated as the number of hosts in a treatment producing parasitoid cocoons divided by the total number of hosts in a treatment, multiplied by 100. Wasps developing from larvae stung as 2nd instars (n=7 for I larvae, n=9 for N larvae) were compared for time of development, adult weight, fecundity, and longevity. Data were subjected to an unequal n, unequal variance t-test (Steel & Torrie 1980) to separate differences between larval treatments (α=0.05).

Acceptability of Progeny from Irradiated Males as Hosts

A single C. marginiventris female was placed onto a cotton leaf in a large, open petri dish. The leaf had feeding damage from the previous night and 10 unstung S. exigua larvae. The following behavioral events were recorded: (1) time before flying off the ‘patch’ (area containing a group of feeding larvae); (2) number of stings before flying off a patch (maximum = five); (3) number of ‘walkbys’ (walking past an unstung host within one half wasp body length); and (4) number of ‘rejections’ (antennating unstung host without stinging). Comparisons between larval treatments were made when host larvae were early 1st instars, late 2nd instars, early 3rd instars, and mid 3rd instars. Behavioral responses from these “no-choice” experiments were recorded from ten wasps for each larval treatment and each host age. Comparisons between treatments were made using the t-test (Steel & Torrie 1980).

Oviposition Preference Test for Cotesia marginiventris

Four cotton plants (2-3 months old, DPL 90) were placed in a cage in the greenhouse. I and N larvae were used as host treatments. A host “patch” for each treatment was established by conﬁning 40 1st instars in a circle (4 cm diam) on the surface of a leaf and allowing the larvae to feed for one day. Within the cage, there were two host patches for each treatment and one patch per plant. Eight female C. marginiventris (2-3 days old) were released in the cage and allowed to search for and parasitize larvae for approximately 4 h. Host larvae were removed from the cage and dissected to determine the number parasitized. This test was replicated ﬁve times. Percent parasitism was calculated as the number of hosts in a treatment containing one or more parasitoid eggs divided by the total hosts in the treatment, multiplied by 100. Because there were no differences between the percent parasitism among patches within each larval treatment, patches for each treatment were pooled for analysis. Comparisons between larval treatments were made using the pooled t-test (Steel & Torrie 1980).

292 Florida Entomologist 79(3) September, 1996

TABLE 1. DAYS TO EMERGENCE, ADULT WEIGHT, FECUNDITY, AND LONGEVITY OF COTE- SIA MARGINIVENTRIS DEVELOPING IN PROGENY OF IRRADIATED (I) (100 GY) AND NON-IRRADIATED (N) SPODOPTERA EXIGUA MALES CROSSED WITH NOR- MAL FEMALES, PARASITIZED AS SECOND INSTARS.

Mean (S. D.)1

Parent Cross of Days To Wasp Wt No. Eggs Longevity Host Larvae Emergence (mg) Laid Days

N& × N( 10.8 a 1.03 a 70.7 a 17.9 a (0.68) (0.08) (30.1) (1.8) N& × N( 11.2 a 0.91 b 94.0 a 16.7 a (0.47) (0.09) (45.3) (1.6)

1Means followed by the same letter are not signiﬁcantly different (P < 0.05) (t-test).

RESULTS AND DISCUSSION

Suitability of Progeny from Irradiated Males as Hosts

Results from this study revealed that progeny of irradiated male S. exigua were generally suitable hosts for C. marginiventris. Wasps developing in progeny of irradiated males had a mean weight that was significantly (P >0.05) lower than wasps developing in progeny of nonirradiated males. However, parasitoid development, longevity, and fecundity were not significantly influenced by the host larval treatment (Table 1). First and second instars of S. exigua were more suitable as hosts for C. marginiventris than third instars, but host suitability within instars was not significantly affected by the larval treatment (Table 2). The percentage of stung larvae that produced C. marginiventris cocoons was similar for both larval treatments, regardless of the host age.

TABLE 2. EFFECT OF LARVAL AGE OF PROGENY OF IRRADIATED (I) (100 GY) AND NON-IR- RADIATED (N) SPODOPTERA EXIGUA MALES CROSSED WITH NORMAL FEMALES AND AS SUITABLE HOSTS FOR COTESIA MARGINIVENTRIS

Mean ± S.E. % Stung Larvae Producing a Cocoon (n)1

Parental cross Early 1st Late 2nd Early 3rd Mid 3rd of host larvae Instar Instar Instar Instar

N& × N( 66.7 ± 11 90 ± 6 39 ± 9 30 ± 9 (18) (29) (28) (30) N& × N( 83.3 ± 9 87.1 ± 6 44 ± 9 29 ± 9 (18) (31) (27) (29)

1There was no signiﬁcant difference due to larval treatment (P ≤ 0.05) (t-test). Armyworm Symposium ’96: Carpenter et al. 293 0.5 0.2 0.7 0.4 ± ± ± ± NONIRRADI-

AND 0.2 1.0 0.3 0.5 0.4 1.9 0.3 2.5 2 ) ± ± ± ± Y 0.4 0.8 0.4 2.3 0.4 2.1 (I)(100 G ± ± ± 0.2 0.7 0.5 3.5 0.4 3.6 IRRADIATED

± ± ± OF

0.2 0.5 0.1 0 0 0.6 0.3 3.5 ± ± ± PROGENY

0.3 0.3 0.1 0.2 0.5 2.3 ± ± ± S.E. Response to Host Age and Larval Treatment Age and Larval Response to Host S.E. ± FEMALES

MARGINIVENTRIS

0.4 0.2 0.2 5 5 3.2 ± ± Mean NORMAL

OTESIA C

WITH

OF 0.4 2.4 0.3 4.7

± ± NINININI Early 1st Instar Late 2nd Instar Early 3rd Instar Mid 3rd Instar 0.05) (t-test). ≤ CROSSED

RESPONSE

N MALES

MEAN

THE

EXIGUA

AGE

HOST PODOPTERA

S 1 OF

(N) FFECT ATED 3. E Walkby = walking past an unstung host within one-half wasp body length. Rejection = antennating unstung host without stinging body length. past an unstung host within one-half wasp = walking Walkby no significant difference due to larval treatment (P There was 1 2 (max. = 5)(max. 10 4.5 ABLE Rejection 10 0 0 0.5 Walkby 10 2.6 No. of stings before flying off No. Time before flying off (min.) 10 — — 1.9 Behavioral Event Behavioral T 294 Florida Entomologist 79(3) September, 1996

TABLE 4. COTESIA MARGINIVENTRIS OVIPOSITION PREFERENCE BETWEEN PROGENY OF IRRADIATED (I) (100 GY) AND NON-IRRADIATED (N) SPODOPTERA EXIGUA MALES CROSSED WITH NORMAL FEMALES

Parental Cross of Host Mean % Parasitism of Larvae n Larval Patch (S.D.)1

N& × N( 10 23.1 a (12) N& × N( 10 31.4 a (16)

1Means followed by the same letter are not signiﬁcantly different (P ≤ 0.05) (t-test).

Acceptability of Progeny from Irradiated Males as Hosts

Host acceptability was not signiﬁcantly affected by the larval treatment (Table 3). The behavior of female C. marginiventris foraging in larval patches was similar for both larval treatments, regardless of the host age.

Oviposition Preference Test for Cotesia marginiventris

Cotesia marginiventris females showed no oviposition preference between irradiated and nonirradiated male S. exigua progeny. The mean percentage of larvae parasitized within N larvae and I larvae patches did not differ signiﬁcantly (Table 4).

Fully successful integration of F1 sterility and parasitoids into a pest management approach can occur only if parasitoid strategies do not negatively impact irradiated insects and their progeny more than those of the wild population, and if F1 sterility does not negatively impact the efficacy of parasitoids. Results from these studies indicate that C. marginiventris and F1 sterility in S. exigua are compatible control strategies. The compatibility of these two strategies is congruent with the findings of Mannion et al. (1994, 1995). Carpenter (1993) suggested several scenarios in which the integration of compatible strategies such as F1 sterility and parasitoids might be used to control lepidopteran pest populations. For example, sterile S. exigua larvae could be field-reared on early season host plants or nursery crops. Cotesia marginiventris (native and/or released) could use these sterile larvae as hosts and, thereby, increase the parasitoid’s early season population. Other natural enemies of S. exigua may also use these sterile hosts. Larvae that escaped the natural enemies would produce sterile adult S. exigua that would reduce the reproductive potential of the next generation of S. exigua. Cotesia marginiventris is considered the dominant parasitoid of S. exigua in the eastern half of the United States (Tingle et al. 1978, Ruberson et al. 1994). This parasitoid is part of a large natural enemy complex that has the capacity to suppress S. exigua populations in cotton. However, S. exigua can become a serious pest of cotton, especially when the natural enemy complex has been disrupted (Ruberson et al. 1994). When S. exigua populations escalate, growers often are reluctant to postpone insecticide applications until the natural enemy complex has brought the S. exigua population under control. Pedigo (1995) suggested that the development of new integrated pest management programs should provide a special focus on the identification of preventative tactics. Ruberson (1994) emphasized the usefulness of conserving natural enemies for effective suppression of S. exigua. Because F1 sterility and C. mar- Armyworm Symposium ’96: Carpenter et al. 295

giniventris are compatible and may provide synergistic effects, further studies are warranted to test the practicality and efﬁcacy of integrating these two tactics for controlling S. exigua.

REFERENCES CITED

ANONYMOUS. 1993. Proc. Intl. symposium on management of insect pests: Nuclear and related molecular and genetic techniques. Jointly organized by the Intl. Atomic Energy Agency and the Food and Agric. Organization of the U.N., Vi- enna, 19-23 October 1992. IAEA-SM-327/35. 1993. BURTON, R. L. 1969. Mass rearing the corn earworm in the laboratory. U.S. Dep. Agric. Agric. Res. Serv. ARS (Ser.) 33-134, Washington, D.C. CARPENTER, J. E. 1993. Integration of inherited sterility and other pest management strategies for Helicoverpa zea: Status and potential. Proceedings, Intl. symposium on management of insect pests: Nuclear and related molecular and genetic techniques. Jointly organized by the Intl. Atomic Energy Agency and the Food and Agric. Organization of the U.N., Vienna, 19-23 October 1992. IAEA- SM-327/35, pp. 363-370. CARPENTER, J. E., AND GROSS, H. R. 1993. Suppression of feral Heliothis zea (Lepi- doptera: Noctuidae) populations following the infusion of inherited sterility from released substerile males. Environ. Entomol. 22: 1084-1091. KNIPLING, E. F. 1970. Suppression of pest Lepidoptera by releasing partially sterile males: A theoretical appraisal. BioScience 20: 456-470. KNIPLING, E. F. 1992. Principles of insect parasitism analyzed from new perspectives: Practical implications for regulating insect populations by biological means. Agricultural Handbook No. 693, United States Department of Agriculture, Washington, DC. LACHANCE, L. E. 1985. Genetic methods for the control of lepidopteran species. USDA, Agric. Res. Serv. ARS. 28. 40 pp. MANNION, C. M., J. E. CARPENTER, AND H. R. GROSS. 1994. Potential of the combined use of inherited sterility and a parasitoid, Archytas marmoratus (Diptera: Ta- chinidae), for managing Helicoverpa zea (Lepidoptera: Noctuidae). Environ. Entomol. 23: 41-46. MANNION, C. M., J. E. CARPENTER, AND H. R. GROSS. 1995. Integration of inherited sterility and a parasitoid, Archytas marmoratus (Diptera: Tachinidae), for managing Helicoverpa zea (Lepidoptera: Noctuidae): Acceptability and suitability of hosts. Environ. Entomol. 24: 1679-1684. PEDIGO, L. P. 1995. Closing the gap between IPM theory and practice. J. Agric. Ento- mol. 12: 171-181. RUBERSON, J. R., G. A. HERZOG, W. R. LAMBERT, AND W. J. LEWIS. 1994. Management of the beet armyworm (Lepidoptera: Noctuidae) in cotton: role of natural enemies. Florida Entomol. 77: 440-435. SMITH, R. H., AND B. L. FREEMAN. 1994. Alabama plan for the management of beet armyworms. Alabama Cooperative Extension Service, Auburn University Circu- lar Anr-842, 4pp. SPRENKEL, R. K., AND T. A. AUSTIN. 1994. 1994 Report of the Cooperative Beet Army- worm Trapping Program. NFREC Extension Report 94-6. 50pp. STEEL, R. G. D., AND J. H. TORRIE. 1980. Principles and procedures of statistics: a bi- ometrical approach, 2nd ed. McGraw-Hill, New York. TINGLE, F. C., T. R. ASHLEY, AND E. R. MITCHELL. 1978. Parasites of Spodoptera exigua, S. eridania (Lepidoptera: Noctuidae) and Herpetogramma bipunctalis (Lepidoptera: Pyralidae) collected from Amaranthus hybridus in ﬁeld corn. En- tomophaga 23: 343-347. WAKAMURA, S., AND M. TAKAI. 1992. Control of the beet armyworm in open ﬁelds with sex pheromone. pp.115-125 in N. S. Talekar [ed.]. Diamondback moth and other crucifer pests. Proc. Intern. Workshop, Tainan, Taiwan, 10-14 December 1990. Asian Vegetable Res. and D. V. Center Pub. No. 92-368.

296 Florida Entomologist 79(3) September, 1996

FACULTATIVE EGG-LARVAL PARASITISM OF THE BEET ARMYWORM, SPODOPTERA EXIGUA (LEPIDOPTERA: NOCTUIDAE) BY COTESIA MARGINIVENTRIS (HYMENOPTERA: BRACONIDAE)

JOHN R. RUBERSON1 AND JAMES B. WHITFIELD2 1Department of Entomology, University of Georgia, Tifton GA 31793

2Department of Entomology, University of Arkansas, Fayetteville AR 72701

ABSTRACT

The braconid parasitoid Cotesia marginiventris (Cresson) has long been known to be a larval parasitoid of numerous lepidopteran species. Recent field observations, however, indicated that C. marginiventris is also capable of functioning as an egg-larval parasitoid of the beet armyworm, Spodoptera exigua (Hübner). These field observations were corroborated by laboratory observations, demonstrating that C. marginiventris is capable of ovipositing in S. exigua eggs, and of successfully developing and emerging from host larvae hatching from stung eggs. The mechanisms used by the parasitoids to locate host egg masses in the field were not determined. These results lend support to phylogenetic hypotheses of the Braconidae that indicate a close relationship between the Cheloninae and the microgastroid taxa.

Key Words: Cotesia marginiventris, Spodoptera exigua, egg-larval parasitism, parasitoid, Braconidae, Microgastrinae

RESUMEN

Desde hace tiempo se conoce que el bracónido Cotesia marginiventris (Cresson) es un parasitoide de numerosas especies de lepidópteros. Sin embargo, observaciones re- cientes de campo indicaron que C. marginiventris es además capaz de funcionar como un parasitoide huevo-larval del gusano de la remolacha, Spodoptera exigua (Hübner). Tales observaciones de campo fueron corroboradas en el laboratorio, demostrando que C. marginiventris es capaz de ovopositar en huevos de S. exigua y emerger exitosa- mente de las larvas del hospedero eclosionadas de huevos parasitados. Los mecanis- mos usados por los parasitoides para localizar las masas de huevos del hospedante en el campo no fueron determinados. Estos resultados soportan la hipótesis ﬁlogenética de que en los Braconidae hay una estrecha relación entre los Cheloninae y los taxa mi- crogastroides.

The parasitoid Cotesia marginiventris (Cresson) is a common and important parasitoid in many agricultural systems (e.g., Kok & McEvoy 1989, McCutcheon et al. 1990, Ruberson et al. 1994). It is capable of attacking a wide range of hosts, chieﬂy from the lepidopteran family Noctuidae (Krombein et al. 1979), although the suitability of hosts varies with the species and age of the host attacked (A. Datema & J. Ru- berson, unpublished data). Among the braconid subfamily Microgastrinae, to which C. marginiventris belongs, larval parasitism is assumed to be the typical behavior, although at least one species, Cotesia hyphantriae (Riley), appears to be an egg-larval parasitoid (Tadic'

Armyworm Symposium ’96: Ruberson and Whitﬁeld 297

1958). Several species of known larval parasitoids in this subfamily have been demonstrated in laboratory tests to be capable of facultatively parasitizing host eggs and emerging from the larvae (Johannson 1951, Wilbert 1960). But Johannson (1951), working with Cotesia glomerata (L.), observed that the females only did so when held in close proximity to the eggs, and concluded that in nature only first-instar larvae of Pieris brassicae L. are attacked by this parasitoid. Thus, it appears that at least some species are capable of successfully parasitizing both host eggs and larvae, although the occurrence of such events in the field has never been documented. C. marginiventris has been clearly shown to be a larval parasitoid (e.g., Boling & Pitre 1970, Kun- nalaca & Mueller 1979, Braman & Yeargan 1991), but the extent to which this parasitoid could function as an egg-larval parasitoid has not been examined, if indeed it has ever been considered. Although C. marginiventris has historically been considered a larval parasitoid, our recent field observations, supported by laboratory data reported below, indicate that this parasitoid, while typically a larval parasitoid, is also capable of parasitizing eggs of the beet armyworm, Spodoptera exigua (Hübner) and emerging from the larvae. This paper presents data from field and laboratory studies demonstrating that C. marginiventris is capable of facultatively parasitizing beet armyworm eggs, and that such an event may not be unusual in the field. We will conclude by considering this behavior in light of phylogenetic relationships within the Braconidae.

MATERIALS AND METHODS

Field Collections

Sampling of beet armyworm egg masses was incidentally undertaken as a component of other projects designed to characterize and quantify the impact of the parasitoid complex attacking this pest in cotton (see Ruberson et al. 1994). Most of the collections in these studies focused on larvae, but occasionally egg masses were collected, chieﬂy to assess the impact of the egg-larval parasitoid Chelonus insularis Cresson. At the time egg masses were collected in the respective cotton ﬁelds, larval populations of beet armyworms were very low. In 1993, a total of 75 egg masses were collected on 3 different dates from 3 different locations [2 in Tift County (1 northern and 1 southern) and 1 in Laurens County, GA]. In 1995, a single egg mass was collected from a third location in Tift County, GA. After each egg mass had hatched, 30 randomly-selected larvae from each egg mass were individually placed in diet cups with a semisynthetic diet (Burton 1969) and held in the laboratory. These larvae were examined daily for parasitoid emergence.

Laboratory Trial

A single experiment was run in the laboratory to determine whether female C. marginiventris would sting eggs of the beet armyworm and if the parasitoid’s offspring could successfully develop after oviposition in the host egg. Female parasitoids were obtained from a laboratory culture that had originated from parasitized beet armyworm larvae collected in cotton ﬁelds. The culture had been in the laboratory approximately one year at the time of the experiment and had been maintained using beet armyworm larvae as hosts. Three parasitoids were each given 1 egg mass (2-d old) and their behavior observed for 1 hour. The parasitoids were then removed, and the egg masses held until larvae hatched, at which time 30 randomly-selected larvae

298 Florida Entomologist 79(3) September, 1996 from each egg mass were placed individually in diet cups as above. The larvae were examined daily for parasitoid emergence.

RESULTS AND DISCUSSION

Initial parasitoid specimens collected in 1993 appeared to be C. marginiventris, based on the appearance of the cocoons and the emerged parasitoids, but were dis- carded before they could be determined to species. Nevertheless, the identification of the parasitoids was not absolutely certain at that time. Parasitoids emerging from the egg mass collected in 1995 were determined to be C. marginiventris by JBW. We assume, therefore, with considerable confidence that the parasitoids reared from egg masses in 1993 were also C. marginiventris. Laboratory data (see below) lend further support to the validity of this assumption. Field sampling indicated that egg-larval parasitism of beet armyworms by C. marginiventris may not be an unusual phenomenon in the field (Table 1). In the largest sample taken (19 August 1993 in Tift County), 23% of the collected egg masses yielded larvae parasitized by C. marginiventris. Indeed, some parasitism was found in samples of egg masses from all locations. Rates of parasitism within parasitized egg masses ranged from 15.4% to 100%, but typically were on the order of 30-40% (Table 1). These levels of parasitism suggest either that individual female C. marginiventris parasitize the egg masses heavily after they locate them, or that multiple females are discovering and exploiting the egg mass (i.e., exhibiting an aggregative response). Beet armyworm egg masses typically consist of approximately 100 eggs, although the number can range from 40 to over 250 (Ruberson et al. 1994); therefore, parasitism rates of 30-40% in large egg masses may represent a substantial reproductive invest- ment by female C. marginiventris which Braman & Yeargan (1991) demonstrated to have a realized lifetime fecundity of 362.7 offspring. In the laboratory test, 2 of the females exhibited considerable interest in their respective egg masses, repeatedly probing the egg masses with their ovipositors during the observation period. The third female showed no interest. Johannson (1951) and Wilbert (1960) observed similar behavior with C. glomerata and Cotesia pieridis (Bouche) females, respectively. Only 7 larvae from the 2 stung egg masses [5 (16.7%) from one egg mass and 2 (6.7%) from the other] yielded C. marginiventris despite the

TABLE 1. PREVALENCE OF EGG-LARVAL PARASITISM BY COTESIA MARGINIVENTRIS AMONG FIELD-COLLECTED EGG MASSES OF THE BEET ARMYWORM, SPODOPTERA EXIGUA.

No. Egg Masses No. Egg Masses Mean % Parasitism Collection Date/Locale Collected Parasitized within Egg Masses1

Tift Co., GA 29 July 1993 (Site 1) 2 2 32.8 ± 5.80 19 August 1993 (Site 2) 61 14 42.1 ± 22.64 3 July 1995 1 1 26.7

Laurens Co., GA 4 September 1993 11 2 31.7 ± 7.07

1With SD; means are for parasitized egg masses only.

Armyworm Symposium ’96: Ruberson and Whitfield 299 high level of apparent ovipositional activity by the females. Nevertheless, these results demonstrate that C. marginiventris is capable of successfully functioning as an egg-larval parasitoid. Age of the host eggs may also influence the success of parasitism—Johannson (1951) noted that only host eggs nearing eclosion were suitable for subsequent development of C. glomerata. A similar situation may also occur for C. marginiventris, but this was not considered in the present experiments in which eggs of intermediate age (2-d old) were used. The facultative parasitization of host eggs by C. marginiventris raises some interesting questions regarding the foraging behavior of this parasitoid. A body of literature has demonstrated that plant kairomones activated by the feeding of host larvae are key foraging cues for this parasitoid (Loke et al. 1983, Turlings et al. 1989, 1990, 1991). However, such cues are lacking on plants with only egg masses present and are limited or absent in fields with low or no larval populations. The prevalence of parasitism observed in our field collections suggests that the parasitoids were quite successful at locating egg masses, even when cues induced by larval feeding were rare. What cues are being used to locate the host egg masses? It is possible that volatiles are released from the large mass of scales that the ovipositing female beet armyworm deposits on the egg mass; perhaps volatiles similar to those left after oviposition by other lepidopteran species (Beevers et al. 1981, Noldus & Van Lenteren 1983). It may also be a result of beet armyworm sex pheromone remnants on the egg mass or leaf, as Noldus et al. (1991) found with the parasitoid Trichogramma evanescens West- wood. Further, the plant itself may respond to some minor disruption of the leaf cuticle resulting from oviposition, by releasing some volatile. Another possible explanation is that female parasitoids were drawn into the field by the feeding of the few larvae present in the field and, thereafter, exploited the egg masses. Whatever the

Fig. 1. Phylogeny of the microgastroid lineage of Braconidae, based on morphological data from Whitﬁeld & Mason (1994), with the known distribution of egg-larval parasitism shown. Biological data from Shaw & Huddleston (1991) and Whitﬁeld & Mason (1994).

300 Florida Entomologist 79(3) September, 1996 cues, however, it is quite clear that larval damage is not the only source of cues to which C. marginiventris is capable of responding in close-range host location. The confirmation that at least some Cotesia spp. are indeed capable of ovipositing into host eggs is of comparative physiological and phylogenetic interest. It has been suspected for some time that the Microgastrinae (including Cotesia) are likely to be closely related to the Cheloninae, the members of which all typically oviposit into host eggs and emerge from the host larvae (Fig. 1). Many other biological similarities (in addition to the structural similarities reviewed by Tobias (1967) between the two subfamilies have now been noted: typically they have three endoparasitic larval instars (Shaw & Huddleston 1991), they both have associations with polydnaviruses for host immune suppression (Fleming 1992, Stoltz & Whitfield 1992), and both groups attack largely overlapping groups of Lepidoptera. Another subfamily, the Adeliinae, has been proposed as being the actual sister-group to the Cheloninae (e.g., Nixon 1965, Du- darenko 1974). Recent phylogenetic work based on comparative morphology (Whit- field & Mason 1994) appears to establish fairly firm sister-group relationship between both Adeliinae and Cheloninae and a close relationship between these two subfamilies and the “microgastroid” complex of subfamilies, including the Microgastrinae (Fig. 1). Ongoing phylogenetic work based on mitochondrial DNA sequences (Whit- field, in prep.) also appears to confirm these relationships. In the Cheloninae (and predictably perhaps also the Adeliinae, see Wharton 1994), the parasitoid eggs hatch relatively soon after oviposition, but development of the first instar is delayed (Ullyett 1949, Narayanan et al. 1961, Powers & Oatman 1984, Bühler et al. 1985, Kawakami 1985, Shaw & Huddleston 1991). The details of the developmental and immunological interactions and the effects of venoms and polydnaviruses appear to differ to some degree between the studied species of chelonines (Jones 1985, 1987, Leluk & Jones 1989) and microgastrines (reviewed by Lavine & Beckage 1995), but too few species have been studied for firm conclusions to be drawn. It would be interesting indeed to examine in further detail the aspects in which the two groups differ or resemble one another in their physiological responses to the common problem of egg-larval parasitism.

ACKNOWLEDGMENTS We appreciate the assistance of Melissa Dykes, Julie McFarland, Russ Ottens, Ray Wilson, and Daniel West in collecting beet armyworm egg masses. The reviews of Drs. Robert M. McPherson and James D. Dutcher (Univ. of Georgia, Tifton) are also appreciated. This research was supported, in part, by the Georgia Agricultural Commodity Commission for Cotton and Cotton Incorporated.

REFERENCES CITED

BEEVERS, M., W. J. LEWIS, H. R. GROSS, JR., AND D. A. NORDLUND. 1981. Kairomones and their use for management of entomophagous insects. X. Laboratory studies on manipulation of host-ﬁnding behavior of Trichogramma pretiosum Riley with a kairomone extracted from Heliothis zea (Boddie) moth scales. J. Chem. Ecol. 7: 635-648. BOLING, J. C., AND H. N. PITRE. 1970. Life history of Apanteles marginiventris with descriptions of immature stages. J. Kansas Entomol. Soc. 43: 465-470. BRAMAN, S. K., AND K. V. YEARGAN. 1991. Reproductive strategies of primary parasitoids of the green cloverworm (Lepidoptera: Noctuidae). Environ. Entomol. 20: 349-353. BÜHLER, A., T. N. HANSLIK, AND B. D. HAMMOCK. 1985. Effects of parasitization of Trichoplusia ni by Chelonus sp. Physiol. Entomol. 10: 383-394.

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BURTON, R. L. 1969. Mass rearing the corn earworm in the laboratory. USDA-ARS 33- 134. DUDARENKO, G. P. 1974. Formation of the abdominal carapace in braconids (Hy- menoptera, Braconidae). Entomol. Rev. 53: 80-90. FLEMING, J. G. W. 1992. Polydnaviruses: mutualists and pathogens. Annu. Rev. Ento- mol. 37: 401-425. JOHANNSON, A. S. 1951. Studies on the relation between Apanteles glomeratus L. (Hym., Braconidae) and Pieris brassicae L. (Lepid., Pieridae). Norsk Entomol. Tids. 8: 145-186. JONES, D. 1985. Endocrine interaction between host (Lepidoptera) and parasite (Che- loninae: Hymenoptera): is the host or parasite in control? Ann. Entomol. Soc. America 78: 141-148. JONES, D. 1987. Material from adult female Chelonus sp. directs expression of altered developmental programme of host Lepidoptera. J. Insect Physiol. 33: 129-134. KAWAKAMI, T. 1985. Development of the immature stages of Ascogaster reticulatus Watanabe (Hymenoptera: Braconidae), an egg-larval parasitoid of the smaller teat tortrix moth, Adoxophyes sp. (Lepidoptera: Tortricidae). Appl. Entomol. Zool. 20: 380-386. KUNNALACA, S., AND A. J. MUELLER. 1979. A laboratory study of Apanteles marginiventris, a parasite of green cloverworm. Environ. Entomol. 8: 365-368. KOK, L. T., AND T. J. MCAVOY. 1989. Fall broccoli pests and their parasites in Virginia. J. Entomol. Sci. 24: 258-265. KROMBEIN, K. V., P. D. HURD, JR., D. R. SMITH, AND B. D. BURKS. 1979. Catalog of Hy- menoptera of America north of Mexico. Smithsonian Instit. Press, Washington D.C. LAVINE, M. D., AND N. E. BECKAGE. 1995. Polydnaviruses: potent mediators of host insect immune dysfunction. Parasit. Today 11: 368-378. LELUK, J., AND D. JONES. 1989. Chelonus sp. near curvimaculatus venom proteins: analysis of their potential role and processing during development of host Tri- choplusia ni. Arch. Insect Biochem. Physiol. 10: 1-12. LOKE, W. H., T. R. ASHLEY, AND R. I. SAILER. 1983. Influence of fall armyworm, Spodoptera frugiperda, (Lepidoptera: Noctuidae) larvae and corn plant damage on host finding in Apanteles marginiventris (Hymenoptera: Braconidae). Environ. Entomol. 12: 911-915. MCCUTCHEON, G. S., S. G. TURNIPSEED, AND M. J. SULLIVAN. 1990. Parasitization of lepidopterans as affected by nematicide-insecticide use in soybean. J. Econ. En- tomol. 83: 1002-1007. NARAYANAN, E. S., B. R. SUBBA RAO, AND K. R. THAKARE. 1961. The biology and some aspects of morphology of the immature stages of Chelonus narayanai Subba Rao (Braconidae: Hymenoptera). Proc. Natl. Inst. Sci. India (B) 27: 68-82. NIXON, G. E. J. 1965. A reclassification of the tribe Microgasterini (Hymenoptera: Braconidae). Bull. British Mus. Nat. Hist., Entomol. Suppl. 2: 1-284. NOLDUS, L. P. J. J., AND J. C. VAN LENTEREN. 1983. Kairomonal effects on searching for eggs of Pieris brassicae, Pieris rapae and Mamestra brassicae of the parasite Trichogramma evanescens Westwood. Med. Fac. Landbouww. Rijksuniv. Gent 48/2: 183-194. NOLDUS, L. P. J. J., R. P. J. POTTING, AND H. E. BARENDREGT. 1991. Moth sex pheromone adsorption to leaf surface: bridge in time for chemical spies. Physiol. En- tomol. 16: 329-344. POWERS, N. R., AND E. R. OATMAN. 1984. Biology and temperature responses of Che- lonus kelliae and Chelonus phthorimaeae (Hymenoptera: Braconidae) and their host, the potato tuberworm, Phthorimaea operculella (Lepidoptera: Gelechiidae). Hilgardia 52: 1-32. RUBERSON, J. R., G. A. HERZOG, W. R. LAMBERT, AND W. J. LEWIS. 1994. Management of the beet armyworm in cotton: role of natural enemies. Florida Entomol. 77: 440-453. 302 Florida Entomologist 79(3) September, 1996

SHAW, M. R., AND T. HUDDLESTON. 1991. Classification and biology of braconid wasps (Hymenoptera: Braconidae). Royal Entomological Society of London, London. STOLTZ, D. B., AND J. B. WHITFIELD. 1992. Viruses and virus-like entities in the parasitic Hymenoptera. J. Hymenop. Res. 1: 125-139. TADIC', M. D. 1958. Apanteles hyphantriae Riley, an egg parasite of the fall webworm. Proc. X Internatl. Congr. Entomol. 4: 859-861. TOBIAS, V. 1967. A review of the classification, phylogeny and evolution of the family Braconidae (Hymenoptera). Entomol. Rev. 46: 387-399. TURLINGS, T. C. J., J. H. TUMLINSON, W. J. LEWIS, AND L. E. M. VET. 1989. Beneficial arthropod behavior mediated by airborne semiochemicals. VIII. Learning of host-related odors induced by a brief contact experience with host by-products in Cotesia marginiventris (Cresson), a generalist larval parasitoid. J. Insect Be- hav. 2: 217-225. TURLINGS, T. C. J., J. H. TUMLINSON, AND W. J. LEWIS. 1990. Exploitation of herbi- vore-induced plant odors by host-seeking parasitic wasps. Science 250: 1251- 1253. TURLINGS, T. C. J., J. H. TUMLINSON, AND W. J. LEWIS. 1991. Larval-damaged plants: source of volatile synomones that guide the parasitoid Cotesia marginiventris to the micro-habitat of its hosts. Entomol. Exp. Appl. 58: 75-82. ULLYETT, G. C. 1949. Distribution of progeny by Chelonus texanus Cress. (Hy- menoptera: Braconidae). Canadian Entomol. 81: 25-44. WHARTON, R. A. 1994. Bionomics of the Braconidae. Annu. Rev. Entomol. 38: 121-143. WHITFIELD, J. B., AND W. R. M. MASON. 1994. Mendesellinae, a new subfamily of braconid wasps (Hymenoptera, Braconidae) with a review of relationships within the microgastroid assemblage. System. Entomol. 19: 61-76. WILBERT, H. 1960. Apanteles pieridis (Bouche)(Hym., Braconidae), ein Parasit von Aporia crataegi (L.)(Lep., Pieridae). Entomophaga 5: 183-211.

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302 Florida Entomologist 79(3) September, 1996

GROWTH INHIBITION OF FALL ARMYWORM (LEPIDOPTERA: NOCTUIDAE) LARVAE REARED ON LEAF DIETS OF NON-HOST PLANTS

B. R. WISEMAN, J. E. CARPENTER AND G. S. WHEELER1 Insect Biology and Population Management Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture Tifton, GA 31793-0748

1Aquatic Weed Research, Agricultural Research Service, U. S. Department of Agriculture, Fort Lauderdale, FL 33314

ABSTRACT

Chemists and natural plant product scientists have shown an interest in compounds from exotic plants that are considered non-hosts for the fall armyworm, Spodoptera frugiperda (J. E. Smith), but may serve as sources of materials that reduce feeding and growth of herbivorous insects. We report here results of laboratory bioassays with neonate and fifth instar fall armyworm fed on a standard diet alone and on an amended diet with celufil or leaves from dogwood, Cornus florida L., hydrangea, Hydrangea macrophylla (Thunb.) Seringe, black cherry, Prunus serotina Ehrh., or Bradford pear, Pyrus calleryna Decne. The neonates had reduced growth

Armyworm Symposium ’96: Wiseman et al. 303 and frass production when fed diets containing leaves of dogwood, hydrangea, black cherry and Bradford pear while fifth-instars had reduced consumption and weight gain when fed the hydrangea leaf-diet. These results suggest a toxic component in the leaves of hydrangea that reduces the development of the fall armyworm neonates and fifth instars. The results also indicate that the leaves of dogwood, black cherry and Bradford pear have growth inhibitors present in their leaves that adversely affect growth of neonate fall armyworm. Because fifth instars performed similarly to controls when fed dogwood, cherry or pear leaf-diets, older larvae may be able to over- come the plants natural defenses better than neonates.

Key Words: Spodoptera frugiperda, antibiosis, deterrent, growth inhibition, protein

RESUMEN

Los químicos y los estudiosos de los productos de plantas naturales han mostrado interés en complejos derivados de plantas exóticas no consideradas como hospedantes de Spodoptera frugiperda (J. E. Smith) pero que pueden servir como fuente de mate- riales que reducen la capacidad de alimentarse y el crecimiento en los insectos herbí- voros. Reportamos aquí los resutados de bioensayos de laboratorio con S. frugiperda neonatas y del quinto instar alimentadas en una dieta estándar solamente y con una dieta con hojas de Cornus florida L., Hydrangea macrophyla (Thunb.) Seringe, Pru- nus serotina Ehrh. o Pyrus calleryna Decne. Los neonatos mostraron reducción del crecimineto y de la producción de excretas cuando fueron alimentados con dietas con- teniendo hojas de C. florida L., H. macrophylla, P. serotina y P. calleryna, mientras que el quinto instar tuvo reducción del consumo de alimento y de la ganancia de peso cuando fue alimentado con una dieta de H. macrophylla. Estos resultados suguieren que un componente tóxico en las hojas de H. macrophylla reduce el desarrollo de los neonatos y quintos instares de S. frugiperda. Los resultados además indican que las hojas de C. florida, P. serotina y P. calleryna tienen inhibidores de creciemiento presentes en sus hojas que afectan adversamente el crecimiento de los neonatos. Debido a que los quintos instares se comportaron como los testigos cuando se alimentaron con C. florida, P. serotina o P. calleryna, las larvas más viejas parecen ser capaces de ven- cer las defensas naturales de las plantas mejor que los neonatos.

The fall armyworm, Spodoptera frugiperda (J. E. Smith), continues to be an important economic pest as was emphasized by participants in the 1996 Armyworm Sym- posium at the annual meeting of the Southeastern Branch of the Entomological Society of America. One of the important controls for the fall armyworm has been plant resistance. This has led to questions such as: (1) Why don’t larvae of the fall armyworm feed on all plant species and (2) could fall armyworm larvae feed on a non- host plant in no-choice situations? Doskotch et al. (1977) stated that it is evident from ﬁeld observations of the Gypsy moth, Lymantria dispar (L.), that phytophagous insects utilize very few plant species as hosts, although many plant species may be acceptable to the Gypsy moth species. Bernays (1983) suggested that the use of natural feeding deterrents is an intuitively satisfying but logistically questionable tactic in crop pest management. Possibly, in the near future, chemicals from non-host plants will be exploited as new environmentally safe pesticides that can be used in biotech- nology to render domestic crop plants to a non-host status. Warthen et al. (1982) and Wiseman et al. (1986) searched a number of non-host plants and found several antifeedant responses by larvae of the fall armyworm. Both research groups used one non-host plant in common, the dogwood tree, Cornus ﬂorida

304 Florida Entomologist 79(3) September, 1996

L. Warthen et al (1982) evaluated twigs while Wiseman et al. (1986) evaluated leaves for response to larvae of the fall armyworm. Wiseman et al. (1986) also found non-host plants, such as Jalapeno peppers and banana leaves (unpublished) on which the larvae of the fall armyworm performed well when plant materials were mixed in diets. The dogwood is a small forest tree, common in the Southeastern United States. Weiner (1982) reported that the Delaware Indians boiled the bark of the dogwood in water and used the solution to reduce fever. Hostettmann et al. (1978) found a mol- luscicidal saponin in the bark of the dogwood and that Biomphalaria glabratus snails were killed within 24 h by a 6 to 12-ppm solution of the saponins. Miller (1978) found that homogenized leaf solutions of dogwood applied to the soil were toxic to the root lesion nematode, Pratylenchus penetrans (Cobb) Chitwood and Oteifa. Villani & Gould (1985) found that crude plant extracts of dogwood leaves did not deter feeding by the wireworm, Melanotus communis Gyll. Warthen et al. (1982) showed that 1.5 g of dogwood twigs in 100 g of diet resulted in about 30% mortality of fall armyworm larvae. However, when Wiseman et al. (1986) used 50 g of fresh dogwood leaves in 400 ml of diet, 8-day-old fall armyworm larvae weighed an average of only 0.4 mg. In addition, none of the larvae fed the dogwood leaf diet pupated. Chemists and natural product scientists have shown an interest in some of the exotic plant materials that were reported by Wiseman et al. (1986). Other plant species whose leaves have compounds that also have possibilities as feeding deterrents against larvae of the fall armyworm are: hydrangea, Hydrangea macrophylla (Thunb.) Seringe, black cherry, Prunus serotina Ehrh., and Bradford pear, Pyrus calleryna Decne. Therefore, we report results of feeding tests with leaves of these plant species incorporated in artiﬁcial diets to determine their effects on fall armyworm larval performance.

MATERIALS AND METHODS

Fall armyworm larvae were obtained from a culture maintained on a modified pinto-bean diet (Burton & Perkins 1989) at the Insect Biology and Population Man- agement Research Laboratory, Tifton, GA. Four experiments each were conducted in a randomized complete-block design with 30 replications and 1 cup per replicate. An extra set of treatments of 15 cups each was set up to measure the decrease in moisture of each diet during the test period. Controls for each experiment were regular pinto bean diet (Burton & Perkins 1989) and pinto bean diet diluted by combining 3 ml of prepared diet with 2 ml of water and mixed at a rate of 50 mg of celufil per ml of dilute diet. Celufil, a fine mesh cellulose filler with minimum nutritive value, binds with laboratory diets and does not readily absorb water. Diets prepared with celufil as an inert material for the controls remain fluid and are easily dispensed. For each experiment and leaf material diet, the pinto bean diet was diluted by combining 3 ml of prepared diet with 2 ml water and 50 mg of ground (1 mm screen) oven-dried leaves per ml dilute diet. All experiments were held in a controlled environmental room maintained at 28 ± 2°C and 65 ± 2% RH with a photoperiod of 14:10 (L:D) h.

Experiment 1

Treatments consisted of dogwood and hydrangea leaves mixed in dilute pinto bean diet, regular pinto bean diet, and a dilute bean diet plus 50 mg celuﬁl per ml dilute diet. The diet treatments were dispensed about 10 ml per cup into 30-ml plastic diet cups. The diets were allowed to cool for about 2 h, after which 1 neonate fall army-

Armyworm Symposium ’96: Wiseman et al. 305 worm was introduced into each cup and the cup was capped. Weights of larvae and cumulative frass produced (weighed immediately after collection from each cup) were recorded at 9 d.

Experiment 2 Treatments consisted of 50 mg each of oven-dried cherry and pear leaves mixed per ml dilute pinto bean diet, regular pinto bean diet, and a dilute pinto bean diet plus 50 mg celuﬁl per ml dilute diet. The diet treatments were dispensed into 30-ml plastic diet cups of about 10 ml per cup. The diets were allowed to cool for about 2 h, after which 1 neonate fall armyworm was introduced into each cup and the cup was capped. Weights of larvae and cumulative frass produced (weighed immediately after collection from each cup) were recorded at 8 d; the same larvae, frass accumulation and diet consumption were weighed again 24 h later (9 d).

Experiments 3 and 4 Treatments consisted of 50 mg each of oven-dried dogwood or hydrangea leaves per ml dilute pinto bean diet (Experiment 3) and cherry or pear leaves (Experiment 4). Regular pinto bean diet and a dilute pinto bean diet plus 50 mg celufil per ml dilute diet served as controls. Treatments of diets and procedures were as described above except that fifth instar fall armyworm were used. Larvae were reared to the fifth instar on the regular pinto bean diet. Initial weights of diets and larvae were recorded as the larvae were placed on the diets. Weight of diets, larvae, and fresh frass was recorded after 24 h. Weight of dry frass and diets was recorded for each after they were dried in an oven at about 50°C for 7 d. The percentage of nitrogen in the dry diets and frass from the latter 2 experiments was determined by an FP-228 nitrogen determi- nator (Model 601-700, Leco, St. Joseph, MI). Protein content was estimated by the formula 6.25 X % nitrogen (Helrich 1990). Data were analyzed by PROC GLM analysis (SAS Institute 1989) and significantly different means were separated by least significant difference (P ≤ 0.05) (SAS Insti- tute 1989).

RESULTS AND DISCUSSION

Experiment 1 Weight of fall armyworm larvae (Table 1) after having fed on dogwood- and hydrangea-leaf diets for 9 d was significantly (P ≤ 0.05) different from the weight of larvae that were fed the regular pinto bean diet or diluted diet plus celufil. In fact, none of the larvae fed hydrangea-leaf diet were alive at 9 d. The dilute diet with celufil is capable of supporting larval growth and development equivalent to the regular pinto bean diet. Therefore, addition of leaf materials did not simply dilute the diet and cause a reduction in growth, i. e. the dogwood-leaf diet. But, since the larvae died on the hydrangea-leaf diet, the presence of a toxic factor was indicated. Frass production during the 9 d feeding was significantly less for larvae that fed on dogwood- and hydrangea-leaf diets than for larvae that fed on the two control diets.

Experiment 2

Larvae weighed signiﬁcantly (P ≤ 0.05) less at 8 and 9 d when fed cherry- or pear-leaf diets than when they were fed on the control diets (Table 2). Frass production for larvae

306 Florida Entomologist 79(3) September, 1996

TABLE 1. WEIGHT OF FALL ARMYWORM LARVAE AND FRASS FROM NEONATES REARED ON DIETS CONTAINING DOGWOOD OR HYDRANGEA LEAVES

Weight (mg) ± SEM at 9 Days

Food Source Larvaea Frass1

Reg. bean diet 208.8 ± 8.3a 192.3 ± 12.8b Diet + celuﬁl 193.0 ± 8.1a 347.1 ± 12.3a Diet + dogwood 11.6 ± 8.3b 11.6 ± 12.5c Diet + hydrangea 0.0 ± 8.1b 0.0 ± 12.3c

1Means within a column followed by the same letter are not significantly different (P ≤ 0.05 LSD; SAS Insti- tute 1989). SEM = standard error of mean was based on pooled error mean square in the analysis. that were fed these diets was significantly (P ≤ 0.05) less at both 8 and 9 d than that for larvae fed the control diets. Both consumption and weight gain during the 24-h feeding period between days 8 and 9 were significantly (P ≤ 0.05) less for the larvae fed the black cherry- and Bradford pear-leaf diets than for larvae that fed on the control diets.

Experiments 3 and 4

The initial weights of the fifth instars for the various treatments were not significantly different before they were transferred to the test diets (Tables 3 and 4). How- ever, after 24-h, larvae fed the hydrangea-leaf diet weighed significantly (P ≤ 0.05) less than larvae that were fed the dogwood-leaf diet or the control diets. The mean weight of larvae that were fed the dogwood diet was not different from the weight of larvae fed the control diets. Larvae fed the regular pinto bean diet gained significantly (P ≤ 0.05) more weight during the 24-h feeding period than did the larvae fed the diet plus celufil or the dogwood-leaf diet. Weight gain during the 24-h feeding period was significantly (P ≤ 0.05) less for larvae fed the hydrangea-leaf diet than for larvae fed the other three treatment diets. Larvae consumed more diet and excreted more frass when fed the diet plus celufil and dogwood-leaf diet than for larvae fed the regular pinto bean diet. Consumption and weight of frass were significantly less for the larvae that fed on the hydrangea-leaf diet than for any of the other three treatments. Per- centage nitrogen of the diet or frass as an indication of the amount of protein in the diets or frass were also significantly (P ≤ 0.05) different among treatments. The regular diet had the highest percentage of nitrogen in the diet but an intermediate amount in the frass. The diet plus the celufil had the least nitrogen in the diet and the frass. The hydrangea-leaf diet had an intermediate percentage nitrogen in the diet but the greatest percentage nitrogen in the frass. Larvae fed the regular pinto bean diet utilized more of the protein consumed than did those that were fed on the other diets. Larvae feeding on the diet plus the celufil consumed more diet than larvae fed the regular pinto bean diet. Nitrogen content was lower in the diet plus celufil and in frass from larvae fed the diet plus celufil, indicating that the larvae that were fed this diet had to consume more diet and excrete less nitrogen to obtain the necessary protein for the growth. However, since the larvae excreted more frass, they also excreted more nitrogen (5.04 versus 3.46 mg) than did larvae that were fed the regular pinto bean diet. Larvae that fed on the dogwood-leaf diet excreted more than twice as much frass as those fed the regular diet. Larvae fed the regular pinto bean diet and dogwood-leaf diet consumed almost equal amounts of pro-

Armyworm Symposium ’96: Wiseman et al. 307 PEAR

13a 13b 13c 13c ± ± ± ± in 24 hrs Weight Gain Weight BRADFORD

3 31a 32a 201 120 31b 1 32c 0 ± ± ± ± CHERRY

(mg) Consumption BLACK

18a 19a 362 322 18b 134 18b 0 ± ± ± ± 2 CONTAINING

SEM ± DIETS

13b 13a 272 307 13c 18 13d 0 ± ± ± ± Weight (mg) Weight REARED

0.05 LSD; SAS Institute 1989). 0.05 LSD; ≤

LARVAE P

[(100 - % moisture loss)/100]. × 14a 14b 290 498 14c 0 14c 35 ± ± ± ± ARMYWORM

FALL

Larvae Frass FOR

6a 6a 394 319 6b 1 6b 4 ± ± ± ± 8 Days 9 Days 8 Days 9 Days CONSUMPTION

LEAF

AND

1 EIGHT LEAVES 2. W Means within a column followed by the same letter are not significantly different ( based on the pooled error mean square in analysis. SEM = standard error of mean was Diet consumption = (initial fresh weight of diet - final diet) 1 2 3 ABLE Reg. Bean Diet CheckReg. 204 Bean Diet + Celufil Check leaves cherry Black 200 1 Bradford pear leaves 4 Treatment T 308 Florida Entomologist 79(3) September, 1996 DIETS 2

0.1c 0.1a 0.1b 0.1b ON

± ± ± ± SEM REARED

LARVAE

0.01c 1.6 0.01b 2.8 0.01b 2.2 0.01a 2.4 ± ± ± ± Percent Nitrogen + Percent ARMYWORM

FALL

19c 3.3 18a 3.3 18a 2.9 18b 3.9 ± ± ± ± INSTAR - FIFTH

c 2 FOR

43c 79 41a 319 41a 315 41b 144 ± ± ± ± SEM (mg) (mg) Frass Diet Frass ± FRASS

Consumption 0.05 LSD; SAS Institute 1989). 0.05 LSD; AND ≤

P DIET

[(100 - % moisture loss)/100]. × OF

11c 425 10ab 880 10b 859 10a 745 ± ± ± ± 1 Larval Weight (mg) Larval Weight Larval Wt After 24 hrs CONTENT

LEAVES

5a 191 5a 357 5a 351 5a 379 ± ± ± ± NITROGEN

Initial HYDRANGEA

Larval Wt AND

DOGWOOD

CONSUMPTION , EIGHT CONTAINING 3. W Means within a column followed by the same letter are not significantly different ( SEM = standard error of mean was based on the pooled error mean square in analysis. SEM = standard error of mean was Diet consumption = (initial fresh weight of diet - final diet) 1 2 3 ABLE Hydrangea leaves 161 Dogwood leaves 165 Bean diet + celufil check 158 Treatment Reg. bean diet checkReg. 163 T Armyworm Symposium ’96: Wiseman et al. 309 DIETS 2

ON 0.04a 0.04b 0.04b 0.04a

± ± ± ± SEM ± REARED

LARVAE

0.01c 2.1 0.01b 2.0 0.01a 2.0 0.01d 1.4 ± ± ± ± Percent Nitrogen Percent ARMYWORM

FALL

17c 4.0 17b 3.4 17a 3.5 17b 2.8 ± ± ± ± INSTAR - FIFTH

3 2 FOR

19b 378 19a 560 19b 403 19b 259 ± ± ± ± SEM (mg) (mg) Frass Diet Frass ± FRASS

Consumption 0.05 LSD; SAS Institute 1989). 0.05 LSD; AND ≤

P 1 DIET

[(100 - % moisture loss)/100]. × OF

9bc 731 9ab 914 9c 716 9a 760 LEAVES ± ± ± ±

Larval Weight (mg) Larval Weight Larval Wt PEAR After 24 hrs

CONTENT

RADFORD 3a 301 3a 324 3a 296 3a 345 B ± ± ± ± NITROGEN

Initial Larval Wt AND

CHERRY

BLACK

CONSUMPTION , EIGHT CONTAINING 4. W Means within a column followed by the same letter are not significantly different ( based on the pooled error mean square in analysis. SEM = standard error of mean was Diet consumption = (initial fresh weight of diet - final diet) 1 2 3 ABLE Bradford pear leaves 142 Black cherry leaves cherry Black 145 Bean diet + celufil check 137 Treatment Reg. bean diet checkReg. 144 T 310 Florida Entomologist 79(3) September, 1996 tein (29 versus 28 mg), but excreted more than twice as much nitrogen (3.45 versus 7.02 mg). Fifth-instar fall armyworms fed the hydrangea-leaf diet consumed less than one-half the amount of diet consumed by larvae fed on the dogwood-leaf diet. And, the nitrogen consumption was about one-half as much. However, the nitrogen excreted by larvae fed the hydrangea-leaf diet was less than one-third as much as that excreted by larvae fed the dogwood-leaf diet and similar to the nitrogen excreted by larvae fed the regular pinto bean diet. Weight of the larvae that fed on the pinto bean diet was significantly (P ≤ 0.05) greater after 24-h than the weight of larvae fed the diet plus celufil and pear-leaf diet (Table 4). Larvae fed the cherry-leaf diet weighed significantly more than larvae that fed on the diet plus celufil diet. However, consumption was significantly (P ≤ 0.05) greater for larvae fed the cherry-leaf diet than for larvae that fed on the other treatment diets. Frass production was also significantly (P ≤ 0.05) greater for larvae fed the cherry-leaf diet than for the other treatments. Frass production was the least from larvae that fed on the regular diet. Percentage nitrogen in the pinto bean diet was significantly higher than the percentage nitrogen in the cherry, pear or celufil diets. Per- centage nitrogen was lowest in the celufil diet as well as in the frass produced. The consumption of nitrogen was highest by larvae fed the cherry-leaf diet (32.0 mg) and least by larvae fed the celufil diet (20.0 mg). However, percentage nitrogen in the frass produced by larvae fed the diets of regular bean, cherry and Bradford pear-leaf were not significantly different. The amount of nitrogen in the frass varied considerably from 11.2 mg for larvae that fed on the cherry leaf diets to those that fed on the pear- leaf diet (7.94 mg), regular diet (5.18 mg) and celufil check (5.64 mg). In summary, fall armyworm neonates did not perform well after feeding on the dogwood, hydrangea, black cherry and Bradford pear-leaf diets. Larvae may have exhibited compensatory feeding when offered the celufil and dogwood-leaf diets as their consumption increased over larvae that fed on the regular pinto bean diet. The results also indicated that the leaves of dogwood, black cherry and Bradford pear have growth inhibitors present in their leaves that adversely affect the growth of neonate fall armyworm. Fall armyworm fifth instars had reduced consumption and weight gain when fed a hydrangea-leaf diet. These results suggested either a severe growth inhibitor or a toxic component in the leaves of hydrangea that caused total mortality as neonates and reduced the performance of the fall armyworm fifth instars. Larvae that fed on the cherry- and pear-leaf diets excreted much higher amounts of nitrogen than larvae that fed on either control diets, indicating a poor assimilation of the protein in these diets. Lastly, these non-hosts offer good possibilities for the discovery of new chemicals that could be used in the management of the fall armyworm.

ACKNOWLEDGMENTS We thank Johnny Skinner, Charles Mullis, and Robert Caldwell for their technical support.

REFERENCES CITED

BERNAYS, E. A. 1983. Antifeedants in crop pest management. pp. 259-269 in D. C. Whitehead and W. S. Bowers [eds.], Current themes in tropical science. Vol. 2. Natural products for innovative pest management. Pergamon Press. New York. BURTON, R. L., AND W. D. PERKINS. 1989. Rearing the corn earworm and fall armyworm for maize resistance studies, pp. 37-45 In Toward insect resistant maize for the third world: Proceedings of the international symposium on methodologies for developing host plant resistance to maize insects. International Maize and Wheat Improvement Center (CIMMYT). El Batan, Mexico, D. F. Armyworm Symposium ’96: Wiseman et al. 311

DOSKOTCH, R. W., T. M. ODELL, AND P. A. GODWIN. 1977. Feeding responses of Gypsy moth larvae, Lymantria dispar, to extracts of plant leaves. Environ. Entomol. 6: 563-566. HELRICH, K. 1990. Ofﬁcial methods of analysis. Assoc. Off. Anal. Chem. 1: 71-72. HOSTETTMANN, K., M. HOSTETTMANN-KALDAS, AND K. NAKANISHI. 1978. Mollusci- cidal saponins from Cornus ﬂorida L. Helvetica Chemica Acta. 61: 1990-1995. MILLER, P. M. 1978. Toxicity of homogenized leaves of woody and herbaceous plants to root lesion nematodes in water and in soil. J. American Soc. Hort. Sci. 103: 78-81. SAS INSTITUTE. 1989. SAS/STAT user’s guide, version 6, 4th ed., vol. 1. SAS Institute, Cary, NC. VILLANI, M., AND F. GOULD. 1985. Screening of crude extracts as feeding deterrents of the wireworm, Melanotus communis. Entomol. exp. appl. 37: 69-75. WARTHEN, J. D., JR., R. E. REDFERN, E. C. UEBEL, AND G. D. MILL, JR. 1982. Antifeed- ant screening of thirty-nine local plants with fall armyworm larvae. J. Environ. Sci. Health A17: 885-895. WEINER, M. A. 1982. Earth medicine-earth foods. Plant remedies, drugs, and natural forms of the North American Indians. MacMillian, New York. WISEMAN, B. R., R. E. LYNCH, K. L. MIKOLAJCZAK, AND R. C. GUELDNER. 1986. Ad- vancements in the use of a laboratory bioassay for basic host plant resistance studies. Florida Entomol. 69: 559-565.

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Armyworm Symposium ’96: All et al. 311

CONTROLLING FALL ARMYWORM (LEPIDOPTERA:NOCTUIDAE) INFESTATIONS IN WHORL STAGE CORN WITH GENETICALLY MODIFIED BACILLUS THURINGIENSIS FORMULATIONS

J. N. ALL1, J. D. STANCIL1, T. B. JOHNSON2 AND R. GOUGER2 1Department of Entomology University of Georgia, Athens, GA 30602

2Ecogen Inc., Langhorne, PA 19047

ABSTRACT

Development of more potent strains of Bacillus thuringiensis (Berliner) (Bt) using recombinant DNA technology may lead to improved insecticidal products for controlling pests like the fall armyworm, Spodoptera frugiperda (J. E. Smith), which have been difficult to manage with conventional Bt-based products. EG1999, a variant Bt strain developed by recombinant DNA technology from EG2348 (the active ingredient of the bioinsecticide Condor®), showed improved control of FAW infestations in whorl stage field corn in 1994 (oil flowable and granular formulations) and sweetcorn in 1995 [wettable granule (ECX9526) (WG)] and granular [ECX9526 (G) formulations] as compared with Javelin® (WG), an insecticide derived from a naturally occurring Bt strain, and methomyl (Lannate® LV). In 1994, leaf samples taken 1 h, 1, 2, 3, 5, and 7 d after spraying and assayed in the laboratory with fall armyworm showed that the Bt products (recombinant and natural) had less than 48 h residual toxicity to 1st instar larvae. In 1994 freshly hatched FAW were placed on plants in A-frame cages to evaluate this as a method to establish artificial field infestations for use when natural

312 Florida Entomologist 79(3) September, 1996

FAW populations are low. The plant damage and insecticide control in the cages were similar to the heavy natural infestations outside the cages.

Key Words: Spodoptera frugiperda, Bt formulations, sweetcorn

RESUMEN

El desarrollo de cepas más potentes de Bacillus thuringiensis (Berliner) (Bt) mediante el uso de la tecnología del DNA recombinante puede contribuir a la mejora de los productos insecticidas para el control de plagas tales como Spodoptera frugiperda (J. E. Smith), que han sido difíciles de manejar con productos comerciales a base de Bt. EG1999, una variante de una cepa de Bt desorrollada mediante la tecnología del DNA recombinante a partir de EG2348 (el ingrediente activo del insecticida Condor®) mos- tró un mejor control de S. frugiperda en maíz en estado de cogollo en 1994 (formulaciones de aceite ﬂowable y granulado), en maíz dulce en 1995 [gránulos humedecibles (ECX9526 (WG)) y formulación granular (ECX9526 (G))] al compararse con Javelin® (WG), un insecticida derivado de una cepa natural de Bt, y con methomyl (Lannate® LV). En 1994 las muestras tomadas a 1 hora, y a los 1, 2, 3, 5, y 7 días depués de la aplicación y ensayadas en el laboratorio con S. frugiperda mostraron que los productos de Bt (recombinante y natural) tenían una toxicidad residual al primer instar me- nor de 24 horas. En 1994 larvas recién eclosionadas fueron puestas sobre plantas en jaulas para evaluar un método para establecer infestaciones de campo cuando las poblaciones naturales de S. frugiperda eran bajas. El daño a las plantas y el control con insecticida dentro de las jaulas fueron similares a las fuertes infestaciones naturales que hubo fuera de las jaulas.

Naturally occurring Bacillus thuringiensis (Berliner) (Bt) based insecticides have had low to moderate effectiveness against the fall armyworm (FAW), Spodoptera frugiperda (J. E. Smith) (Gardner & Fuxa 1980; Krieg & Langenbruch 1981; Teague 1993). Biopesticide products containing Bt continue to be improved with the use of genetic technologies. ECX9399 (Ecogen, Inc., Langhorne, PA) showed improved toxicity as compared with conventional Bt products to FAW in corn field tests in three southeastern states (All et al. 1994). ECX9399 was an oil flowable (OF) formulation of a recombinant DNA variant (EG1999) of EG2348, the active strain in the Bt product Condor®. The present research evaluates field efficacy and stability of EG1999 in an (OF), wettable granule (WG), and granular (G) formulation for FAW control in whorl stage field corn (1994) and sweetcorn (1995).

MATERIALS AND METHODS

The field tests were conducted near Athens at the University of Georgia Plant Sci- ences Farm using Dekalb 689 variety field corn in August 1994 and Silverqueen variety sweetcorn in August 1995. Plots were 4 rows × 6 m long with 95 cm row width and 1 m alleys and were arranged in a randomized complete block experimental design with 4 replications. Prior to spraying, a FAW baited pheromone trap was monitored weekly for moth flights and the field was sampled for larvae and damage by examining 50 plants at random at 3 to 5 day intervals. Spraying was initiated when 50% of the plants showed damage by larvae (scarification of leaves). In both years plants were in the two leaf stage, about 25 cm tall, when insecticide application was initiated. Sprays were ap-

Armyworm Symposium ’96: All et al. 313

2 plied with a CO2 backpack sprayer in 190 liter/ha spray volume, at 3.2 kg/cm pressure, with a TG3 nozzle about 20 cm over the plant whorl. Granular formulations were applied in an 18 cm wide band over the row using a Noble® granular spreader and small plot procedures described in All & Hammes (1977). Four applications of the recombinant DNA Bt insecticides were made at 7-day intervals. In 1994 (OF) and (G) formulations of ECX9426 were tested and in 1995 (WG) and (G) formulations of ECX9526 were evaluated (see Table 1). Methomyl (Lannate® LV) was used as a conventional insecticide standard in 1994 and 1995, and a Bt (subspecies kurstaki) standard, Javelin® (WG), was used in 1994 at recommended rates for a conventional Bt standard. In 1994 artificial infestations using neonate FAW were conducted by placing an A- frame cage 120 cm long × 60 cm wide × 105 cm high covered with lumite insect screen (32 × 32 mesh) on top of a treated 5 × 10 cm wood base that had been buried 2.5 cm deep in the middle of one row of each plot. The cage covered 6 to 8 plants and was removed at the time of spraying. After the sprays dried, 5 freshly hatched FAW larvae (obtained from the Insect Biology and Population Management Research Laboratory, USDA-ARS, Tifton GA) were placed in the whorl of each plant; the plants were infested again 3 days later. Infestations were continued after each spray application. Efficacy was determined at selected intervals by examining all plants and rating damage as 1 = no damage or very slight (<5%) defoliation, no feeding or excrement in the whorl (“no whorl damage”, NWD); 2 = light (5-10% defoliation), NWD; 3 = moderate defoliation (up to 20%), NWD; 4 = light to moderate defoliation (up to 20%) and light whorl damage (WD); 5 = moderate to heavy defoliation (20-40%) and moderate WD; 6 = heavy (>40%) defoliation and severe WD; and 7 = only midribs of leaves left, whorl destroyed. Five (1994) or 10 (1995) plants in each plot were examined for dead or live larvae and their size was classified as 1 (instar 1 or 2); 2 (instar 3 or 4), or 3 (instar 5 or 6). One month after the final rating, the remaining plants within each plot and in the cages (1994) were harvested and weighed. Samples of the plants were then dried for 10 days at 88°C to obtain biomass estimates of the plants, and the weight data were converted to dry weight. In 1994 a study was conducted to determine the toxicity and residual effects of the spray treatments on FAW larvae. Samples were taken from the midsection of leaves in each plot about 1 h after the first spray application, as well as 1, 2, 3, 5, and 7 d post spraying. Leaves (each treatment replicated 8 times) were cut into 5 cm sections and placed in petri dishes containing moistened filter paper. Each leaf sample was infested with 5 neonate larvae and the plates were sealed with parafilm and placed in an incubator (31°C) for 24 h. The plates were then unsealed and larval mortality was determined and defoliation rated on an ascending scale of 1 = no feeding to 6 = heavy feeding. Surviving larvae were then placed in individual diet cups containing pinto bean diet and their status was visually monitored for 7 d. Final mortality counts were made, larval size measured under a dissecting microscope, and the larvae dried for 8 d at 88°C and weighed. The data were analyzed using analysis of variance and Dun- can’s new multiple range analysis procedures on SAS microcomputer based statistical analysis software (SAS 1985).

RESULTS AND DISCUSSION

Natural infestations were higher in 1994 than 1995, but untreated check plants suffered serious damage (damage rating of 6.3 in natural infestations and 6.7 in cages in 1994 as compared with 4.1 natural infestation in 1995) both years. When plants

314 Florida Entomologist 79(3) September, 1996

TABLE 1. DAMAGE RATINGS OF FAW INFESTATIONS IN WHORL STAGE FIELD CORN (1994) AND SWEETCORN (1995) TREATED WITH RECOMBINANT DNA BT FOR- MULATIONS AND SELECTED CONVENTIONAL INSECTICIDE STANDARDS.

% Control2 No. Live Larvae/Plant2

Cage Cage Fields Plants Plants Field Plants Plants Treatment and Rate1 18 d 28 d 21 d 18 d 28 d 21 d

1994 ECX9426 OF 1.2 32 a 36 a 52.5 a 3.0 ab 1.7 a 6.0 a ECX9426 OF 2.4 64 b 44 a 79.1 b 2.3 ab 1.0 b 2.0 ab ECX9426 G 5.6 32 a 28 a 50.0 a 2.5 ab 1.6 ab 4.8 ab ECX9426 G 11.3 68 b 72 b 79.9 b 1.6 bc 0.5 bc 2.0 ab Javelin® WG 1.1 40 a 28 a 31.3 a 3.5 a 1.0 bc 4.0 ab Methomyl 0.5 80 c 64 b 86.1 b 0.5 c 0.5 c 1.8 b

1995

ECX9526 WG 1.1 60 a 33 b — 0.3 a 0.45 a — ECX9526 WG 2.4 67 a 52 a — 0.3 a 0.53 a — ECX9526 G 5.6 20 b 3 b — 0.3 a 0.70 a — ECX9526 G 11.3 20 b 13 b — 0.2 a 0.55 a — Methomyl 0.5 72 a 69 a — 0.1 b 0.38 a —

1Rates of ECX9426 (OF) are presented as volume (liters) of formulated product per ha, ECX9526 (WG), Jav- elin® (WG), ECX9426 (G) and ECX9526 (G) as weight (kg) of formulated product per ha and rate of methomyl as weight (kg) of active ingredient per ha. 2Means followed by the same letter within a column and year are not significantly different in Duncan’s new multiple range test (P < 0.05). Percent control values for treatment are based on damage ratings compared with untreated checks. were examined for dead and live larvae, insects of all sizes were found in all of the treatments. Table 1 shows that in 1994 the higher rate of both the ECX9426 (OF) and (G) formulations gave superior percent control [1 - (treatment damage rating/check rating) × 100] as compared to Javelin® (WG) at a rate of 1.1 kg/ha. However, only the (G) formulation was statistically similar to methomyl at 0.5 kg active ingredient per ha in the 28 d sample. The high 1994 natural infestation rate resulted in levels of plant damage similar to the injury that occurred in the infestations in cages. Relative efficacy of the various treatments in the cages was similar to their performance on the non-caged plants in plots subjected to natural infestations. These results indicate that use of artificial infestations in A-frame cages could be a useful method to simulate high natural infestations in situations where indigenous FAW populations are low. In 1995 the percent FAW control at the 2.4 kg rate of ECX9526 (WG) was similar to that provided by methomyl, but efficacy of ECX9526 (G) was reduced substantially compared with a similar formulation used in 1994 (Table 1). Overall, weather was drier in 1995 during the test period, and this may have negatively influenced FAW control by the (G) formulation by reducing dispersion of the bacteria within the whorls.

Armyworm Symposium ’96: All et al. 315

In the 1994 leaf sample assay of the two ECX9426 (OF) rates, Javelin® (WG), and methomyl, all of the treatments were highly toxic to larvae exposed to leaves taken one hour following spraying (Table 2). The granular formulation of ECX9426 was not tested because of poor leaf coverage. Toxicity of all of the Bt products dropped dramatically on the leaves collected 24 h after spraying, demonstrating a rapid loss in residual potency under field conditions. FAW mortality on Bt sprayed leaves was reduced even further 2 days after spraying and was less than 8% (statistically similar to the check leaves) on days 3, 5, and 7 after spraying (for this reason only data up to day 3 are presented in Table 2). Methomyl residues on sprayed leaves were highly toxic to FAW larvae up to the 2 day sample, but substantially less mortality occurred on leaves collected on day 3. Toxicity of methomyl residues was less than 5% on days 5 and 7 and was not significantly different from mortality on check leaves after day 3. The growth of larvae surviving from exposure on treated leaves and then placed on diet for 7 d was determined as an indicator of physiological inhibition resulting from insecticide toxicity. Weight and length measurements followed similar trends, therefore only size measurements are presented in Table 3. Survivors from all of the insecticide leaf samples taken on the same day as spraying were significantly smaller than the larvae exposed on check leaves after 7 d on diet. The size of survivors was reduced on the high, but not the low, rate of ECX9426 (OF) on 24 and 48 h leaf samples; whereas the size of survivors from the Javelin® treatment was not reduced in the 24 h leaf sample or thereafter. Reduced FAW growth by survivors on methomyl treated leaves occurred for up to 3 d after spraying, but not from leaves collected at 5 and 7 d after spraying. These results indicate that residues of ECX9426 (OF) sprayed on corn leaves at a rate of 2.4 kg/ha reduce growth of FAW survivors and are significantly more effective than Javelin® (WG) at 1.1 kg/ha. However, the data also demonstrate the weak residual activity (less than 2 d) of Bt products on corn leaves for FAW.

TABLE 2. FAW LARVAL MORTALITY IN 1994 FOLLOWING FEEDING ON CORN LEAF SAM- PLES COLLECTED AT SELECTED INTERVALS AFTER TREATMENT WITH BT-BASED AND CONVENTIONAL INSECTICIDE PRODUCTS.

% Mortality1 2 Period Treatment and Rate Between Spray and Observation ECX9426 (OF) Javelin® Methomyl Collection Time 1.2 2.4 1.1 0.5 Check

1 h 24 h 47.5 c 55.0 c 72.5 b 97.5 a 0.0 d 7 d 65.0 c 90.0 ab 82.5 b 100.0 a 0.0 d 1 d 24 h 25.0 b 22.5 b 2.5 c 90.0 a 0.0 c 7 d 35.0 c 65.0 b 5.0 d 95.0 a 7.5 d 2 d 24 h 12.5 bc 22.5 b 7.5 c 97.5 a 0.0 c 7 d 15.0 bc 30.0 b 15.0 bc 97.5 a 0.0 c 3 d 24 h 0.0 b 2.5 b 5.0 ab 17.5 a 2.5 b 7 d 2.5 b 7.5 ab 7.5 ab 20.0 a 2.5 b

1Means followed by the same letter across a row are not signiﬁcantly different in Duncan’s new multiple range test (P < 0.05). 2Rates of ECX9426 (OF) are presented as volume (liters) of formulated product per ha, Javelin® (WG) as weight (kg) of formulated product per ha and methomyl as weight (kg) of active ingredient per ha.

316 Florida Entomologist 79(3) September, 1996

TABLE 3. GROWTH (AFTER 7 DAYS ON DIET) OF FAW LARVAE SURVIVORS FROM CORN LEAF SAMPLES COLLECTED AT SELECTED INTERVALS FOLLOWING SPRAYING WITH BT-BASED AND CONVENTIONAL INSECTICIDE PRODUCTS.

Mean Size (cm) of Survivors of 7 D1 Treatment and Rate2

Leaf Collection ECX9426 (OF) Javelin® Methomyl Following Spray 1.1 2.4 1.1 0.5 Check

1 h 1.65 b 1.20 bc 1.33 c 1.00 d 3.00 a 1 d 2.25 ab 1.88 bc 2.95 a 1.10 d 2.83 a 2 d 2.70 ab 2.38 b 2.70 ab 1.05 c 3.00 a 3 d 2.90 ab 2.83 ab 2.78 ab 2.60 b 2.95 a 5 d 2.93 ab 3.00 a 2.93 ab 2.78 b 2.95 ab 7 d 2.75 a 2.75 a 2.90 a 2.73 a 2.95 a

Silage (dry wt) yield in the treatments paralleled efficacy trends. In 1994, plant production in check and Javelin® plots was 5229 and 6250 kg/ha, respectively, and these were significantly less than yields in the methomyl plots (9408 kg/ha). Yield of the ECX9426 (OF) treatments was 6232 and 6698 for the 1.2 and 2.4 kg/ha rates, respectively. In comparison, the ECX9426 (G) treatments yielded 7619 and 6904 kg/ha, respectively, at the 5.6 and 11.3 kg/ha rates, but these were not statistically different from the other treatments. Yield in 1995 was 6368 kg/ha in check plots, which was significantly less than all of the treatments except the 2.4 kg/ha rate of ECX9526 (G) which averaged 7375 kg/ha. Methomyl at 0.5 kg active ingredient per ha had highest yield with 15,943 kg/ha yield in dry plant matter followed by ECX9526 (WG), 2.4 kg/ ha (15,765 kg/ha), > ECX9526 (WG) 1.2 kg/ha (11,606 kg/ha), > ECX9526 (G) (11,445 kg/ha), all of which were statistically similar to each other (Duncan’s new multiple range test (P < 0.05)), but significantly higher than the yield in check plots. Overall, the two years of tests with four formulated Bt products (ECX9426 (OF), ECX9526 (WG), ECX9426 (G) and ECX9526 (G)) derived from a recombinant DNA strain EG1999 demonstrated that at the rates tested the materials provided improved control of FAW infestations in corn compared with a formulation of an indigenous Bt strain (Javelin®). Under certain circumstances, the recombinant DNA Bt products produced control of FAW similar to that of a conventional insecticide (methomyl). It appears that the recombinant DNA Bt products have low residual potency which has been a problem with most Bt-based insecticides (Krieg & Langenbruch 1981). These tests verified 1993 research with EG1999 formulations tested in three southeastern states (All et al. 1994). Ecogen Corp. is currently pursuing registration of an EG1999 product under a tentative trade name of Crystar®.

REFERENCES CITED

ALL, J. N., AND G. G. HAMMES. 1977. Application technique for screening granular insecticides in row crops. J. Georgia Entomol. Soc. 12: 90-92. Armyworm Symposium ’96: All et al. 317

ALL, J. N., J. D. STANCIL, T. B. JOHNSON, AND R. GOUGER. 1994. A genetically-modi- ﬁed Bacillus thuringiensis product effective for control of the fall armyworm (Lepidoptera: Noctuidae) on corn. Florida Entomol. 77: 437-440. GARDNER, W. A., AND J. R. FUXA. 1980. Pathogens for the suppression of the fall armyworm. Florida Entomol. 63: 439-447. KRIEG, A., AND G. A LANGENBRUCH. 1981. Susceptibility of arthropod species to Ba- cillus thuringiensis, pp. 837-896 in H. D. Burges [ed.], Microbial control of pests and plant diseases 1970-1980. Academic, New York. SAS INSTITUTE. 1985. SAS Users Guide: Statistics, Version 6 ed. SAS Institute, Cary, NC. TEAGUE, T. G. 1993. Control of fall armyworm in sweet corn with Bacillus thuringiensis, 1991. Insecticide & Acaricide Tests 18: 127-128.

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Armyworm Symposium ’96: Davis et al. 317

INSECT COLONY, PLANTING DATE, AND PLANT GROWTH STAGE EFFECTS ON SCREENING MAIZE FOR LEAF-FEEDING RESISTANCE TO FALL ARMYWORM (LEPIDOPTERA: NOCTUIDAE)

F. M. DAVIS1, B. R. WISEMAN2, W. P. WILLIAMS1, AND N. W. WIDSTROM2 USDA, ARS, Crop Science Research Laboratory Mississippi State, MS 39762

1Crop Science Research Laboratory, USDA, ARS, Mississippi State, MS 39762

2IBPMRL, USDA, ARS, Tifton, GA 31793

ABSTRACT Field experiments were conducted at Mississippi State, MS and Tifton, GA to determine effects of laboratory insect colony, planting date, and plant growth stage on screening maize, Zea mays L., for leaf-feeding resistance to the fall armyworm (FAW), Spodoptera frugiperda (J. E. Smith). The experiments were conducted using a randomized complete block design with treatments in a factorial arrangement with 6 replications. Treatments consisted of 2 insect colonies, an early and a late planting period, 2 plant growth stages, and 4 single cross maize hybrids (2 susceptible and 2 resistant to leaf-feeding by FAW) at each location. Each plant in an experiment was infested with 30 neonate FAW larvae when the plants of the second planting within each planting period reached the V4 (Tifton) or V8 (Mississippi State) stage. Each plant was visually scored for leaf damage 7 and 14 days after infestation. Statistical analyses revealed interactions among factors resulting in inferences having to be made using nonmarginal means. Signiﬁcant differences in rating scores within each factor (insect colony, planting date, and plant growth stage) were found for some comparisons. However, none of these factors appreciably altered our ability to distinguish between resistant and susceptible genotypes which is the objective of screening. Key Words: Spodoptera frugiperda, plant resistance, maize hybrids, screening

RESUMEN Se llevaron a cabo experimentos de campo en el estado de Mississippi y en Tifton, Georgia, para determinar los efectos de una colonia de insectos de laboratorio, fechas

318 Florida Entomologist 79(3) September, 1996 de simbra y estado de crecimiento de las plantas en el tamizaje de plantas de maíz, Zea mays L., resistentes al daño causado por Spodoptera frugiperda (J. E. Smith). Los experimentos fueron conducidos usando un diseño de bloques completamente aleato- rizados con los tratamientos ordenados en un diseño factorial con 6 réplicas. Los tratamientos consistieron en dos colonias de insectos, un periodo de siembra temprano y otro tardío, dos estados de crecimiento, y cuatro cruces sencillos de maíz híbrido (dos susceptibles y dos resistentes al gusano) en cada localidad. Cada planta en cada ex- perimento fue infestada con 30 larvas neonatas de S. frugiperda cuando las plantas de la segunda siembra dentro de cada período de siembra alcanzaron los estados V4 (Tif- ton) or V8 (Mississippi). Cada planta fue visualmente evaluada en cuanto a daño a las hojas a los 7 y 14 días de la infestación. Los análisis estadísticos revelaron interaccio- nes entre los factores, lo que dió como resutado el tener que hacer inferencias mediante el uso de medias no marginales. En algunas comparaciones fueron encontradas diferencias signiﬁcativas en el valor de cada factor (colonia de insectos, fecha de siembra, y estado de crecimiento de las plantas). Sin embargo, ninguno de esos factores permitió distinguir entre los genotipos resistentes y susceptibles, lo cual fue el obje- tivo del tamizaje.

The fall armyworm (FAW), Spodoptera frugiperda (J. E. Smith), is a polyphagous insect that attacks many crops in the Americas (Ashley et al. 1989). In the southeastern United States, the FAW possess a serious threat to maize, Zea mays L., production, especially to late-season plantings (Scott et al. 1977). FAW often attack late- season plantings of maize from seedling to mature stages of growth. Protection of these plantings by multiple applications of insecticides is not practical. Therefore, more economical management tactics must be pursued. The best tactic would be to grow maize that naturally resists the feeding of the pest. Entomologists and plant geneticists (USDA-ARS) located at Mississippi State, MS and Tifton, GA have screened maize for leaf-feeding resistance to the FAW for many years. Field screening techniques have been developed that have resulted in identiﬁ- cation, development, and release of FAW leaf-feeding resistant maize germplasm (Davis et al. 1989, Williams & Davis 1989, and Widstrom et al. 1993). Occasionally, results from our screening experiments have been poorer than expected or desired. When this happens, the research program suffers from loss of researcher(s) time, funds, seed, and progress. The researchers at Tifton have expressed concern over the virulence of their FAW culture since infusion of wild genes is not routinely done. Also, they have experienced poor establishment of FAW on both susceptible and resistant genotypes when the screening experiments were planted late in the growing season. Both research groups have observed that smaller plants are often more heavily damaged than larger plants of the same genotype. In 1993, the researchers at Mississippi State experienced a poor screening year with the FAW. Possible causes were insect colony, limited insect supply, and procedures used to process eggs and larvae for ﬁeld infesting or some combination of these factors. Environment is also a possible factor, but is not under the control of the researcher(s). The present study was conducted to determine what effects insect colony, date of planting (early vs late), and plant growth stage when infested may have on screening maize for FAW leaf-feeding resistance.

MATERIALS AND METHODS

Field experiments were conducted at Mississippi State, MS and Tifton, GA during the 1995 growing season.

Armyworm Symposium ’96: Davis et al. 319

Insect Colony. At both Tifton and Mississippi State, screening for leaf-feeding resistance requires that each plant within the experiments be infested with approximately the same number of neonate FAW to provide a uniform infestation of the pest. Neonates for infesting were obtained from laboratory colonies. Eggs from the Missis- sippi colony were shipped by overnight mail to Tifton when plants there reached the appropriate growth stage and vice versa for Georgia colony eggs to Mississippi. Per- sonnel at each location have developed their own procedures for laboratory rearing of FAW (Burton & Perkins 1989, Davis 1989). Their procedures differ primarily in larval diets, handling of adults and eggs, and genetic maintenance of the laboratory colonies. The Georgia colony has been maintained on a modiﬁed pinto bean diet for about six years in the laboratory without any infusion of wild genes (personal communication: W. D. Perkins), whereas, the Mississippi colony, which is reared on a wheatgerm- casein diet, has an infusion of wild genes almost every year. Planting Dates. Researchers at both locations have utilized early and late plantings for screening. Sometimes inclement weather dictates later plantings and, on occasion early and a late plantings of the same maize genotypes are used so that experiments can be repeated within the same growing season. In our experiments, there were two planting dates within each of the early and late planting periods. This was done so that two whorl stages of growth would be available at the time of infestation. Planting dates were as follows: early - Georgia (Apr. 5 and 19), Mississippi (Apr. 6 and 20), late - Georgia (May 2 and 10), Mississippi (May 3 and 17). Plant Growth Stages. Entomologists and plant geneticists normally select a preferred growth stage to infest when screening for resistance. At Tifton, the researchers have selected the V4 stage, whereas, at Mississippi State the preferred stage is from

V6-8 (based on the system devised by Ritchie & Hanway [1982] for identifying stages of maize development). Sometimes, infestation is not possible at the desired plant stage for various reasons. For example, if insect supplies are inadequate when plants are ready, the plants will have grown to advanced stages before infestation can occur. The infestations for the early and late planting periods were made when the plants of the second planting within each period reached the V8 stage at Mississippi State and the V4 stage at Tifton. Growth stage of the plants of the ﬁrst planting within each planting period was recorded the day of infestation. Maize Hybrids. Two susceptible and two resistant single cross maize hybrids were selected to study the above factors and their effects on identifying different levels of susceptibility. The susceptible hybrids were ‘AB24E × Mp305’ and ‘SC229 × Tx601’, and the resistant hybrids were ‘Mp496 × Mp708’ and ‘Mp704 × Mp707’. Experimental Design. The experiments were conducted using a randomized complete block design with treatments in a factorial arrangement with six replications. Treatments were 2 insect cultures, early and late plantings, 2 plant growth stages, and 4 single cross maize hybrids. The treatments were represented in each replication by a single plot of maize 6.1 m long and 0.9 m apart. Plots were thinned to approximately 20 plants before infestation. Accepted recommended agronomic practices were followed at both locations. Infestation Technique. Each plant was infested with 30 neonate FAW mixed in 20/ 40 size corn cob grits and delivered into each whorl by a hand-held plastic device (Mihm 1983; Davis et al. 1989) when the plants of the second planting of each planting period (early or late) reached the speciﬁed growth stage. Plants of both plantings within each planting period were infested on the same day. The infestation dates were as follows: Mississippi State - early period plantings on May 25 and late period plantings on June 16; Tifton - early period plantings on May 12 and late period plantings on June 6.

320 Florida Entomologist 79(3) September, 1996

Data. Each plant within a plot was visually scored for the degree of leaf-feeding damage on the 7th and 14th day after infestation. The rating systems used were the ones developed by Davis et al. (1992) speciﬁcally for the FAW. Both 7- and 14-day rating scales are based on the type and number of feeding lesions present on the leaves. The scores range from 0 to 9 with 0 indicating no damage and 9 indicating heavy damage. The two rating scales are highly correlated in their ability to separate resistant from susceptible genotypes. The 7-day scale minimizes the effect of leaf damage caused by migrating mid- instar FAW larvae, whereas, the 14-day scale gives the researcher a view of the total feeding damage caused by the larvae and provides an op- portunity to determine if the plants show any late resistance response to the insect that would not be apparent at 7 days after infestation. Statistical Analyses. Plot means were used in the analyses. Experiments were analyzed by location because of the differences in growth stage at the time of infestation. Data from the early and late planting periods were combined by the day (7 or 14) in which leaf-feeding damage scores were taken and analyzed by use of ANOVA (SAS 1989). Means were separated by least signiﬁcant difference test at the 5% level of probability.

RESULTS

Mississippi State

An error was made when infesting the plants of the early planting period that resulted in only 3 of the 6 replications being used in the statistical analyses. Three factor interactions were significant for the analyses of both 7- and 14-day leaf-feeding damage rating scores. Therefore, inferences have been made utilizing nonmarginal means. Insect Colony. Differences between FAW colonies for the degree of leaf-feeding damage were determined by comparing means within planting date periods, within growth stages infested, and within and among hybrids. Differences in 7 days rating scores between colonies occurred only within the susceptible hybrid ‘Ab24E × Mp305’ for the early planting period (Table 1). In this case, the Mississippi colony produced significantly more damage (at both leaf stages) than the Georgia colony. In the late planting period, significant differences in leaf damage scores occurred for 6 of the 8 comparisons between colonies within hybrids and growth stages. Each time, the Mis- sissippi colony produced more damage than the Georgia colony. However, both colonies ranked the hybrids somewhat similarly when comparisons were made between cultures within growth stages and planting dates (Table 1). Similar results were obtained between colonies when 14-day rating scores were analyzed (Table 2). Planting Date. The effect of planting date periods on screening results was determined by comparing means from 7- and 14-day ratings within growth stages at infestation, within insect colonies, and within and among hybrids. When comparisons were made between mean 7-day leaf-feeding damage scores of early and late planting periods (within the V8 stage and within insect colonies and hybrids), only 1 of the 8 comparisons showed a significant difference (Table 1). However, when the same comparisons were made for plants infested in the V10 stage, 5 of the 8 comparisons were significantly different. The rating scores were higher for the early planting period. Comparisons among hybrids (between early and late planting periods, within growth stage and insect colony) revealed that the separation of susceptible from resistant hybrids was basically the same for both planting periods.

Armyworm Symposium ’96: Davis et al. 321 (R) 0.5 0.2 0.5 0.2 10 ± ± ± ± V RESISTANT [ 0.5 0.8 5.0 0.9 4.0 0.3 3.5 1.8 8 ± ± ± ± V PLANTS

MAIZE

0.4 0.3 0.4 6.6 0.4 6.7 4.4 4.2 SD). 10 ± ± ± ± ± WHORL

X OF

, MS ( STAGES 0.4 0.2 5.9 1.0 4.7 0.6 3.7 2.3 8

TATE ± ± ± ± V S TWO

ISSISSIPPI M 0.8 0.2 0.2 7.8 0.5 7.7 6.0 5.1 10 AT ± ± ± ±

V INFESTATION

EACH

AFTER

0.5 0.8 5.5 0.1 5.6 0.5 3.9 2.9 8 ± ± ± ± V DAYS 7 ARMYWORMS

FALL

TAKEN

Insect Colony Insect Colony 0.4 0.9 0.5 7.2 0.5 7.7 5.1 4.4 10 ± ± ± ± V Early Planting Period Late Planting Period SCORES

NEONATE Growth Stage at Infestation Growth Stage at Infestation MS GA MS GA 30 RATING 0.4 0.4 7.1 0.1 6.2 0.1 4.5 2.9 8

± ± ± ± WITH V (S)] 1. For comparisons among means within early planting = 0.8 For 1. comparisons among means within late planting = 0.6 For 2. comparisons among means between plantings = 0.7 For 3. DAMAGE

FEEDING - SUSCEPTIBLE

EAF AND Mp708 (R) Mp707 (R) 5.9 4.6 Mp305 (S) 8.2 Tx601 (S) 8.0 × × × × 1. L LSD (0.05) values: ABLE SC229 Mp496 Mp704 Maize Hybrid Ab24E T

322 Florida Entomologist 79(3) September, 1996 (R) 0.7 0.6 1.1 0.5 10 ± ± ± ± V RESISTANT [ 0.7 0.4 6.5 1.1 5.2 0.4 4.5 4.6 8 ± ± ± ± V PLANTS

MAIZE

0.6 0.6 0.7 8.1 0.6 8.1 6.2 5.5 SD). 10 ± ± ± ± ± WHORL

V X OF

, MS ( STAGES

0.3 0.4 6.2 0.6 5.5 4.0 0.2 7.3 8 TATE ± ± ± ± V S TWO

ISSISSIPPI M 0.8 0.3 0.8 8.8 7.5 6.6 0.3 8.9 10 AT ± ± ± ±

V INFESTATION

EACH

AFTER

0.2 0.3 8.0 0.8 5.8 4.8 0.9 7.2 8 ± ± ± ± V DAYS ARMYWORMS 14

FALL

TAKEN

Insect Colony Insect Colony 0.4 0.6 0.6 7.8 6.9 5.3 0.2 7.2 10 ± ± ± ± V Early Planting Period Late Planting Period SCORES

NEONATE Growth Stage at Infestation Growth Stage at Infestation MS GA MS GA 30 RATING 0.1 0.1 7.8 0.8 6.5 5.5 0.1 8.0 8

± ± ± ± WITH V (S)] 1. For comparisons among means within early planting = 1.0 For 1. comparisons among means within late planting = 0.7 For 2. comparisons among means between early and late plantings = 0.9 For 3. DAMAGE

FEEDING - SUSCEPTIBLE

EAF AND Mp708 (R) Mp707 (R) 7.0 5.9 Mp305 (S) 8.2 Tx601 (S) 7.9 × × × × 2. L LSD (0.05) values: ABLE SC229 Mp496 Mp704 Maize Hybrid Ab24E T Armyworm Symposium ’96: Davis et al. 323

The results of the 14 day ratings were similar to those obtained for the 7-day ratings (Table 2). Signiﬁcant differences between early and late planting periods (within hybrids, insect colonies, and growth stages) occurred only when the plants were in the

V10 leaf stage at infestation. Again, plants of the early planting period suffered more damage than those of the late planting period. Plant Growth Stage. The effect of plant growth stage on screening results (damage rating scores) was determined by comparing means from 7- and 14-day ratings within planting date periods, within insect colonies, and within and among hybrids. Regard- less of planting date period or insect colony, all comparisons between growth stages within hybrids were signiﬁcantly different for the 7 day ratings (Table 1). In each

case, the V10 plants sustained less damage than the V8 plants. Comparisons among hybrids within growth stage, planting date period, and insect colony revealed that resistant hybrids were separated from susceptible hybrids regardless of growth stage at infestation. When the same comparisons were made for 14-day ratings (Table 2), only 1 of 8 comparisons were signiﬁcant during the early planting period, whereas all comparisons in the late planting period showed signiﬁcant differences between growth stages.

Again, the V10 plants suffered less damage than the V8 plants. In general, the susceptible and resistant hybrids separated out as expected when comparisons were made between growth stages within insect colony and planting date period. However, the separation, especially of ‘SC229 × Tx601’ and ‘Mp496 × Mp708’, was not as clearcut as occurred for 7-day ratings.

Tifton Interactions among factors were also significant at Tifton. There was a significant planting date period × insect colony interaction for the 7-day rating scores and a significant planting date period × hybrid × insect colony interaction for the 14-day rating scores. Since these interactions were encountered, our inferences were made using nonmarginal means. The same comparisons as described for interpreting the Missis- sippi State data were used to study the effects that insect colony, planting date period, and plant growth stage may have on screening results. Insect Colony. Only 3 of 16 comparisons between insect colonies (within planting date period and plant growth stage) were significant when the 7-day rating data were analyzed (Table 3). These significant comparisons were observed only in the early planting period and involved only the resistant hybrids. In each case, the Mississippi colony caused more damage than the Georgia colony. The hybrids were separated as to level of susceptibility similarly by either insect colony. Results similar to those found for 7-day ratings occurred when the 14-day ratings were analyzed (Table 4). Significant differences between cultures occurred only in the early planting period and within the resistant hybrids. Again, the Mississippi colony caused more damage than the Georgia colony. No differences were observed between insect colonies for separation of susceptible from resistant hybrids in the late planting period. However, a few differences between insect colonies did occur in the early planting period. For example, when plants were

infested in the V4 stage, there were no signiﬁcant differences in rating scores among the hybrids infested with the Mississippi colony, whereas hybrids infested at the same growth stage with the Georgia colony separated out according to the expected resistant and susceptible categories. Planting Date. Only 2 of 16 comparisons within insect colonies, plant growth stages, and hybrids showed signiﬁcant differences between planting date periods when 7-day ratings were analyzed (Table 3). Both occurred within the resistant hy- 324 Florida Entomologist 79(3) September, 1996 (R) 2.4 1.4 1.0 0.8 8 ± ± ± ± V RESISTANT [ 0.8 0.6 6.0 0.8 6.5 0.7 3.7 1.7 4 ± ± ± ± V PLANTS

MAIZE

2.1 1.9 0.8 8.2 1.4 8.0 7.2 5.0 8 ± ± ± ± WHORL V

SD). ± STAGES 0.6 0.8 6.3 1.0 2.8 2.3 0.9 7.0

± ± ± ± X V TWO

, GA ( OF

IFTON T 0.6 2.5 0.8 8.0 7.8 5.8 1.1 8.0 8 AT ± ± ± ±

V INFESTATION

EACH

AFTER

0.5 0.5 6.0 1.7 3.7 1.7 0.4 6.0 4 ± ± ± ± V DAYS 7 ARMYWORMS

FALL

TAKEN

Insect Colony Insect Colony 0.6 2.1 0.9 8.7 8.5 6.0 0.6 8.8 8 ± ± ± ± V Early Planting Period Late Planting Period SCORES

NEONATE Growth Stage at Infestation Growth Stage at Infestation MS GA MS GA 30 RATING 0.0 0.4 7.0 1.0 6.3 3.0 0.0 7.0 4

± ± ± ± WITH V (S)] DAMAGE

FEEDING - SUSCEPTIBLE

EAF AND Mp708 (R) Mp707 (R) 8.8 7.5 Mp305 (S) 9.0 Tx601 (S) 9.0 × × × × 3. L LSD (0.05) value = 1.3 ABLE SC229 Mp496 Mp704 Maize Hybrid Ab24E T Armyworm Symposium ’96: Davis et al. 325 (R) 0.8 0.4 1.3 1.3 8 ± ± ± ± V RESISTANT [ 0.0 0.0 8.3 1.3 8.8 1.1 7.0 3.8 4 ± ± ± ± V PLANTS

MAIZE

0.4 0.0 1.0 9.0 1.5 9.0 7.0 6.4 8 ± ± ± ± WHORL V

SD). STAGES ±

0.4 0.8 9.0 0.6 6.3 3.8 0.0 8.8

4 ± ± ± ± X V TWO

, GA ( OF

IFTON T 0.0 1.1 0.8 8.8 7.2 7.0 0.0 9.0 8 AT ± ± ± ±

V INFESTATION

EACH

AFTER

0.0 1.7 9.0 1.5 7.5 5.7 0.0 9.0 4 ± ± ± ± V DAYS ARMYWORMS 14

FALL

TAKEN

Insect Colony Insect Colony 0.0 0.5 0.8 9.0 6.8 7.0 0.0 9.0 8 ± ± ± ± V Early Planting Period Late Planting Period SCORES

NEONATE Growth Stage at Infestation Growth Stage at Infestation MS GA MS GA 30 RATING 0.0 0.4 9.0 0.8 8.7 6.5 0.0 9.0 4

± ± ± ± WITH V (S)] DAMAGE

FEEDING - SUSCEPTIBLE

EAF AND Mp708 (R) Mp707 (R) 8.2 8.3 Mp305 (S) 9.0 Tx601 (S) 9.0 × × × × 4. L LSD (0.05) value = 0.9 ABLE SC229 Mp496 Mp704 Maize Hybrid Ab24E T 326 Florida Entomologist 79(3) September, 1996 brids with lower rating scores being given to the plants of the late planting. Generally, the 7-day ratings from early and late planting periods resulted in no differences being observed as to separation of hybrids into resistant and susceptible categories. Four of the 16 comparisons between planting date periods were signiﬁcant when 14-day ratings were analyzed (Table 4). The differences all occurred within the resistant hybrids. As with the 7-day ratings, lower rating scores were attained in the late planting period plants. Planting date did not appreciably affect the separation of hybrids into susceptible and resistant categories (Table 4).

Plant Growth Stage. When comparisons were made between V4 and V8 growth stages within insect colonies, planting date periods, and hybrids (7-day ratings), all but one of the comparisons were significant (Table 3). The V4 stage plants at infestation received higher rating scores than those infested at the V8 stage. There were some differences that occurred at the 7-day rating as to separation of hybrids into resistant and susceptible categories. These differences involved the resistant hybrid ‘Mp496 × Mp708’ when infested primarily at the V4 stage. In these cases, no significant differences in rating scores were detected between this hybrid and the two susceptible hybrids. Fewer differences between growth stages were detected when the 14-day ratings were analyzed (Table 4). The only significant differences were found within the resistant hybrid ‘Mp704 × Mp707’. This difference was significant regardless of planting date or insect colony. In each case, the V8 stage plants sustained less feeding damage. The separation of resistant from susceptible hybrids was not as clearcut when plants were infested at the V4 stage than when infested at the V8 stage.

DISCUSSION

Concern over the effect that continuous laboratory rearing of an insect on an artificial diet can have on field screening for resistance has been expressed by many plant resistance researchers. Guthrie et al. (1974) found that European corn borer, Ostrinia nubilalis (Hübner), laboratory colonies of up to one year in age (approximately 12 generations) could be used to successfully screen maize for resistance to this pest. How- ever, European corn borer colonies maintained in the laboratory for longer periods of time were observed to begin losing their virulence. By the 46th laboratory generation, the borer larvae were found to cause little damage to field grown maize plants and were deemed to be no longer usable for screening purposes. Quisenberry & Whitford (1988) observed differences in damage to bermudagrass cultivars depending upon the strain of FAW (bermudagrass/rice strain vs the maize strain) and the diet used to maintain the laboratory colonies. They suggested that FAW strain and laboratory diet be considerations when screening for bermudagrass resistance. In our study, some significant differences in damage rating scores within hybrids were detected between FAW colonies. When this occurred, the Mississippi colony (with routine infusion of wild genes) was always found to cause more leaf damage than the Georgia colony. Both laboratory colonies were capable of causing sufficient damage so that resistant genotypes could be separated from susceptible genotypes. Our results did show that a FAW colony can be maintained in the laboratory on an artificial diet for at least 6 years (approximately 72 generations) without losing its ability to cause sufficient leaf damage to separate maize genotypes appropriately as to susceptibility level. However, the Georgia colony appears to have lost some of its virulence when compared to the Mississippi colony. Also, our results showed that planting date (early vs late) was not a factor at either location in affecting the successful separation of resistant from susceptible genotypes. Armyworm Symposium ’96: Davis et al. 327

Therefore, a researcher could expect similar results if a screening experiment was repeated in the same year. Williams et al. (1983) suggested that resistance mechanisms operating in FAW resistant maize plants may have been more strongly expressed at the 10-leaf stage than the 5-leaf stage. Videla et al. (1992) reported that FAW resistant maize was resistant to this pest throughout the vegetative growth stages based on larval growth and survival. However, no consistent differences between larval growth or survival were found among plant growth stages within the test single cross maize hybrids. Wiseman & Isenhour (1993) observed that commercial maize hybrids in the 12-leaf stage, when infested with neonate FAW, tolerated damage by this pest better than plants at the 8- leaf stage at infestation. Our results, especially the 7-day ratings at both locations (Tables 1 and 3), show that FAW feeding damage occurred less on the older than on the younger plants for both resistant and susceptible hybrids. In general, this difference in growth stage susceptibility did not alter the separation of resistant from susceptible hybrids. However, the best separations among hybrids seemed to occur when the plants were V8 stage when infested. Researchers should be aware that rating scores within experiments could reﬂect differences in plant stage because of dissimilar growth rates among genotypes. Also, plants of different sizes within a plot caused by variation in germination can result in a somewhat inaccurate rating score. When thinning or infesting plants within a plot, plants not in the appropriate stage should be eliminated. These data on differences in rating scores between plant growth stages conﬁrm the suggestion by Williams et al. (1983) concerning the increased expression of resistant mechanisms in

V8 and older stage plants. Future research on elucidating the factors responsible for this resistance should consider comparing magnitude of factor expression in plants of different growth stages. In conclusion, signiﬁcant differences in rating scores within each factor (insect colony, planting date period, and plant growth stage) were found for some comparisons. However, none of these factors (at least in this study) were found to appreciably alter the separation of resistant from susceptible genotypes, which is the objective of screening. When a screening failure occurs, it is important to determine, if possible, the factor or factors responsible. Under other circumstances, the three factors studied here may play a role. Since these data do not present a clear picture on the effect of insect colo- nization on the ability of resulting FAW larvae to damage maize plants, we suggest a regular infusion of wild genes into the laboratory colonies. Other factors should also be investigated, such as breakdowns in the proper procedures for handling eggs and neonate larvae for ﬁeld infesting. We consider one of the best ways to avoid screening failures is to have an ample supply of healthy, highly virulent larvae at the time the plants reach the desired growth stage. Further, it is recommended that plants be infested at or near the V8 stage for the best separation of test genotypes for leaf-feeding damage.

ACKNOWLEDGMENTS

We thank Johnny Skinner, Charles Mullis, and Mitchell Cook of the Insect Biology and Population Management Research Laboratory and Thomas Oswalt and Susan Wolf of the Crop Science Research Laboratory for their technical support in this study. Also, appreciation is extended to our secretary, Edna Carraway, for preparation of this manuscript. This article is a contribution of the Crop Science Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture in cooperation with 328 Florida Entomologist 79(3) September, 1996 the Mississippi Agricultural and Forestry Experiment Station. It is published with approval of both agencies as Journal No. 8914 of the Mississippi Agricultural and For- estry Experiment Station.

REFERENCES CITED

ASHLEY, T. R., B. R. WISEMAN, F. M. DAVIS, AND K. L. ANDREWS. 1989. The fall armyworm: A bibliography. Florida Entomologist 72: 150-202. BURTON, R. L., AND W. D. PERKINS. 1989. Rearing the corn earworm and fall armyworm for maize resistance studies, p. 37-45 in Toward insect resistant maize for the third world: Proceedings of the international symposium on methodologies for developing host plant resistance to maize insects. Mexico, D.F.: CIMMYT. DAVIS, F. M. 1989. Rearing the southwestern corn borer and fall armyworm at Mis- sissippi State, p. 27-36 in Toward insect resistant maize for the third world: Proceedings of the international symposium on methodologies for developing host plant resistance to maize insects. Mexico, D.F.: CIMMYT. DAVIS, F. M., W. P. WILLIAMS, AND B. R. WISEMAN. 1989. Methods used to screen maize for and to determine mechanisms of resistance to the southwestern corn borer and fall armyworm, p. 101-108 in Toward insect resistant maize for the third world: Proceedings of the international symposium on methodologies for developing host plant resistance to maize insects. Mexico, D.F.: CIMMYT. DAVIS, F. M., S. S. NG, AND W. P. WILLIAMS. 1992. Visual rating scales for screening whorl-stage corn for resistance to fall armyworm. Mississippi Agric. and For- estry Exp. Stn. Tech. Bull. 186. 9 pp. GUTHRIE, W. D., Y. S. RATHONE, D. F. COX, AND G. L. REED. 1974. European corn borer: Virulence on corn plants of larvae reared for different generations on a meridic diet. J. Econ. Entomol. 67: 605-606. MIHM, J. A. 1983. Techniques for efﬁcient mass rearing and infestation of fall armyworm, Spodoptera frugiperda (J. E. Smith), for host plant resistance studies. CIMMYT, Mexico. QUISENBERRY, S. S., AND F. WHITFORD. 1988. Evaluation of bermudagrass resistance to fall armyworm (Lepidoptera: Noctuidae) inﬂuence of host strain and dietary conditioning. J. Econ. Entomol. 81: 1463-1468. RITCHIE, S. L., AND J. J. HANWAY. 1982. How a corn plant develops. Iowa State Uni- versity Crop Ext. Serv. Spec. Rpt. 48. 21 pp. SAS INSTITUTE. 1989. SAS/STAT users guide version 6, fourth edition, Vol. 1. SAS In- stitute, Cary, N.C. SCOTT, G. E., F. M. DAVIS, G. L. BELAND, W. P. WILLIAMS, AND S. B. KING. 1977. Host- plant resistance is necessary for late-planted corn. Mississippi Agric. and For- estry Exp. Stn. Res. Rpt. 13. 4 pp. VIDELA, G. L., F. M. DAVIS, W. P. WILLIAMS, AND S. S. NG. 1992. Fall armyworm (Lep- idoptera: Noctuidae) larval growth and survivorship on susceptible and resistant corn at different vegetative growth stages. J. Econ. Entomol. 85: 2486- 2491. WIDSTROM, N. W., W. P. WILLIAMS, B. R. WISEMAN, AND F. M. DAVIS. 1993. Registra- tion of GT- FAWCC(5) maize germplasm. Crop Sci. 33: 1422. WILLIAMS, W. P., AND F. M. DAVIS. 1989. Breeding for resistance in maize to southwestern corn borer and fall armyworm, p. 207-210 in Toward insect resistant maize for the third world: Proceedings of the international symposium on methodologies for developing host plant resistance to maize insects. Mexico, D.F.: CIMMYT. WILLIAMS, W. P., F. M. DAVIS, AND B. R. WISEMAN. 1983. Fall armyworm resistance in corn and its suppression of larval survival and growth. Agron. J. 75: 831-832. WISEMAN, B. R., AND D. J. ISENHOUR. 1993. Response of four commercial corn hybrids to infestations of fall armyworm and corn earworm (Lepidoptera: Noctuidae). Florida Entomologist 76: 283-292.

Armyworm Symposium ’96: Wiseman et al. 329

RESISTANCE OF A MAIZE POPULATION, FAWCC(C5), TO FALL ARMYWORM LARVAE (LEPIDOPTERA: NOCTUIDAE)

B. R. WISEMAN, F. M. DAVIS1, W. P. WILLIAMS1 AND N. W. WIDSTROM USDA, ARS, IBPMRL Tifton, GA 31793

1USDA, ARS, Plant Science Research Laboratory, Mississippi State, MS. 39762

ABSTRACT

Field tests at Mississippi State, MS, and Tifton, GA, were conducted to evaluate the effect of resistance of a maize, Zea mays (L.), germplasm population, ‘GT- FAWCC(C5)’, to feeding by larvae of the fall armyworm, Spodoptera frugiperda (J. E. Smith). Plants of selected maize entries were infested at the 8 and 12 leaf stage with two applications of 15 larvae per plant. Resistance traits measured were leaf damage at 7 and 14 days after infestation and number and weight of surviving larvae per plant at 7 and 10 days after infestation. Leaf damage ratings at both 7 and 14 days after infestation and the number and weight of surviving larvae per plant on GT- FAWCC(5) at 7 and 10 days after infestation on GT-FAWCC(C5) equalled the number and weight of surviving larvae on ‘MpSWCB-4’, the resistant check. Both the resistant check and GT-FAWCC(C5) were signiﬁcantly more resistant to whorl damage than the susceptible check, ‘Ab24E × SC229’, for all resistance traits. It is evident that antibiosis (low weight) and nonpreference (fewer larvae per plant and fewer larvae preferring leaf samples) mechanisms of resistance are present in the GT-FAWCC(C5) population as well as for MpSWCB-4.

Key Words: Leaf-feeding resistance, antibiosis, nonpreference, artiﬁcial infestations

RESUMEN

Fueron realizados estudios de campo en el estado de Mississippi y en Tifton, Geor- gia, para evaluar la resistencia de una población de germoplasma de maíz, Zea mays “GT-FAWCC(C5)” al daño producido por larvas de Spodoptera frugiperda (J. E. Smith). Plantas de entradas seleccionadas de maíz fueron infestadas en el estado de 8-12 hojas con dos aplicaciones de 15 larvas por planta. Las variables de resistencia medidas fueron el daño a las hojas y el número y peso de las larvas sobrevivientes por planta a los 7 y 14 días después de la infestación. El daño foliar a los 7 y 14 días des- pués de la infestación y el número y peso de las larvas sobrevivientes por planta en GT-FAWCC(C5) a los 7 y 10 días después de la infestación igualaron al número y peso de las larvas sobrevivientes en “MpSWCB-4”, el testigo resistente. Tanto el testigo resistente como el GTT-FAWCC(C5) fueron signiﬁcativamente más resistentes al daño del cogollo que el testigo susceptible, “Ab24E × SC229”. Es evidente que los mecanis- mos de resistencia mediante antibiosis (bajo peso) y no preferencia (pocas larvas por planta y pocas larvas preﬁriendo muestras de hojas) están presentes en las poblaciones de GT-FAWCC(C5) y MpSWCB-4.

Maize, Zea mays (L.), is a crop upon which fall armyworm (FAW), Spodoptera frugiperda (J. E. Smith), infestations often reach devastating levels in the southeastern United States. In 1975, losses in Georgia were estimated at over 20 million dollars

330 Florida Entomologist 79(3) September, 1996

(Sparks 1979). Yield losses attributed to the fall armyworm for the U. S. have been estimated at 2% annually (Wiseman & Morrison 1981). Wiseman & Isenhour (1993) showed that commercial maize hybrids suffered greater yield losses (32.4%) when manually infested with 2 applications of 20 neonates per plant at the 8-leaf stage than at the 12-leaf stage (15.4%). Maize is the most valuable ﬁeld cereal crop in the U. S. (Anonymous 1995). Al- though the number of acres planted with maize and the value of the crop ﬂuctuate annually, 72.9 million acres were harvested in 1994 in the U. S. with a production of 10.1 billion bushels and a value of 22.2 billion dollars (Anonymous, 1995). Thus, the continued development and release of new maize germplasm with resistance to the FAW is important. Understanding the mechanisms of resistance or the biological effects of resistant cultivars on the FAW is important in managing the insect pest. Therefore, the objective of this study was to determine leaf damage at 7 and 14 days after infestation (DAI) and biological responses of FAW when feeding on a newly released resistant germplasm population, ‘GT-FAWCC(C5)’ (Widstrom et al. 1993).

MATERIALS AND METHODS

Two dent maize entries, (resistant ‘MpSWCB-4’ or susceptible ‘Ab24E × SC229’), were selected for comparison with GT-FAWCC(C5) because of their response to FAW in ﬁeld tests (Scott & Davis 1981; Widstrom et al. 1993). The three entries were seeded in separate experiments on 5 April, 1995 at Tifton, GA and on 17 April, 1995 at Mis- sissippi State, MS. Test plots consisted of 3 rows 6.1 m long and 0.9 m apart. Plants were thinned to about 30 cm apart. Recommended agronomic practices were followed at both locations. A split plot design with 6 replications was used with whole plots being leaf-stage at the time of infestation: 8-leaf stage (V4 at Tifton and V8 at Mississippi State) or 12- leaf stage (V8 at Tifton and V12 at Mississippi State) (Ritchie & Hanway 1982). Sub- plots consisted of maize entries. At Tifton, leaf stage was determined by counting the total number of emerged leaves, and at Mississippi State, leaf stage was determined by counting only leaves with their collars exposed. Whole plots were bordered with 2 rows of a commercial maize hybrid. Each plant within the 8- or 12-leaf stage treatments was infested with 2 applications of 15 neonate FAW on the same day (Wiseman 1989). By infesting with two applications, noninfested plants within the experiments do not exist. From row one and two of each sub-plot, 10 plants were harvested at soil level at both 7 and 10 DAI and taken to the laboratory to determine the number and weight of surviving larvae per plant. Plants on the third row of each subplot were rated at 7 and 14 DAI using a visual rating scale of 0-9 (Davis 1992) where 0 = no damage and 9 = whorl destroyed. FAW larvae used to infest plants were obtained from colonies maintained at the Insect Biology and Population Management Research Laboratory, Tifton, GA and the Crop Science Research Laboratory, Mississippi State, MS, respectively (Perkins 1979; Davis 1989). A laboratory test was designed to determine nonpreference for leaf samples of the three maize entries. Whorl samples were obtained from the 8- leaf stage at Missis- sippi State on May 25, 1995, taken to the laboratory and excised into disks of about 2 cm diam (Davis et al. 1989). The leaf disks of each entry were randomly placed on the outer inside edge of a large dish (15.3 cm diam, 2.7 cm deep). One FAW egg mass in the blackhead stage containing about 50 eggs was placed in the center of each dish and the dish was placed in darkness. The experiment was arranged as a randomized complete block with 22 replications. The experiment was held in a controlled environ-

Armyworm Symposium ’96: Wiseman et al. 331 ment room maintained at 27˚ C and 50-60% RH. Total number of larvae found on each maize entry leaf section (if larvae were not actually on the leaf section at the time of recording, they were not counted) per dish was recorded 24 h after egg hatch. Damage ratings, the number and weight of surviving larvae and number of larvae preferring a leaf sample were analyzed by PROC GLM (SAS Institute 1989). When signiﬁcant differences were indicated, means were separated by least signiﬁcant differences (LSD) at P = 0.05 (SAS Institute 1989).

RESULTS AND DISCUSSION A significant (P ≤ 0.05) leaf-stage × entry interaction was found for the 7-day leaf damage ratings at Mississippi State, but not at Tifton (Table 1). Plants from all three entries at Mississippi State and two (Ab24E × SC229 and MpSWCB-4) at Tifton received more damage at the 8-leaf stage than at the 12-leaf stage. Both, GT- FAWCC(C5) and MpSWCB-4 had significantly (P ≤ 0.05) less damage 7 DAI in the 8- and 12-leaf stages than Ab24E × SC229 at both locations. GT-FAWCC(C5) was significantly less damaged at the 8-leaf stage than MpSWCB-4 at Tifton. A significant (P ≤ 0.05) leaf-stage × entry interaction also was found for leaf damage ratings 14 DAI at Mississippi State, but not at Tifton (Table 1). At Tifton, plants of all entries infested at the 8-leaf stage yielded significantly higher damage ratings (8.2 vs 4.7) than plants of all entries at the 12-leaf stage. Plants from all three entries at both locations received more damage at the 8-leaf stage than at the 12-leaf stage. Both GT-FAWCC(C5) and MpSWCB-4 were damaged significantly (P ≤ 0.05) less 14 DAI in the 8- and 12-leaf stages than Ab24E × SC229 at Mississippi State and Tifton. MpSWCB-4 was damaged significantly less than GT-FAWCC(C5) at Mississippi State at the 8-leaf stage but not at Tifton. A significant (P ≤ 0.05) leaf-stage × entry interaction was found for the number of larvae recovered per plant 7 DAI at each location (Table 2). Significant differences in the number of larvae per plant were found 7 DAI between the 8- and 12-leaf stages for each entry at each location. In each case, plants infested at the 12-leaf stage had fewer larvae 7 DAI than plants infested at the 8-leaf stage of each entry. Both GT- FAWCC(C5) and MpSWCB-4 had significantly fewer larvae per plant than the susceptible check, Ab24E × SC229, at both leaf stages and locations. A significant (P ≤ 0.05) interaction (leaf-stage × entry) was found for number of larvae recovered per plant 10 DAI at Tifton, but not at Mississippi State (Table 2). The mean number of larvae recovered 10 DAI at Mississippi State at the 8-leaf stage was significantly higher (4.7 vs 3.0) than the mean number recovered at the 12-leaf stage. Significant differences between the number of larvae per plant 10 DAI at the 8- and 12-leaf stages for each entry occurred at Mississippi State, but differences between plant growth stages were noted only for GT-FAWCC(C5) at the Tifton location. GT- FAWCC(C5) and MpSWCB-4 had significantly fewer larvae per plant than the susceptible check, Ab24E × SC229, at the 8-and 12-leaf stages at Mississippi State, these differences were noted only for the 12-leaf stage at Tifton. Total weight of larvae per plant recovered 7 DAI from plants of entries infested at the 8-leaf stage at Tifton was significantly (P ≤ 0.05) greater than weight of larvae recovered from plants of entries infested at the 12-leaf stage (Table 3). No differences in weight of larvae were found between the 8- and 12-leaf stages at Mississippi State. Larvae feeding on and collected from 8- or 12-leaf stage plants of GT-FAWCC(C5) and MpSWCB-4 weighed significantly less than larvae feeding on and collected from Ab24E × SC229 at both locations. Total weight of larvae per plant recovered 10 DAI from plants of entries infested at the 8-leaf stage at Mississippi State (164 vs 79) was significantly (P ≤ 0.05) heavier

332 Florida Entomologist 79(3) September, 1996 ARMYWORM

FALL

FIFTEEN

0.05). SEM = standard error of the 0.05). ≤ OF

P APPLICATIONS

1 TWO

WITH

Damage Rating x INFESTATION

AFTER

DAYS 14 7DAI 14 DAI AND 7 AT

MAIZE

FOR

Mississippi State Tifton Mississippi State Tifton 8 Leaf 12 Leaf 8 Leaf 12 Leaf 8 Leaf 12 Leaf 8 Leaf 12 Leaf RATINGS

. PLANT DAMAGE

- PER

LEAF

EAN LARVAE SC229 7.2 a * 6.7 a 8.3 a * 6.0 a 8.6 a * 7.3 a 8.8 a * 7.8 a × 1. M Mean damage ratings within a column not followed by the same letter or horizontal means separated * are significantly different ( 1 ABLE GT-FAWCC(C5)MpSWCB-4SEM 5.0 b * 4.2 b 3.2 b * 3.7 b 3.0 c 0.11 5.5 b 2.7 b 0.11 * 3.0 b 6.4 b 0.56 * 5.1 c 4.2 b * 0.56 4.5 b 7.3 b 0.27 * 7.7 b 3.7 c * 0.27 4.7 b 0.31 0.31 Entry Ab24E least significant mean was based on pooled error mean square in the analysis (SAS Institute 1989). least significant mean was T

Armyworm Symposium ’96: Wiseman et al. 333 FALL

FIFTEEN

0.05). SEM = standard error of the 0.05). ≤

P APPLICATIONS

TWO

1 AFTER

DAYS 10 AND 7 AT

No. Larvae Recovered at No. x ENTRIES

MAIZE

7 DAI 10 DAI PLANT

PER

. RECOVERED

Mississippi State Tifton Mississippi State Tifton PLANT 8 Leaf 12 Leaf 8 Leaf 12 Leaf 8 Leaf 12 Leaf 8 Leaf 12 Leaf

PER

LARVAE

NUMBER

EAN ARMYWORM SC229 11.8 a * 6.4 a 11.8 a * 9.9 a 7.1 a * 4.4 a 4.2 a 4.9 a × 2. M Mean number of larvae within a column not followed by the same letter or horizontal means separated * are significantly different ( 1 ABLE GT-FAWCC(C5)MpSWCB-4SEM 6.0 b * 2.9 c 0.7 b * 1.1 b 6.8 b 0.81 * 6.1 b 1.9 b 0.81 * 2.4 b 4.1 b 0.71 * 4.4 b 2.2 b * 0.71 2.2 b 3.5 a * 0.74 3.9 a 1.9 b 0.74 2.9 b 0.26 0.26 Ab24E least significant mean was based on pooled error mean square in the analysis (SAS Institute 1989). least significant mean was Entry T

334 Florida Entomologist 79(3) September, 1996 AND

PLANT

PER

LARVAE

FIFTEEN

2 APPLICATIONS

TWO

. WITH

0.05). SEM = standard error of the least signiﬁcant mean was based on pooled SEM = standard error of the least signiﬁcant mean was 0.05). ≤

P Weight per Plant Weight x GENOTYPES

INFESTED

MAIZE

LARVAE

7 DAI 10 DAI SUSCEPTIBLE

ARMYWORM

AND

FALL

FOR

RESISTANT

Mississippi State Tifton Mississippi State Tifton PLANT

8 Leaf 12 Leaf 8 Leaf 12 Leaf 8 Leaf 12 Leaf 8 Leaf 12 Leaf ON

PER

1 ) DAYS MG 10 ( AND 7 WEIGHT

FOR

EAN FED SC229 129 a 141 a 158 a 201 a 370 a * 223 a 387 a 420 a × 3. M Total weight of larvae per plant. Total Mean weight of larvae within a column not followed by the same letter are signiﬁcantly different ( 1 2 ABLE GT-FAWCC(C5)MpSWCB-4SEM 19 b 9 b 1 b 7.8 27 b 9 b 39 b 7.8 65 b 16.5 65 b 88 b 56 b 16.5 3 b 25.1 120 b 12 b 203 c 114 b 25.1 166 b 19.7 19.7 Ab24E Entry error mean square in the analysis (SAS Institute 1989). T Armyworm Symposium ’96: Wiseman et al. 335

than weight of larvae recovered from plants of entries infested at the 12-leaf stage, but not at Tifton (Table 3). Weight of larvae per plant was significantly more for larvae feeding on, and collected from, Ab24E × SC229 from the 8-leaf stage at Mississippi State than from the 12-leaf stage. The total weight of larvae per plant or weight per larva from both GT-FAWCC(C5) and MpSWCB-4 was significantly less 10 DAI than the weight of larvae from the susceptible check (Ab24E × SC229) for both the 8- and 12-leaf stages and locations. Results from the laboratory nonpreference study indicated that leaf sections of GT- FAWCC(C5) were less preferred by FAW neonate than those for Ab24E × SC229 [Ab24E × SC229 15.46a larvae per dish; MpSWCB-4 10.09ab larvae per dish and GT- FAWCC(C5) 7.96b larvae per dish; number of larvae followed by the same letter are not significantly different P ≤ 0.05]. MpSWCB-4 and AB24E × SC229 were equally preferred as was MpSWCB-4 and GT-FAWCC(C5). Wiseman et al. (1981) reported that FAW larvae preferred MpSWCB-4 similar to the susceptible check but possessed higher antibiosis than the susceptible check and that ‘Antigua 2D-118’ was more nonpreferred by FAW larvae than MpSWCB-4. This appears to be the case here for GT-FAWCC(C5) except that both MpSWCB-4 and GT-FAWCC(C5) are similar in antibiosis. In summary, though the 8- and 12- leaf stages at Mississippi State and Tifton were measured differently, the leaf damage ratings 7 and 14 DAI, number and weight of larvae per plant at 7 and 10 DAI at both locations for GT-FAWCC(C5) equalled those of MpSWCB-4, the resistant check. MpSWCB-4 and GT-FAWCC(C5) were significantly more resistant than the susceptible check, Ab24E × SC229, when weight per larva was also calculated. It is evident that antibiosis (low total weight of larvae per plant or weight per larva) and nonpreference (less establishment per plant and numbers preferring leaf samples) mechanisms of resistance are present in the GT- FAWCC(C5).

ACKNOWLEDGMENTS

We thank Johnny Skinner, Charles Mullis, and Mitchell Cook of the Insect Biology and Population Management Research Laboratory and Thomas Oswalt and Paul Buckley of the Crop Science Research Laboratory for their technical support in this study.

REFERENCES CITED

ANONYMOUS. 1995. The world of corn: Agriculture’s innovators. National corn growers association. National corn development foundation. 39 p. DAVIS, F. M. 1989. Rearing the southwestern corn borer and fall armyworms at Mis- sissippi State, pp. 27-36 in Toward insect resistant maize for the third world: Proceedings of the international symposium on methodologies for developing host plant resistance to maize insects. Mexico, D. F.: CIMMYT. 327 p. DAVIS, F. M., S. S. NG, AND W. P. WILLIAMS. 1989. Mechanisms of resistance in corn to leaf feeding by Southwestern corn borer and European corn borer (Lepi- doptera: Noctuidae). J. Econ. Entomol. 82: 919-922. DAVIS, F. M., S. S. NG, AND W. P. WILLIAMS. 1992. Visual rating scales for screening whorl-stage corn for resistance to fall armyworm. Mississippi Agricultural & Forestry Experiment Station. Technical Bulletin 186. 9 pp. PERKINS. W. D. 1979. Laboratory rearing of the fall armyworm. Florida Entomol. 62: 87-91. RITCHIE, S. W., AND J. J. HANWAY. 1982. How a corn plant develops. Iowa State Uni- versity Coop. Ext. Ser. Spec. Rpt. 48. 21 pp. 336 Florida Entomologist 79(3) September, 1996

SAS INSTITUTE. 1989. SAS/STAT user’s guide version 6, fourth edition, Vol. 1. SAS In- stitute, Cary, N. C. SCOTT, G. E., AND F. M. DAVIS. 1981. Registration of MpSWCB-4 population of maize. (Reg. No. GP 87). Crop Science 21: 148. SPARKS, A. N. 1979. A review of the biology of the fall armyworm. Florida Entomol. 62: 82-87. WIDSTROM, N. W., W. P. WILLIAMS, B. R. WISEMAN, AND F. M. DAVIS. 1993. Registra- tion of GT-FAWCC(C5) maize germplasm. Crop Science 33: 1422. WISEMAN, B. R. 1989. Technological advances for determining resistance in maize to insects, pp. 94-100 in Toward insect resistant maize for the third world: Pro- ceedings of the international symposium on methodologies for developing host plant resistance to maize insects. Mexico, D. F.: CIMMYT. 327 p. WISEMAN, B. R., AND D. J. ISENHOUR. 1993. Response of four commercial corn hybrids to infestations of fall armyworm and corn earworm (Lepidoptera: Noctuidae). Florida Entomol. 76: 283-292. WISEMAN, B. R., AND W. P. MORRISON. 1981. Components for management of ﬁeld corn and grain sorghum insects and mites in the United States. USDA-ARS ARM-S-18. 18p. WISEMAN, B. R., W. P. WILLIAMS, AND F. M. DAVIS. 1981. Fall armyworm: Resistance mechanisms in selected corns. J. Econ. Entomol. 74: 622-624.

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336 Florida Entomologist 79(3) September, 1996

BEET ARMYWORM (LEPIDOPTERA: NOCTUIDAE) CONTROL ON COTTON IN LOUISIANA

V. J. MASCARENHAS1, B. R. LEONARD2, E. BURRIS3 AND J. B. GRAVES1 Louisiana State University Agricultural Center

1Department of Entomology, Baton Rouge, LA 70803;

2Macon Ridge Location of the Northeast Research Station, Winnsboro, LA 71295; and

3Northeast Research Station, St. Joseph, LA 71366.

ABSTRACT

Efficacy of selected labeled and experimental insecticides against beet armyworm, Spodoptera exigua (Hübner), populations from Louisiana were determined in both a laboratory diet bioassay and in replicated field plots. Significantly higher LC50’s for chlorpyrifos and thiodicarb were observed for one of two field-collected strains relative to a laboratory-reference strain in the laboratory diet bioassays. No significant differences in susceptibility between the reference strain and field-collected strains were observed for chlorfenapyr (proposed common name), spinosad or tebufenozide. For the reference strain, LC50’s (ppm) for tebufenozide, spinosad, chlorfenapyr, chlorpyrifos, and thiodicarb were 2.6, 2.8, 4.8, 4.9, and 319.8, respectively. In two field tests, all three experimental insecticides (chlorfenapyr, spinosad, and tebufenozide) as well as chlorpyrifos significantly reduced the numbers of beet armyworm larvae relative to the untreated control at all sampling periods (3, 5, 7, and 10 days after treatment), except for Test 2 at 3 days after treatment. Thiodicarb provided satisfactory control of

Armyworm Symposium ’96: Mascarenhas et al. 337 larvae in Test 1; however, in Test 2 thiodicarb did not signiﬁcantly reduce the numbers of beet armyworm compared with the untreated control. The microbial insecticide Spod-X provided inadequate larval control in both tests.

Key Words: Beet armyworm, insecticides, insecticide efﬁcacy, insecticide bioassay

RESUMEN

Fue determinada la eficacia de insecticidas registrados y experimentales contra poblaciones del gusano de la remolacha, Spodoptera exigua (Hübner). Los ensayos se realizaron en el laboratorio, con dietas, y en parcelas replicadas en el campo. En los ensayos de laboratorio fueron observadas CL50 significativamente más altas para chlorpyrifos y thiodicarb en una de las dos cepas colectadas en el campo en relación con una cepa de referencia de laboratorio. No fueron halladas diferencias significati- vas entre la cepa de referencia y las colectadas en el campo, en cuanto a la suscepti- bilidad a chlorfenapyr (nombre común propuesto), spinosad o tebufenozide. Para la cepa de referencia, las CL50 para tebufenozide, spinosad, chlorfenapyr, chlorpyrifos y thiodicarb fueron 2.6, 2.8, 4.8, 4.9 y 319.8 ppm, respectivamente. En dos ensayos de campo, los tres insecticidas experimentales (chlorfenapyr, spinosad, y tebufenozide), así como chlorpyrifos, redujeron significativamente los números de larvas del gusano de la remolacha en relación con el testigo sin tratar en todos los muestreos (3, 5, 7 y 10 días después del tratamiento), excepto en el ensayo 2 a los tres días después del tratamiento. Thiodicarb produjo un control satisfactorio de las larvas en el ensayo 1; sin embargo, en el ensayo 2 thiodicarb no redujo significativamente los números del gusano de la remolacha comparados con los del testigo no tratado. El insecticida micro- biano Spod-X produjo un control larval inadecuado en ambas pruebas.

The beet armyworm, Spodoptera exigua (Hübner), has historically been viewed as a secondary pest of cotton in most of the southeastern United States. However, population outbreaks experienced in the 1980’s and early 1990’s in Alabama, Georgia, Lou- isiana, Mississippi (Douce & McPherson 1991, Burris et al. 1994, Layton 1994, Smith 1994), and more recently in Texas (Arrillago 1995) have demonstrated the potential damage associated with this pest and the ineffective control provided by most currently labeled insecticides. During the outbreak of 1993, beet armyworms infested 60% of the total cotton acreage in the mid-south and southeastern states, with approximately 35% of this acreage having infestations above an economic level (Smith 1994). The economic impact of beet armyworm infestations varies from region to region and includes both the direct yield loss caused by insect injury and the high production costs associated with frequent and costly insecticide usage. In some regions of the southeast, such as Alabama and Mississippi, where the beet armyworm outbreak of 1993 was particularly devastating, large cotton acreages were abandoned after the control costs exceeded $250-370 per ha (Layton 1994, Smith 1994). The studies reported here were designed to evaluate the susceptibility of ﬁeld populations of beet armyworm to standard and experimental insecticides in laboratory and ﬁeld experiments.

MATERIALS AND METHODS

Laboratory and ﬁeld experiments were conducted to evaluate the efﬁcacy of standard and experimental insecticides against the beet armyworm. Insecticides tested

338 Florida Entomologist 79(3) September, 1996 included commercial formulations of two currently recommended insecticides, thiodicarb [Larvin® 3.2F (flowable powder), Rhone-Poulenc Ag. Co., Research Triangle Park, North Carolina] and chlorpyrifos [Lorsban® 4EC (emulsifiable concentrate), Dow- Elanco, Indianapolis, Indiana], as well as three experimental compounds, tebufenozide (Confirm® 2F, Rohm & Haas Co., Philadelphia, Pennsylvania), chlorfenapyr (Pirate® 3F, American Cyanamid Co., Wayne, New Jersey) and spinosad (Tracer® 4F, DowElanco, Indianapolis, Indiana). Spod-X® (Crop Genetics Interna- tional, Wilmington, Delaware), a NPV (nuclear polyhedrosis virus) product which is labeled for beet armyworm control in cotton, was included in the field tests.

Diet Bioassay

Susceptibility of a laboratory-reference and two ﬁeld-collected strains of beet armyworms was evaluated using a surface-treated diet bioassay similar to that described by Joyce et al. (1986) and Chandler & Ruberson (1994). Both ﬁeld strains were collected from northeast Louisiana, one near Newellton and the other near St. Joseph.

These strains were bioassayed using individuals from the F2-F4 laboratory-reared generations. Three ml of an artiﬁcial wheat germ/pinto bean diet were pipetted into individual 30 ml diet cups (Schneider Paper Product Inc., New Orleans, Louisiana) and allowed to cool. Serial dilutions were prepared for each insecticide tested, and 100 µl of each concentration (four concentrations and a water control) were individually pipetted onto the surface of the diet and allowed to air dry at room temperature for approximately 1 h. Diet cups were shaken to evenly distribute the insecticide solution over the diet’s surface. One ﬁrst instar (approximately 3-d old) beet armyworm larva was placed on the treated diet and cups were then capped. A minimum of 50 larvae per dose were bioassayed for each insecticide and mortality was observed at 48, 72, 96, and 120 h. Larvae were considered dead if they did not respond to prodding with a paint brush. Bioas- says were conducted under constant light at 22±1°C and 40±5% RH. Data from the diet bioassay were analysed by probit analysis using POLO-PC (LeOra Software

1987). LC50’s of field-collected strains were considered to be significantly different than that of the reference strain if the 95% confidence limits did not overlap. Toxicity ratios (TR) were calculated by dividing the LC50 of a field strain by that of the reference strain. Field strains bioassayed were collected from the same region where field studies were conducted. The reference strain of beet armyworms was obtained from the USDA-ARS, Southern Insect Management Laboratory (SIML) at Stoneville, Missis- sippi.

Field Experiments

Field tests were conducted at the Northeast Research Station (Test 1) near St. Jo- seph, Louisiana and at the Macon Ridge location of the Northeast Research Station (Test 2) near Winnsboro, Louisiana during the summer of 1995. Both tests were arranged in a randomized complete block design with 4 replications. Plots measured four rows (approximately 1 m centers) by 15.25 m. Test 1 was planted to ‘Stoneville LA 887’ cotton on 16 May and Test 2 was planted to ‘DPL 5690’ cotton on 20 June. Larval densities in each block were estimated before insecticide application by taking 6-10 drop cloth (approximately 1 row meter each) samples. Treatments for Test 1 and 2 were applied on 15 and 30 August, respectively, with a high clearance sprayer. In Test 1, the sprayer was calibrated to deliver 93.5 liters total spray volume per ha through Teejet X-12 hollow cone nozzles (2 per row) at 3.6 kg/cm2. In Test 2, the sprayer

Armyworm Symposium ’96: Mascarenhas et al. 339 was calibrated to deliver 105.5 liters total spray volume per ha through Teejet X-8 hollow cone nozzles (2 per row) at 3.1 kg/cm2. Treatment effect was measured by taking 2 drop cloth samples in each plot and counting the number of live larvae. Sampling was done in areas within a row where evidence of a ‘hit’ (recently hatched egg mass) and/or larval feeding was observed. This sampling procedure was adopted because randomly sampling for a clump-distributed pest population would not appropriately reﬂect larval densities in ﬁeld plots. At each sampling period [2, 5, 7, and 10 days after treatment (DAT)], one row of each plot was sampled, so that rows 1, 2, 3, and 4 were sampled at 3, 5, 7, and 10 DAT, respectively. This sampling pattern was used to avoid sampling an individual ‘hit’, which may have been disturbed during an earlier sampling period. Recently deposited egg masses were avoided during the last two sampling dates, because these ‘hits’ represented infestations which would not have received the full treatment effect. With this sampling approach, neonates through second instar larvae were not included in samples taken at 7 and 10 DAT. Total number of live larvae per 0.3 m of row was used in the data analysis. Data were analyzed by ANOVA and means were separated according to Fis- cher’s protected LSD (SAS Institute 1988).

RESULTS

Diet Bioassay

In the thiodicarb bioassays, LC50’s ranged from 320 to 641 ppm (parts per million) and a significantly higher LC50 was obtained with larvae of the Newellton strain compared with the SIML reference strain (Table 1). In the chlorpyrifos bioassays, Newel- lton strain larvae had a significantly higher LC50 than both the St. Joseph and the reference strain. Furthermore, the toxicity ratio for Newellton strain (TR = 7.1) was much higher for chlorpyrifos than for all other insecticides. There were no significant differences in the LC50’s between either of the field-collected strains and the SIML strain for all three experimental insecticides. For the tebufenozide bioassays, LC50’s for all strains evaluated ranged from 2.6 to 5.5 ppm. A similar range in LC50’s was observed for spinosad (2.1-4.8 ppm). Slightly higher LC50’s, which ranged from 4.0 to 6.1 ppm, were obtained in the chlorfenapyr bioassays. Slopes of dosage-mortality lines for chlorfenapyr were steeper compared with slopes for the other chemicals.

Field Experiments

The average number of beet armyworm larvae per 0.3 m of row in Test 1 and 2 prior to application of the various treatments was 5.1 and 12.5, respectively. In Test 1, numbers of beet armyworm larvae were significantly lower than that of the untreated control for all treatments at 3 and 5 DAT, except for Spod-X (Table 2). Similar results were observed at 7 DAT when all treated plots, except for Spod-X and thiodicarb, had fewer live larvae than the untreated control. At the final observation (10 DAT), all treatments had significantly reduced the number of beet armyworm larvae relative to the untreated control. In Test 2, no significant differences among treatments were observed at 3 DAT (Ta- ble 3). At 5 DAT, only the chlorfenapyr and spinosad treatments significantly reduced the number of beet armyworm larvae compared with the untreated control. All treated plots, except for thiodicarb and Spod-X, had significantly fewer larvae than the untreated control at 7 DAT. By 10 DAT, all treatments, except for thiodicarb, had significantly fewer larvae relative to the untreated control (Table 3).

340 Florida Entomologist 79(3) September, 1996 2 TR INSECTICIDES

SELECTED

5.4 1.3 TO

ARMYWORM

4 95% Conf. Limits 95% Conf. BEET

Low High OF

STRAIN

REFERENCE - 50 LABORATORY LC

(ppm) Slope(SE) . A

DIET AND

1 TREATED

COLLECTED TO

- SIML. FIELD 50

values compared with the SIML strain. 50 EXPOSURE TWO

ﬁeld strain/LC 50 DAYS

chlorfenapyr 400 4.9 3.94(0.50) 2.8 6.7 FIVE

USCEPTIBILITY 3 AFTER 1. S N - Indicates number of individual larvae tested. Mississippi. Stoneville, USDA-ARS, SIML - Southern Insect Management Laboratory, TR - Toxicity ratio = LC Toxicity TR - 90% Conﬁdence Limits reported. 1 2 3 4 *Indicates signiﬁcantly different LC ABLE Newellton, LANewellton, LA Joseph, St. MSSIML, LANewellton, LA Joseph, St. chlorpyrifos MSSIML, LANewellton, LA Joseph, St. spinosad 180 MSSIML, 250 LANewellton, 250 tebufenozide 250 250 250 4.8 4.0 250 6.1 275 250 34.2* 1.85(0.48) 3.96(0.68) 2.8 14.5 2.97(0.40) 250 2.6 2.56(0.39) 4.8 1.8 1.78(0.39) 3.44(0.40) 3.2 2.1 2.6 2.26(0.40) 26.6 3.5 1.28(0.22) 1.77(0.23) 1.3 7.7 8.6 6.7 1.82(0.32) 1.5 9.6 2.7 42.3 1.5 24.0 4.5 0.8 1.2 1.2 4.0 7.1 7.9 3.1 3.0 1.7 0.7 SIML, MS SIML, St. Joseph, LA Joseph, St. MSSIML, LANewellton, LA Joseph, St. thiodicarb 250 400 275 319.8 250 4.4 614.3* 1.74(0.34) 641.4 2.69(0.29) 2.78(0.34) 238.0 3.28(0.33) 2.6 510.2 479.9 406.4 728.4 6.2 926.0 1.9 1.7 2.0 Strain Insecticide N T

Armyworm Symposium ’96: Mascarenhas et al. 341

TABLE 2. EFFICACY OF SELECTED INSECTICIDES AGAINST BEET ARMYWORM AT 3, 5, 7, AND 10 DAYS AFTER TREATMENT IN TEST 1 AT THE NORTHEAST RESEARCH STATION, ST. JOSEPH, LOUISIANA.

Numbers of Beet Armyworm Larvae per 30.5 cm

Treatment Rate (kg AI/ha) 3 DAT1 5 DAT 7 DAT 10 DAT

Chlorfenapyr 0.22 0.5 b 0.3 c 0.6 b 0.3 b Chlorpyrifos 1.12 0.9 b 0.5 c 1.2 b 0.5 b Spinosad 0.08 0.7 b 0.2 c 0.4 b 0.2 b Spod-X 185.52 6.1 a 3.7 ab 4.0 a 1.1 b Tebufenozide 0.14 2.1 b 0.6 c 1.1 b 0.5 b Thiodicarb 0.67 1.7 b 2.7 b 1.9 ab 0.9 b Untreated 5.1 a 5.1 a 3.9 a 2.4 a

P > F 0.01 0.01 0.01 0.01

1Means within a column not followed by a common letter are signiﬁcantly different (P = 0.05; LSD). 2Milliliters formulated material per ha.

DISCUSSION

Variation in susceptibility of field-collected strains of beet armyworm to chlorpyrifos have been reported by Chandler & Ruberson (1994); three (Bartow Co., Georgia, Macon Co., Alabama and Yazoo Co., Mississippi) of seven field strains had significantly higher LC50’s than the SIML reference strain. LC50’s of field strains reported by

Chandler & Ruberson were 2- to 29-fold higher than the highest LC50 (Newellton, Lou- isiana) observed in the bioassays reported herein. As with chlorpyrifos, ranges of

LC50’s reported for thiodicarb by Chandler & Ruberson (1994) were larger than those observed in bioassays of Louisiana strains. LC50’s observed for all three experimental compounds generally were much lower than those observed with the standard insecticides tested (Table 1). Significantly higher LC50’s observed with the Newellton field strain in both the chlorpyrifos and thiodicarb bioassays likely are due to the fact that this strain was collected from an area which typically receives high insecticide inputs. Although the St. Joseph strain also was collected from a high-input cotton producing region, the actual collection was made from a field within the Northeast Research Sta- tion, which typically does not receive intensive insecticide applications. Susceptibility of the beet armyworms on the Northeast Research Station (both the St. Joseph and Macon Ridge locations) to standard insecticides was also observed in the field tests in which chlorpyrifos (Tables 2 and 3) and thiodicarb (Table 2) significantly reduced the numbers of beet armyworm larvae relative to the untreated control. Considerable variation in control of beet armyworms with chlorpyrifos and thiodicarb is reported in the literature. Smith (1985) reported 65 and 76% control at 3 DAT in Texas with chlorpyrifos and thiodicarb, respectively. In South Carolina, Sul- livan et al. (1991) reported 90% control with thiodicarb, while in Mississippi Reed et al. (1994) reported less than 50% larval control with either insecticide. In both field tests, chlorfenapyr and spinosad provided excellent and rapid beet armyworm control and performed as well as, or better than, the standard, chlorpyrifos. Efficacy of chlorfenapyr against beet armyworm also has been documented in numerous EUP trials

342 Florida Entomologist 79(3) September, 1996

TABLE 3. EFFICACY OF SELECTED INSECTICIDES AGAINST BEET ARMYWORM AT 3, 5, 7, AND 10 DAT IN TEST 2 AT THE MACON RIDGE RESEARCH STATION, WINNS- BORO, LOUISIANA.

Numbers of Beet Armyworm Larvae per 30.5 cm1

Treatment Rate (kg AI/ha) 3 DAT1 5 DAT 7 DAT 10 DAT

Chlorfenapyr 0.22 2.8 a 0.6 c 1.1 b 0.4 c Chlorpyrifos 1.12 4.7 a 3.6 bc 1.2 b 0.3 c Spinosad 0.08 5.8 a 0.7 c 1.3 b 0.4 c Spod-X 185.5 10.7 a 7.9 a 3.4 ab 0.7 bc Tebufenozide 0.14 14.5 a 1.4 bc 0.4 b 0.7 bc Thiodicarb 0.67 12.6 a 4.4 ab 5.2 a 1.4 ab Untreated — 9.2 a 4.4 ab 5.4 a 1.8 a

P > F 0.41 0.02 0.04 0.05

1Means within a column not followed by a common letter are signiﬁcantly different (P = 0.05; LSD). 2Milliliter formulated material per ha. throughout the southeast (Wiley et al. 1995) where 90-98% control was reported. Tebufenozide, an insect growth regulator, provided satisfactory control of beet armyworm. However, this compound generally had a slower mode of action that required 5 days or more to obtain maximum control. Similar ﬁndings were reported by Furr & Harris (1995) where maximum control (83%) was achieved with tebufenozide at 9 DAT. Although this product has a slightly slower mode of action than chlorfenapyr and spinosad, it appears to be well suited for integration into an overall pest management program. Commercialization of these new compounds for beet armyworm control in cotton may lower the insecticide inputs (kg AI/ha) required to maintain this pest under an economic threshold level.

ACKNOWLEDGMENTS The ﬁnancial support of Cotton Incorporated is gratefully acknowledged. Appreci- ation is expressed to American Cyanamid Company, Crop Genetics International, DowElanco, Rhone-Poulenc Agricultural Company and Rohm & Haas Company for supplying the insecticides. We thank Don Cook, Chad Comeaux, Larry Daigle, Hunter Fife, Joe Pankey, Rosanne Mascarenhas and Karla Torres for their assistance in many aspects of these studies. This manuscript is approved for publication by the Director of the Louisiana Agricultural Experiment Station as Manuscript No. 96-17-0125.

REFERENCES CITED

ARRILLAGO, P. 1995. Farmers want to end boll weevil spraying, pp. 1c and 7c in The Advocate (October 10), Baton Rouge, Louisiana. BURRIS, E., S. MICINSKI, B. R. LEONARD, J. B. GRAVES, AND R. D. BAGWELL. 1994. The performance of cotton insecticides in Louisiana 1994 Louisiana Agric. Expt. Sta. Mimeo Series No. 91, 84 pp. CHANDLER, L. D., AND J. R. RUBERSON. 1994. Comparative toxicity of four commonly used insecticides to ﬁeld-collected beet armyworm larvae from the southeast- Armyworm Symposium ’96: Mascarenhas et al. 343

ern United States, pp. 860- 864 in Proc. Beltwide Cotton Conference, National Cotton Council, Memphis, Tennessee. DOUCE, G. K., AND R. M. MCPHERSON. 1991. Summary of losses from insect damage and cost of control in Georgia, 1989. Georgia Agric. Expt. Sta. Spec. Publ. 70. FURR, E. R., AND F. A. HARRIS. 1995. Evaluation of insecticide efﬁcacy for beet armyworm management in Mississippi, pp. 902- 903 in Proc. Beltwide Cotton Conf., National Cotton Council, Memphis, Tennessee. JOYCE, J. A., R. J. OTTENS, G. A. HERZOG, AND M. H. BASS. 1986. A laboratory bioassay for thiodicarb against the tobacco budworm, bollworm, beet armyworm and fall armyworm. J. Agric. Entomol. 3: 207-212. LEORA SOFTWARE. 1987. POLO-PC a user’s guide to Probit or Logit analysis. LeOra Software, Berkley, California 94707. LAYTON, M. B. 1994. The 1993 beet armyworm outbreak in Mississippi and future management guidelines, pp. 854-856 in Proc. Beltwide Cotton Conf., National Cotton Council, Memphis, Tennessee. REED, J. T., B. LAYTON, AND C. S. JACKSON. 1994. Evaluation of insecticide and insecticide combinations for beet armyworm control. Arthropod Management Tests 19: 237-238. SAS INSTITUTE. 1988. SAS/STAT users guide, version 6.03, [ed.] SAS Institute, Cary, North Carolina. 1028 pp. SMITH, R. H. 1985. Fall and beet armyworm control, pp. 134-136 in Proc. Beltwide Cotton Conf., National Cotton Council, Memphis, Tennessee. SMITH, R. H. 1994. Beet armyworm: a costly caterpillar, pp. 13-14 in Proc. Beltwide Cotton Conf., National Cotton Council, Memphis, Tennessee. SULLIVAN, M. J., S. G. TURNIPSEED, AND T. W. SMITH. 1991. Beet armyworm control in South Carolina, pp. 777 in Proc. Beltwide Cotton Conf., National Cotton Council, Memphis, Tennessee. WILEY, G. L., L. R. DESPAIN, K. KALMIWITZ, T. CAMPBELL, F. WALLS, T. HUNT, AND K. TREACY. 1995. Results of the PirateTM insecticide-miticide EUP program on foliage feeding insects on cotton, pp. 925-928 in Proc. Beltwide Cotton Conf., Na- tional Cotton Council, Memphis, Tennessee.

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Armyworm Symposium ’96: Rogers and Marti 343

BEET ARMYWORM (LEPIDOPTERA: NOCTUIDAE): EFFECTS OF AGE AT FIRST MATING ON REPRODUCTIVE POTENTIAL

C. E. ROGERS AND O. G. MARTI, JR. Insect Biology and Population Management Research Laboratory Agricultural Research Service, U.S. Department of Agriculture, Tifton, GA 31793-0748

ABSTRACT

The effects of age at the first mating on the reproductive potential of the beet armyworm, Spodoptera exigua (Hübner), was studied in the laboratory. Both the fecundity and fertility of eggs laid were significantly affected (P < 0.01) by age of males and females at the time of mating. Delayed mating by females increased longevity but decreased fecundity and fertility (P < 0.05). Delayed mating by males increased longevity (P < 0.05) but decreased the number of spermatophores they transferred to females (P < 0.01). The number of spermatorphores transferred during mating affected female fecundity and fertility (P < 0.01). The optimum age for the first mating

344 Florida Entomologist 79(3) September, 1996 for both males and females was 1-2 days post-emergence. Delaying the ﬁrst mating by either sex beyond 3-4 days post-emergence signiﬁcantly, and adversely, impacted the reproductive potential of the beet armyworm.

Key Words: Spodoptera exigua, fecundity, fertility, longevity

RESUMEN Fueron estudiados en el laboratorio los efectos de la edad en el momento del primer apareo sobre el potencial reproductivo del gusano de la remolacha, Spodoptera exigua (Hübner). La fecundidad y la fertilidad de los huevos puestos fueron signiﬁcativa- mente afectadas (P < 0.01) por la edad de los machos y hembras en el momento del apareo. El retardo en el apareo en las hembras aumentó la longevidad pero disminuyó la fecundidad y la fertilidad (P < 0.05). El retardo en el apareo en los machos aumentó la longevidad (P < 0.05) pero disminuyó el número de espermatóforos transferidos a la hembra (P < 0.01). El número de espermatóforos transferidos a la hembra durante el apareo afectó la fecundidad y la fertilidad de las hembras (P < 0.01). La edad óptima para el primer apareo en machos y hembras fue 1-2 días después de la emergencia. El retado del primer apareo más allá de los 3-4 días de la emergencia redujo signiﬁcati- vamente el potencial reproductivo del gusano de la remolacha en ambos sexos.

The beet armyworm, Spodoptera exigua (Hübner), was introduced into Oregon in 1876 and within a few years had migrated across the southern latitudes of the United States (Mitchell 1979). In recent years, the beet armyworm has become a serious economic pest of vegetables and cotton in the Southeast and mid-South (Douce & McPherson 1991, Layton 1994). The beet armyworm wreaked havoc on the cotton crop in the Lower Rio Grande Valley of Texas in 1995 (Summy et al. 1996). In the absence of heavy insecticide pressure on crops, populations of the beet armyworm often are maintained below economic levels by larval and pupal natural enemies (Ruberson et al. 1994). The beet armyworm has no effective natural enemies of its adult stage; however, it is a good factitious host for the ectoparasitic nematode, Noctuidonema guyanense Remillet and Silvain (Nematoda: Acugutturidae) in the laboratory (Rogers & Marti 1996). Before studying the effects of N. guyanense on the reproductive potential of S. exigua, we investigated the effects of age at mating on the reproductive potential of nematode-free moths.

MATERIALS AND METHODS

Experimental insects were from an established colony of the beet armyworm maintained on a pinto bean-based diet at the ARS Insect Biology and Population Man- agement Research Laboratory, Tifton, GA (Burton 1967). As moths emerged, they were placed individually in a 0.6-liter cardboard container, provided 10% honey solution for nourishment, and maintained at 27°C and 80% RH in a 14:10 h (L:D) photoperiod. To determine the effect of age at ﬁrst mating on fecundity and fertility in the beet armyworm, pairs of different male/female age combinations were established. Tests encompassed 19 trials, each of which contained 19-24 replicated pairs ranging in age from 1 to 9 days post-emergence. Mating pairs were maintained as mentioned above in 0.6-liter cardboard cages with the top and bottom covered with netting and paper towel lids. Cages were lined with waxed paper to facilitate removal and counting of eggs. Cages were examined daily to record data on moth mortality/longevity

Armyworm Symposium ’96: Rogers and Marti 345 and to collect eggs. Eggs were incubated 4 days at 30°C. The number of unhatched eggs and viable larvae was used to compute daily fecundity and fertility for each pair. Moths were maintained in their respective cages until both died. Dead females were dissected to determine the number of harbored spermatophores. All data were subjected to an analysis of variance by the General Linear Model Procedure, and significantly different means were separated by the least significant difference (LSD) test (SAS Institute 1989). Pearson correlation coefficients also were computed to document significant interactions among biological parameters.

RESULTS AND DISCUSSION

Among the 443 pairs, 112 females received no spermatophores, 145 females received a single spermatophore, and one female each received as many as 7, 8, or 9 spermatophores (Table 1). An average of 1.55 spermatophores was transferred to females among all matings. Discounting the 112 pairs with which no spermatophores were transferred, the remaining 331 pairs transferred an average of 2.07 spermatophores. The number of spermatophores transferred during mating was significantly affected by male age (r = -0.69; P < 0.01; F = 7.70; d.f. = 8,424), female age (r = -0.56; P < 0.05; F = 6.45; d.f. = 8,424), and combined pair ages (r = -0.76; P < 0.01; F = 7.87; d.f. = 10,422) at the first mating. Also, the number of spermatophores transferred during mating significantly affected the number of eggs laid (r = 0.73; P < 0.01; F = 1.68; d.f. = 9,423), the percentage of eggs hatching (r = 0.81; P < 0.01; F = 24.66; d.f. = 9,423), and the number of resulting viable larvae (r = 0.84; P < 0.01; F = 23.26; d.f. = 9,423). The lone female receiving 7 spermatophores laid 1,993 eggs and the 6 females receiving 6 spermatophores laid an average of 1,464.8 eggs, of which 99.4 and 66.4% hatched, respectively (Table 1). One female received 9 spermatophores. However, these were small and shriveled, and none of her eggs hatched. Females receiving only 1 or 2 spermatophores laid an average 500.1 to 831.2 eggs, of which 16.3 to 50.9% hatched. Females receiving no spermatophores laid an average of only 263.9 eggs, none of which hatched. Thus, with the beet armyworm, it appears that multiple spermatophores must be received by females for maximum reproductive capacity to be realized. The number of spermatophores transferred during mating seemed to have no effect on the longevity of females (r = -0.37, P > 0.05) or males (r =-0.22, P > 0.05). A correlation between spermatophore transfer at mating and female fecundity and fertility also has been reported for Trichoplusia ni (Hübner) (Noctuidae) (Ward & Landolt 1995), S. frugiperda (J. E. Smith) (Rogers & Marti 1994), and Atteva punctella (Cramer) (Yponomeutidae) (Taylor 1967). It appears that unsuccessful matings (evidenced by transfer of viable sperm) and low fertility among lepidopteran species are not uncommon, especially if the initial mating after emergence is delayed by 3 or more days (Unnithan & Paye 1991, Proshold 1996, Ellis & Steele 1982, Proshold et al. 1982, Lingren et al. 1988). The age of females at their first mating significantly affected fecundity (r = -0.69; P < 0.01; F = 5.84; d.f. = 8,435), fertility (r = -0.69; P < 0.01; F = 15.45; d. f. = 8,435), longevity (r = 0.74; P <= 0.01; F = 8.21; d.f. = 8,431), and the number of viable larvae (r = -0.72; P < 0.01; F = 14.88; d.f. = 8,435). Females mating on the first or second day post-emergence received significantly more spermatophores (P < 0.01; F = 6.45; d.f. = 8,432) than females mating at an older age (Table 2). Percentage hatch of eggs laid by females mating during their first 3 days was significantly higher than for eggs laid by older females. Although mating by females at an early age enhances their fecundity and fertility, it also significantly shortens their life span (Table 2). Females mating by day 3 post-emergence lived an average 12.4 to 17.4 days, while those mating at an

346 Florida Entomologist 79(3) September, 1996 1 S.E.) 0.7 a 0.6 a 0.8 a 1.1 a 1.5 a 1.6 a 2.8 a 6.9 a 6.9 a 6.9 a ±

± ± ± ± ± ± ± ± ± ± Male (x 1 S.E.) Longevity (Days) ±

0.7 a 0.6 a 0.8 a 1.1 a 17.1 1.6 a 14.1 2.9 ab 12.6 2.9 ab 14.6 7.3 a 12.1 13.7 11.0 7.3 a 18.0 13.0 7.3 a 8.0 ± ± ± ± ± ± ± ± ± ± Female (x Female ARMYWORM

1 BEET

2.4 cd 3.2 b 4.4 bc 6.4 b 19.3 6.6 b 16.9 11.9 ab 18.5 29.3 a 16.0 29.3 a 16.9 12.7 19.0 9.0 ± ± ± ± ± ± ± ± S.E.) 29.3 d 14.0 2.8 d 22.3 ± ±

± THE

(x OF

% Eggs Hatching POTENTIAL

1 < 0.05 by the SAS LSD test. 158.0 ab 387.1 a 66.4 99.4 36.6 d 32.2 a 42.8 c 58.4 c 0 84.5 c 16.3 86.6 bc 50.9 44.1 44.7 49.6 387.1 c 387.1 c 78.7 0. P ± ± ± ± ± ± ± ± ± ± S.E.) ±

(x No. Eggs Laid No. REPRODUCTIVE

TRANSFER

SPERMATOPHORE

FFECTS 1. E Means within a column followed by different letters are signiﬁcantly different, Means within a column followed by different letters are signiﬁcantly different, 1 ABLE No. Spermatophores Transferred Spermatophores No. 0 N 112 263.9 12345678 145 82 500.1 44 21 831.2 20 653.2 902.3 6 937.6 1 1464.8 1 1993.0 801.0 9 1 681.0 T

Armyworm Symposium ’96: Rogers and Marti 347 2 1 0.9 a 1.4 a 1.1 a 1.5 a 0.8 ab 1.4 a 0.8 bc 0.7 c 1.4 d ± ± ± ± ± ± ± ± ± S.E.) ±

(x Female Longevity Female . 1 4.5 bc 22.8 4.7 c 20.0 3.8 b 19.9 3.8 a 17.4 3.3 a 6.5 a 16.6 12.4 3.8 c 22.7 6.5 c 21.2 6.5 c 20.3 ± ± ± ± ± ± S.E.) ± ± ± ±

(x ARMYWORM

% Eggs Hatching BEET

THE

1 OF

50.4 d 3.9 86.1 c 11.6 86.1 cd 10.4 86.1 ab 5.3 50.4 bc 24.9 86.1 c 9.8 49.7 b 41.5 44.2 a 86.1 a 44.5 46.8 ± ± ± ± ± ± ± ± ± S.E.) ±

(x No. Eggs Laid No. POTENTIAL

< 0.05 by the SAS LSD test. P REPRODUCTIVE

1 THE

0.2 c 318.8 0.3 b 504.8 0.2 bc 381.2 0.3 c 720.1 0.2 ab 533.7 0.3 c 466.8 0.2 a 0.3 a 0.2 b 885.8 730.0 549.4 S.E.) ± ± ± ± ± ± ± ± ± ON

(x MATING

No. Spermatophores Received No. FIRST

AGE

FEMALE

FFECTS 2. E Means within a column followed by different letters are significantly different, Means within a column followed by different letters are significantly different, Days 1 2 ABLE 9 70 1.0 8 24 1.5 7 45 1.3 6 24 0.7 5 70 1.8 4 24 0.9 Female Age (Days)Female N 1 91 2.4 23 24 72 1.8 1.6 T 348 Florida Entomologist 79(3) September, 1996 older age lived for 20 days or more. The number of eggs laid by females also significantly affected their longevity (r = -0.51; P < = 0.05; F = 1.36; d.f. = 37,402). Also, egg fertility was inversely correlated with female longevity (r = -0.67; P < 0.05; F = 5.32; d.f. = 37,402). Females laying an average of 676.8 or more eggs had an average longevity of up to 12 days, whereas females laying an average of fewer than about 400 eggs lived an average of more than 15 days. The fertility of eggs laid by females living fewer than 12 days averaged 56.4 to 93.7%, whereas the fertility of eggs from females living more than 15 days averaged less than 27%. These relationships among reproductive parameters of females are not unique to S. exigua. These same relationships exist in other lepidopteran species, e.g., Spodoptera littoralis (Boisduval) (Ellis & Steele 1982), Lymantria dispar (L.) (Lymantridae) (Proshold 1996), Chilo partellus (Swinhoe) (Pyralidae) (Unnithan & Paye 1991), Heliothis virescens (F.) (Noctuidae) (Proshold et al. 1982), Pectinophora gossypiella (Saunders) (Lingren et al. 1988), and S. frugiperda (Rogers & Marti 1994). The age of males at first mating significantly affected the number of spermatophores transferred (r = -0.69; P < 0.01; F = 7.70; d.f. = 8.424), the number of eggs laid by their mates (r = -0.59; P < 0.01; F = 14.09; d.f. = 8,435), fertility of mate’s eggs (r = -0.65; P < 0.01; F = 14.09; d.f. = 8,435), the number of resulting viable larvae (r = 0.61; P < 0.01; F = 9.94; d.f. = 8,435), and the longevity of males after their first mating (r = -0.65; P < 0.01; F = 8.77; d.f. = 8,425 (Table 3). Males mating on days 1-4 post-emergence transferred significantly more spermatophores (x = 1.82-2.23) than males mating on days 6-9 (x = 0.7-1.2). Significantly more eggs were laid by females mating with 1- to 3-day old males (x = 701.1-787.4) than females mating with 7- to 9-day old males (x = 343.6-371.6). The percentage of eggs hatching from eggs of females mating with 1- to 3-day old males averaged about 45%, compared with 5.2-19.9% hatching of eggs from females mating with 4- to 9-day old males. Although the total longevity of males was only weakly correlated with mating age (r = 0.30; P > 0.05; F = 3.54; d.f. = 8,425), their longevity after mating averaged 15.8 to 17.6 days for those mating on day 1-3 post-emergence, but only 10.6 to 14.5 days for those mating 4-9 days post-emergence. Female longevity was only weakly correlated (r = 0.35; P > 0.05; F = 5.72; d.f. = 8,431) with male age at mating. Although the reproductive status of males affected the reproductive potential of the beet armyworm, their contributions had less impact than those of females. For the fall armyworm, the age of males at first mating had little impact on the reproductive potential of female mates (Rogers & Marti 1994). However, in H. virescens and L. dispar, the age of males at first mating affected spermatophore and/or sperm transfer to females during mating (Proshold 1996, Proshold & Bernon 1994). In A. punctella, the failure of female fertility is usually due to the failure of males to transfer sperm during mating, but there was no relationship between potency of sperm and age of males at mating (Taylor 1967). In C. partellus, the age of males had no effect on mating activity up to 5 days post-emergence, nor did it affect the fecundity or fertility of female mates (Unnithan & Paye 1991). Just as the age of females and males at mating independently affected the reproductive potential of the beet armyworm, the combined age of mating pairs interacted to significantly affect the number of spermatophores transferred (r = -0.76; P < 0.01; F = 7.87; d.f. = 10,422), the number of eggs laid (r = -0.78; P < 0.01; F = 12.45; d.f. = 10,433), fertility of eggs (r = -0.82; P < 0.01; F = 22.34; d.f. = 10,433), the number of resulting viable larvae (r = 0.81; P < 0.01; F = 19.36; d.f. = 10,433), and the longevity of female partners (r = 0.67; P < 0.01; F = 5.83; d.f. = 10,429). Mating moths with a combined age of 2 days transferred significantly more spermatophores (x = 3.3) than pairs with a combined age of 4 days or more (x = 0.7-2.1). Pairs with a combined age of 2 or Armyworm Symposium ’96: Rogers and Marti 349 2 1 1.1 a 1.1 b 1.1 b 1.6 a 0.8 ab 1.5 a 0.9 b 1.5 c 0.9 b ± ± ± ± ± ± ± ± ± S.E.) ±

(x Female Longevity Female 1 Longevity (Days) 0.8 a 21.2 0.9 a 19.5 0.8 b 18.4 1.3 a 21.2 0.6 a 20.6 1.2 b 23.7 0.7 ab 17.4 1.2 c 12.4 0.7 b 18.0 . ± ± ± ± ± ± ± ± ± S.E.) ±

(x Male Longevity ARMYWORM

BEET

1 THE

4.7 bc 21.3 3.3 b 19.9 6.7 b 18.5 3.8 a 18.8 6.6 a 14.7 3.8 a 18.6 4.9 bc 19.2 4.7 c 17.6 6.6 c 20.6 ± ± ± ± ± ± S.E.) ± ± ± OF ±

(x % Eggs Hatching POTENTIAL

1 2 < 0.05 by the SAS LSD test. 63.8 c 14.6 66.7 c 7.0 64.5 c 5.2 89.2 a 5.3 45.3 bc 20.4 91.2 c 19.9 51.5 ab 44.9 89.2 a 46.8 51.5 a 44.7 P ± ± ± ± ± ± ± ± ± S.E.) ±

Female (x REPRODUCTIVE

No. Eggs Laid by No. THE

1 MATING

0.2 bc 343.6 0.2 c 437.4 0.2 c 371.6 0.3 c 720.1 0.2 ab 535.9 0.3 a 489.8 0.2 a 701.1 0.3 a 730.0 0.2 a 787.4 S.E.) ± ± ± ± ± ± ± ± ± ±

(x FIRST

Transferred AT

No. Spermatophores No. AGE

MALE

FFECTS 3. E Means within a column followed by different letters are signiﬁcantly different, Means within a column followed by different letters are signiﬁcantly different, mated with males of respective age. Females 1 2 ABLE 9 47 1.2 8 43 0.9 7 46 0.9 6 24 0.7 5 93 1.6 4 23 1.8 3 71 2.2 2 24 1.8 1 72 2.2 Male Age (Days) N T 350 Florida Entomologist 79(3) September, 1996

Fig. 1. Fecundity of the beet armyworm as a function of combined age of mating pairs on their ﬁrst mating.

4 days at mating produced significantly more eggs (x? = 907.4) than pairs with a combined age greater than 6 days (x = 238.7-610.9) (Fig. 1). Eggs laid by females from pairs with a combined age of 2 or 4 days at first mating produced eggs having a significantly higher percentage hatching (x = 57.7-61.1%) than eggs from older pairs (x = 1.8-44.4%). If either mate of the pair were aged, the contributions of its younger mate were greatly minimized (Fig. 2). The number of viable larvae produced was significantly greater in mating pairs with a combined age of 2-4 days (x = 637.1-767.7) than the number produced by pairs with a combined age greater than 6 days (x = 9.8- 316.7). Females from pairs having a combined age of 2-6 days at first mating lived significantly fewer days (x = 15.1-16.2) than females of pairs having a combined age greater than 8 days (x = 19.6-21.9). In summary, the reproductive potential of the beet armyworm is significantly impacted by the age of both males and females at their first mating. However, there is a trade-off between enhanced reproductive potential of young moths and longevity of ovipositing females, i.e., more fecund/fertile moths have a shorter life span than females with a reduced reproductive potential. One to 2 days post-emergence is the optimum age for the first mating for both sexes for maximum reproductive potential. Hence, to evaluate the effects of adult mortality factors, e.g., parasitic nematodes, on the reproductive potential of the beet armyworm, it will be necessary to standardize the age of young moths.

REFERENCES CITED

BURTON, R. L. 1967. Mass rearing the fall armyworm in the laboratory. USDA-ARS- 33-177. Govt. Print. Ofﬁce, Washington, DC. DOUCE, G. K., AND R. M. MCPHERSON. 1991. (eds.) Summary of losses from insect damage and costs of control in Georgia, 1989. GA Agric. Exp. Sta. Special Pub- lication 70, 46 pp. Armyworm Symposium ’96: Rogers and Marti 351

Fig. 2. Fertility of beet armyworm eggs as a function of male and female age at the ﬁrst mating of a pair.

ELLIS, P. E., AND G. STEELE. 1982. The effects of delayed mating on the fecundity of females of Spodoptera littoralis (Boisduval) (Lepidoptera: Noctuidae). Bull. En- tomol. Res. 72: 295-302. LAYTON, M. B. 1994. The 1993 beet armyworm outbreak in Mississippi and future management guidelines. Beltwide Cotton Conf., Vol 2: 854-856. LINGREN, P. D., W. B. WARNER, AND T. J. HENNEBERRY. 1988. Inﬂuence of delayed mating on egg production, egg viability, mating, and longevity of female pink bollworm (Lepidoptera: Gelechiidae). Environ. Entomol. 17: 86-89. MITCHELL, E. R. 1979. Migration by Spodoptera exigua and S. frugiperda, North American style, pp. 386-393 in R. L. Rabb and G. E. Kennedy [eds.], Movement of highly mobile insects: concepts and methodology in research. NC State Univ., Raleigh, 456 pp. PROSHOLD, F. I. 1996. Reproductive capacity of laboratory-reared gypsy moths (Lepi- doptera: Lymantriidae): effect of age of female at time of mating. J. Econ. En- tomol. (accepted 1/15/96). PROSHOLD, F. I., AND G. L. BERNON. 1994. Multiple mating in laboratory-reared gypsy moths (Lepidoptera: Lymantriidae). J. Econ. Entomol. 87: 661-666. 352 Florida Entomologist 79(3) September, 1996

PROSHOLD, F. I., C. P. KARPENKO, AND C. K. GRAHAM. 1982. Egg production and oviposition in the tobacco budworm: effect of age at mating. Ann. Entomol. Soc. America 75: 51-55. ROGERS, C. E., AND O. G. MARTI, JR. 1994. Effects of age at ﬁrst mating on the reproductive potential of the fall armyworm (Lepidoptera: Noctuidae). Environ. En- tomol. 23: 322-325. ROGERS, C. E., AND O. G. MARTI, JR. 1996. Beet armyworm (Spodoptera exigua) as a host for the ectoparasitic nematode, Noctuidonema guyanense. J. Agric. Ento- mol. (Accepted 1/22/96). RUBERSON, J. R., G. A. HERZOG, W. R. LAMBERT, AND W. J. LEWIS. 1994. Management of the beet armyworm (Lepidoptera: Noctuidae) in cotton: role of natural enemies. Florida Entomol. 77: 440-453. SAS INSTITUTE. 1989. SAS/STAT User’s guide, version 6, 4th ed., Vol. 1 and Vol. 2. SAS Institute, Cary, NC. SUMMY, K. R., J. R. RAULSTON, D. SPURGEON, AND J. VARGAS. 1996. An analysis of the beet armyworm outbreak on cotton in the Lower Rio Grande Valley of Texas during the 1995 production season. Beltwide Cotton Conf. (accepted 1/7/96). TAYLOR, O. R., JR. 1967. Relationship of multiple mating to fertility in Atteva punctella (Lepidoptera: Yponomeutidae). Ann. Entomol. Soc. America 60: 583-590. UNNITHAN, G. C., AND S. O. PAYE. 1991. Mating longevity, fecundity, and egg fertility of Chilo partellus (Lepidoptera: Pyralidae): effects of delayed or successive matings and their relevance to pheromonal control methods. Environ. Entomol. 20: 150-155. WARD, K. E., AND P. J. LANDOLT. 1995. Inﬂuence of multiple matings on fecundity and longevity of female cabbage looper moths (Lepidoptera: Noctuidae). Ann. Ento- mol. Soc. America 88: 768-772.

Buckingham and Bennett: Biology of Parapoynx diminutalis 353

LABORATORY BIOLOGY OF AN IMMIGRANT ASIAN MOTH, PARAPOYNX DIMINUTALIS (LEPIDOPTERA: PYRALIDAE), ON HYDRILLA VERTICILLATA (HYDROCHARITACEAE)

GARY R. BUCKINGHAM1 AND CHRISTINE A. BENNETT2 1Agricultural Research Service U.S. Department of Agriculture Florida Biological Control Laboratory Gainesville, FL 32614-7100

2Department of Entomology and Nematology Institute of Food and Agricultural Sciences University of Florida, Gainesville, FL 32611

ABSTRACT

The Asian moth Parapoynx diminutalis Snellen is an immigrant in Florida and Panama where it attacks hydrilla, Hydrilla verticillata (L. ﬁl.) Royle, an immigrant submersed weed from Asia. Field populations of P. diminutalis are occasionally heavy on hydrilla but are rarely found on other plant species, including those that are laboratory hosts. Larvae build portable cases from which they feed on leaves and stems. The 7 instars can be differentiated by head capsule widths. Measurements are presented of other immature stages. In the laboratory at 26.7°C, eggs developed in 4-6 d, larvae in 21-35 d, prepupae in 1-2 d, and pupae in 6-7 d. Adults lived 3-5 d at 24.4°C.

Key Words: Biological control, fecundity, degree-days, host plants, Bacillus thuringiensis

RESUMEN

La polilla asiática, Paraponynx diminutalis Snellen es un inmigrante de la Florida y Panamá donde ataca a Hydrilla verticillata (L. ﬁl.) Royle, una hierba sumergida inmigrante de Asia. Las poblaciones de campo de P. diminutalis son ocasionalmente altas en H. verticillata pero raramente son encontradas en otras especies de plantas, incluidas las que son hospedantes de laboratoirio. Las larvas fabrican una funda por- tátil desde la cual se alimentan de las hojas y los tallos. Los siete instares pueden ser diferenciados mediante el ancho de la cápsula cefálica. Las medidas de los estadios inmaduros son presentadas. En el laboratorio, a 26.7°C, los huevos se desarrollaron en 4-6 días, las larvas en 21-35 días, las prepupas en 1-2 días y las pupas en 6-7 días. Los adultos vivieron 3-5 días a 24.4°C.

Parapoynx diminutalis Snellen is an immigrant Asian moth that attacks the submersed aquatic plant hydrilla, Hydrilla verticillata (L. ﬁl.) Royle, in Florida. Hydrilla, also an Asian immigrant, is the state’s most important aquatic weed and is found throughout much of the Southern USA and along the East Coast to Maryland (Lange- land 1990). The native range of hydrilla includes Asia, Australia, the Rift Valley area of Africa, and Europe, where only relict populations occur. The moth was ﬁrst reported in Florida in 1976, when it was discovered in hydrilla plantings at a research station in Ft. Lauderdale (Del Fosse et al. 1976). Subsequently, it was found in north Florida

354 Florida Entomologist 79(3) September, 1996

(Balciunas & Habeck 1981) and in Panama, where it infested hydrilla in the Panama Canal. It has also been reported colonizing glasshouses at aquatic plant nurseries in England and Denmark (Agassiz 1978, 1981). Its native range includes much of Asia from Afghanistan to the Philippines and north to Shanghai, People’s Republic of China, Africa from Ethiopia to South Africa (Speidel 1984) and Australia (Yoshiyasu 1992). Prior to its discovery in Florida, P. diminutalis was the subject of a study in Pa- kistan to determine its potential for introduction into the United States for biological control of hydrilla (Baloch & Sana-Ullah 1974). Baloch & Sana-Ullah (1974) presented a brief description of the biology along with results of preliminary laboratory host range tests. Additional biological studies, apparently with this species, were reported by Varghese & Singh (1976) in Malaysia in support of the USA biological control program. They referred to their subject as Nymphula sp. in the text but as N. diminutalis in the table headings. Nymphula diminutalis is a synonym of P. diminutalis. Chantaraprapha & Litsinger (1986) included a table with life history data for P. diminutalis in their study of the host range in the Philippines. We originally began studies with P. diminutalis collected in Panama and held in quarantine at the Florida Biological Control Laboratory, Division of Plant Industry (DPI), Florida Department of Agriculture and Consumer Services in Gainesville, FL. It was thought to be a little known Neotropical species, P. rugosalis Moschler. Balciu- nas & Center (1981) attempted to study P. rugosalis in Panama before its introduction into our quarantine and reported results of tests with larvae of Parapoynx sp. The species tested by them was probably not P. rugosalis but rather a third, as yet unidenti- ﬁed species. Three subsequent attempts by researchers, including one of us (CAB), to collect and carry to our quarantine laboratory the unidentiﬁed Parapoynx sp. yielded only P. diminutalis. We continued our studies after determining the correct identity of our material in order to better document the laboratory biology and host range of this interesting species. We report here the results of our biological studies. Our host range studies have been reported elsewhere (Buckingham & Bennett 1989).

MATERIALS AND METHODS All experiments were conducted in the quarantine facility at the Florida Biological Control Laboratory from 1980 to 1982. Hydrilla, the laboratory host plant, was collected periodically from several sites in Levy and Alachua counties or was grown in outdoor pools at the laboratory. The majority of test insects were from a colony established from eggs and larvae collected in November 1980 in the Rio Chagres near Gam- boa, Panama, by J. K. Balciunas. Additional insects were collected at Orange and Lochloosa Lakes near Gainesville. Adult and larval specimens have been deposited in the Florida State Collection of Arthropods, DPI, Gainesville and in the National Mu- seum of Natural History, Washington, D.C. The rearing colony was maintained in a quarantine greenhouse in a large wooden cage described by Buckingham & Bennett (1989). Larvae were also reared in 3.8 liter jars ﬁlled with water and hydrilla and capped with nylon organdy. The jars were held either in a greenhouse (12.2-31.1°C, 50-90% RH) or in a temperature- and humidity-controlled room (25 ± 1°C, 50-60% RH). Both had ﬂuorescent lights on 16 h photophase. Plants and water were added when necessary. The rearing material was transferred at least weekly to new jars sterilized with bleach.

Biological Studies All studies were conducted in environmental chambers at 26.7°C, 16 h photophase, unless otherwise indicated. Individual larvae on hydrilla were conﬁned after hatching

Buckingham and Bennett: Biology of Parapoynx diminutalis 355 in small plastic cups, 29.6 ml, with plastic lids. These small cups were used in many of the studies. The cups were examined daily for shed head capsules to determine the number and the durations of the stadia. Head capsule measurements were made with larvae preserved in 75% isopropyl alcohol. All measurements were made with a ste- reomicroscope and an ocular micrometer and are reported as follows: mean ± standard deviation (range, number). Neonate mortality outside of water on both dry and moist filter paper was tested by placing single larvae (n=30) without hydrilla in small plastic cups with lids. Neo- nate mortality in aerated water was tested in small plastic cups which had organdy glued over holes in the bottoms (n=20). The cups sat partially submerged on gravel in water-filled, aerated pans. These were compared with water-filled cups without holes (n=50). The neonates were tested at 22.2 ± 1°C, and those in water were provided with hydrilla. Most larval observations were made in small plastic cups, in 266 ml styrofoam cups, or in 0.95 liter or 3.8 liter jars. Hydrilla and water were added as needed. Larval and larval-pupal development times were determined at various temperatures in environmental chambers with 16 h photophases. Pupal development times were determined by observing pupae removed from cocoons and held on moist cotton in small plastic cups or in plastic petri dishes. Newly emerged females were confined individually with 2 newly emerged males in plexiglass cylinders (42 cm high, 14.5 cm ID) to determine longevity and realized fecundity, the number of eggs laid. We did not record matings, and deaths were recorded daily. Small amounts of water and hydrilla were placed in the bottom of each cylinder, which was capped with nylon organdy. Some females were dissected at death to determine the number of eggs remaining in the ovaries.

RESULTS AND DISCUSSION

Egg Stage

Description. Dorsoventrally compressed; outline circular at deposition, elliptical at maturity; chorion appears smooth at 50×; bright yellow at deposition changing to whitish and then transparent at hatching; length and width of mature egg presented in Table 1. The height of one detached mature egg was 0.26 mm. Development. Eggs were deposited in various size masses on leaves or stems of plants lying at the water surface, on moist filter paper in petri dishes, and on the sides of plexiglass cylinders at the waterline. The egg masses on hydrilla (Table 1) were generally smaller than masses placed on filter paper. Eggs were arranged in uneven rows within a mass. Most of the developing larvae were oriented in the same direction; however, often the larvae in one row, or portions of a row, were oriented in the opposite direction. Larval head position was initially indicated by two small dark eyespots; later the head was light brown with dark mandible tips. The curled embryo developed lying on its side, but within a few hours prior to hatching it actively moved within the egg. The development time is presented in Table 1. Usually all or most eggs in a mass hatched on the same day. Normal egg development occurred both underwater and outside of water on moist filter paper.

Larval Stage

Description. Seven (I-VII) instars: I - whitish almost transparent; no tracheal gills; orange malpighian tubules noticeable; one pair of long anal setae (0.20-0.22mm);

356 Florida Entomologist 79(3) September, 1996 smaller setae in longitudinal rows dorsally and along each side of abdomen; head capsule light brown with dark brown ocelli and epicranial suture; unspotted; mandibles reddish; light brown pronotal shield about equal to width of head capsule (Table 1); length of neonates about 1.00 mm. II - VII - whitish, mature larva turns yellowish just before pupation; tracheal gills present along each side of abdomen, number increasing on successive instars; head capsule light brown with scattered small dark brown spots at base of setae (Fig. 1A.), length increasing with each instar to 7.4-14.1 mm for last instar; instars best separated by widths of head capsules (Table 1). Head capsule widths were not compared between sexes. A detailed description and an illustration of the head capsule of the fourth instar was presented by Yoshiyasu (1985) for Japa- nese specimens. Development. The duration of the larval stage and pupal stage at 26.7°C is presented in Table 1, along with the approximate duration of each stadium. The days required for development from neonate to adult at 4 constant temperatures is presented in Table 2. A plot of the development rates (1/days to develop) against temperatures revealed that 36.1°C was near the upper threshold. Although adults were produced at that temperature, the development rate slowed. The estimated lower threshold for development obtained by the “linear approximation” method (Wilson & Barnett 1983) was 12.7°C. The threshold was identical with linear regression of the 3 lower temperatures (y = -0.0308 + (0.00243x), r = 0.99). The estimated degree-days for mean development times and for the ranges calculated using 12.7°C were 424 (351-551), 388 (294-490), 418 (340-483), and 494 (397-608), respectively, for the four temperatures listed in Table 2. Although these are approximations, they should be useful when planning additional field or laboratory studies with this species. Behavior. Neonates were active crawlers. Although some began feeding immediately, most wandered about the containers before settling on plants. It was not unusual to find that some had crawled from the water and out of containers that were not covered. Neonates fed on leaves, either by scraping the surface and rendering the leaf transparent, or by completely eating portions of the leaf. Most fed without a shelter but some made simple shelters by cutting small (about 2mm long) pieces of leaf and attaching them to the leaf surface. Most second instars made these simple shelters, and all later instars constructed tubular cases by tying together pieces of leaves or stems. Their cases might be mistaken for those of some caddisflies. Balciunas & Minno (1985) included P. diminutalis in a key to the larval cases of insects on hydrilla in the United States. Larvae generally fed on leaves by partially exiting from the cases they carried with them. At night, however, larvae were observed crawling without cases. On one occasion, many naked larvae were clustered together near the surface in an aerated jar that had a fluorescent light lying across the mouth. This suggested an attraction to light, but we did not test this hypothesis. Leaves were eaten most readily, but portions of the stem were also eaten during heavy feeding. Mortality. Neonates often crawled from their containers, but they were unable to develop outside water. All died within 1 h in dry cups, whereas in cups with moist filter paper all survived for 23.5 h. Survival in moist cups without hydrilla was 83% at 32 h, 57% at 46.5 h, 30% at 55 h, and 0 at 72 h. Two of an additional three neonates tested in moist cups with hydrilla were still alive at 72 h but both were dead at 6 d. These relatively long periods of survival out of water suggest that neonates might successfully disperse with hydrilla carried on boat trailers, boat propellers, etc. When neonates were held in water without food, forty-two percent were alive after 6 d in the greenhouse at 14.3-26.7°C, but none was alive after 14 d. Early instar mortality (within the first 2 weeks) ranged from 30 to 100% and was greater than, or equal to, 50% in ten of 21 diverse experiments where individual lar-

Buckingham and Bennett: Biology of Parapoynx diminutalis 357

TABLE 1. BIOLOGICAL STATISTICS FOR P. DIMINUTALIS.

Statistic n Mean SD Range

Measurements

Mature egg (mm) Length 10 0.44 0.02 0.42-0.48 Width 10 0.34 0.02 0.32-0.34 Egg mass size (Nos.) 112 29.7 19.4 2-99 Larval head capsule (mm) Instar I 18 0.21 0.01 0.20-0.24 II 5 0.29 0.02 0.26-0.30 III 1 0.42 IV 8 0.62 0.03 0.60-0.66 V 16 0.81 0.05 0.75-0.90 VI 30 1.02 0.02 0.98-1.06 VII 18 1.15 0.04 1.10-1.20 Female pupa (mm) Length 11 7.82 0.34 7.14-8.33 Width 11 2.09 0.10 1.96-2.30 Width at spiracles 11 2.31 0.09 2.21-2.47 Male pupa (mm) Length 10 6.63 0.20 6.14-6.80 Width 10 1.67 0.11 1.45-1.79 Width at spiracles 10 1.84 0.11 1.62-1.96 Wingspan (mm) Female 10 18.12 0.98 16.50-19.67 Male 10 13.40 0.93 12.34-14.84

Development Times (Days @ 26.7°C)

Egg 4-6 Larva + Pupa 17 27.1 3.8 21-351 Prepupa 1-2 Pupa 10 6-7 Total (Estimated) 25-41

Longevity (Days)

22.2-25.6°C Female 33 5.2 2.1 1-10 Male 26 5.9 3.2 1-17

1Instar I=4, II=3, III=3-5, IV=2-3, V=2-5, VI=3-8, VIII=3-9 days.

358 Florida Entomologist 79(3) September, 1996

TABLE 1. (CONTINUED) BIOLOGICAL STATISTICS FOR P. DIMINUTALIS.

Statistic n Mean SD Range

24.4°C Female 3 4.3 0.6 4-5 Male 6 4.0 1.4 3-5

1Instar I=4, II=3, III=3-5, IV=2-3, V=2-5, VI=3-8, VIII=3-9 days. vae were monitored. There were no obvious causes for this; however, initial mortality (4-9 d) was generally less [41 ± 30% (0-100, 18 experiments)] when eggs containing active larvae were transferred to the test containers rather than neonates [61 ± 20% (30-90, 6)]. In various experiments (Buckingham & Bennett 1989), more adults were generally produced in the greenhouse than in the laboratory. Aeration of the water did not signiﬁcantly improve survival in the small containers with individual neonates and hydrilla. After 5 days, 16 of 20 neonates were alive in aerated cups versus 34 of 50 in non-aerated cups (X2, p=.48). After 13 days, 15 larvae were still alive in the aerated cups versus 30 in the non-aerated cups (p=.36). There was no apparent increase in survival in various other experiments in which the small cups were aerated versus those experiments in which they were not aerated. Aeration, however, did decrease mortality in rearing jars containing older larvae with too high a density of hydrilla stems. Several jars were found with moribund larvae that recovered when the water was aerated. This mortality with densely packed hydrilla was probably due to the decrease in oxygen at night (measured at 1.4 ppm) caused by plant respiration. Chantaraprapha & Litsinger (1986) also reported that larval mortality was high (50-60%) during their experiments with this species. Mor- tality was generally higher when large numbers of larvae, especially the early instars, were present. Because there was an abundance of leaves for the early instars, the

Fig. 1. Parapoynx diminutalis Snellen: A) mature larva with characteristic dark spots on the head capsule, B) male.

Buckingham and Bennett: Biology of Parapoynx diminutalis 359

TABLE 2. PERCENTAGE SURVIVAL AND TIME (DAYS) REQUIRED OF P. DIMINUTALIS FOR DEVELOPMENT FROM NEONATE TO ADULT AT FOUR CONSTANT TEMPERATURES.

Temp (°C) % Survival n Mean SD Range

22.2 70 21 44.6 5.1 37-58 26.7 40 20 27.7 3.8 21-35 30.6 60 24 23.4 2.6 19-27 36.1 40 20 21.1 2.8 17-26 higher mortality suggests that larvae killed each other rather than competed for resources. However, no observations were made to conﬁrm that. Larvae are often quite damaging in research plantings of hydrilla in pools or other small protected containers in Florida. We immersed larvae with hydrilla in a commercial preparation of Bacillus thuringiensis, Dipel Hg, (Sunnyland Corp., Sanford FL) at 10% of the dosage recommended as a garden spray and produced 80% mortality within 4 days compared to no mortality in the controls. The surviving larvae in the Dipel Hg did not feed and were dead when examined after 10 days. Larvae were found relatively often on other plant species associated with hydrilla in our research plantings, but in addition to hydrilla, we have found immatures in the ﬁeld only on coon- tail, Ceratophyllum demersum L. (1 larva, 1 pupa), southern naiad, Najas guadalupensis (Sprengel) Magnus (2 larvae), and Illinois pondweed, Potamogeton il- linoensis Morong (5 larvae, 3 pupae). This is in sharp contrast to the 14 plant species in 13 genera that produced adults in our host range tests (Buckingham & Bennett 1989).

Pupal Stage

Description. Similar to those of other species of Parapoynx (Lekic 1971, Virak- tamath et al. 1974, Yoshiyasu 1985). Narrow, elongate, with 3 distinct spiracular tu- bercles along each side and 2 strong dark setae on top of the head. Female differentiated most easily from male by the length of the antennae. Female antennae extending to abdominal segment A4 just anterior to the wing tips; male antennae exceeding the wing tips and extending to A5. Females generally larger than males (Ta- ble 1). The abdomen expands in length when the pupa darkens prior to emergence. Lengths of expanded darkened pupae: female - 8.97 ± 0.05 mm (8.93-9.01, 4); male - 7.9 mm (n=1). The pupa was enclosed in an air-ﬁlled white silken cocoon. The cocoon was ﬁrmly attached along one side to the submersed stem or occasionally was attached at one end and perpendicular to the stem. The cocoons were covered with leaves or bare stem sections and were similar to the larval cases. Pupae obtained air through the cocoon from 1-4 excavations of various sizes made in the stems by the larvae. Air was held between the layers of the multilayered silk cocoon in a broad silvery band surrounding the spiracles. Lekic (1971) illustrated similar holes in the plant associated with cocoons of P. stratiotata L., and Yoshiyasu (1985) illustrated a silvery band in the cocoon of Parapoynx vittalis (Bremer). The length of female cocoons was generally greater than that of male cocoons (Table 1). Development. Prepupae remained immobile in the cocoon for 1-2 d before pupat- ing. Pupal development times are presented in Table 1. Distinct color changes were associated with pupal development. Body color changed from white to yellow while

360 Florida Entomologist 79(3) September, 1996 the eyes darkened to red and then to dark brown. The wing pattern was visible shortly before emergence. The pupa actively moved its abdomen when disturbed during the ﬁrst few days but was immobile during the last few days. Pupae died in the cocoons if the stems were only a few cms long or if the stem became waterlogged. Pupae developed normally when removed from cocoons in water to moist sphagnum or to ﬁlter paper in closed containers. All cocoons were constructed on submersed stems. This contrasts with the observation of Varghese & Singh (1976) that the anterior end of the pupa often remained above the water surface and of Chantaraprapha & Litsinger (1986) that they pupate out of water on plant vegetation. These differences might be due to water level changes exposing the cocoons or to a complex of species rather than one widely distributed species.

Adults Description. Adults are white with sinuous light-brown or tan bands on the wings (Fig. 1B). Variable amounts of black scales mask portions of the bands. The body has transverse tan stripes. Females had a wider wingspan (Table 1), more pointed forewings, a more robust abdomen, and relatively shorter antennae than the males. The tip of the male abdomen has a tuft of white setae that is more noticeable than that of the female. With experience, we could easily distinguish males by this character. De- tailed taxonomic descriptions along with illustrations of the genitalia can be found in Agassiz (1978), Speidel (1984), and Yoshiyasu (1985). The North American species most easily confused with P. diminutalis, especially the male, is Parapoynx allionealis (Walker). However, the light-brown or tan middle (postmedial) band on the forewing of P. diminutalis is much more sinuous than that on P. allionealis, which was illustrated by Monroe (1973). The wing patterns of both sexes of P. diminutalis are similar although the male generally has more black scales on the wings. The amount of black tended to vary inversely in both sexes with the temperature at which the larva was reared and with the type of host plant. Both sexes generally held the wings outspread and pressed to the substrate while resting. When disturbed, however, they often landed with the forewings folded over the hindwings but not overlapping the body. Development and Longevity. The development time from neonate to adult at 26.7°C is presented in Table 1. No cohorts were followed from egg deposition to adult; however, total development time at 26.7°C should be 25-41 days based upon an egg stage of 4-6 d and the larval/pupal times. Mean longevities of males and females were the same (Table 1). In a greenhouse at fluctuating temperatures, mean longevities were slightly longer: females 5.2 ± 2.1 days (n=33, max 8 days), males 5.9 ± 3.2 days (n=26, max 17 days). However, deaths were not recorded on the weekends in that experiment. Fecundity. In oviposition experiments females that had been reared at various temperatures from 21.1°C to 26.7°C laid a mean of 222.9 ± 140.6 eggs (3-524, 26). The mean without four females who laid less than 22 eggs each was 261.2 ± 116.3 eggs (108-524, 22). Fourteen of the preceding 22 females were dissected at death. Eleven had no eggs in their ovaries and three had only 24.7 ± 20.5 eggs (4-45). Differences among individuals in egg deposition apparently reflected true differences in the egg complements except for individuals that laid very few eggs. For example, three females that laid 0, 3, and 19 eggs had 26, 199, and 196 eggs, respectively, in their ovaries at death. Behavior. Adults began emerging in the large greenhouse cage approximately 30 min after dusk and began flying about 30 min later. After emergence, they sat above or on the water surface while their wings expanded. If disturbed before their wings expanded, they ran quickly across the hydrilla or the water surface, their long legs providing speed as they remained high above the substrate. Even though unable to fly Buckingham and Bennett: Biology of Parapoynx diminutalis 361 during these first few minutes, they would not be easy prey for most predators. We observed a few adults emerge shortly before dusk in India in 1985 while we were con- ducting another project, although the majority of adults emerged after dusk. One of us (CAB) also observed pre-dusk emergence in Panama in 1982. Mating was not observed in the greenhouse cage until about 3 h after dusk. Pairs in copula rested facing in opposite directions for at least 30 min. The maximum time in copula was not determined. We did not test females for multiple matings. No females oviposited the night they emerged. In the cage, they oviposited within the first hour after dusk. The female sat above the water surface and usually inserted her ovipositor into the water to oviposit on leaves or stems. Eggs were placed occasionally, just above the water surface. Because of the 1-day preoviposition period, there is probably heavy preoviposi- tional mortality in the large water bodies in Panama and Florida. Hydrilla mats ex- tend considerable distances from shore, thus many moths rest during the day exposed on the mats. Balciunas & Habeck (1981) reported heavy bird predation on moths at a large Florida lake. We have also observed large numbers of detached moth wings on the water surface above hydrilla mats. Although the adult proboscis was greatly reduced, it was apparently functional. Adults that had been held in a refrigerator for several days without water and adults collected at outdoor lights extended their probosci to the water when presented wet leaves and wet paper toweling. We did not test longevity without water, but adults provided sugar water lived no longer than those with water. Adults in the greenhouse cage were attracted to both incandescent white lights and UV blacklights. They did not respond to incandescent red light which was used to observe them without disturbance. The sizes of both the egg and the last instar head capsule of our population were considerably smaller than those reported by Varghese & Singh (1976) in Malaysia. Al- though these differences might be merely populational or technique differences, they suggest that two different species might have been studied. Our 4th instar head capsule width was equal to that of a Japanese 4th instar illustrated by Yoshiyasu (1985). The number of larval instars that we found (7), was different from that reported by Varghese & Singh (1976) (6) and by Chantaraprapha & Litsinger (1986) (4). Fecundity and development times also varied, but those are often greatly influenced by techniques. Taxonomic studies of these populations and others of this widely distributed species appear to be warranted. Parapoynx diminutalis is apparently well established in both Panama and Flor- ida, but its potential to extensively damage or control hydrilla is probably greater in Panama because of the milder climate. In Florida, at least in north-central Florida, near Gainesville, the larval populations are reduced to almost undetectable levels during late winter and early spring probably by the cool water temperatures in autumn and winter and the annual late winter decline in hydrilla. However, some populations near Gainesville rebounded quickly in some years, so that by late summer the defoliation of surface hydrilla stems was extensive. Early summer augmentation of moth populations by the release of immatures or adults might allow the populations to increase fast enough to at least slow the growth of hydrilla. Extensive defoliation might also increase the plants sensitivity to herbicides or pathogens. If plants attacked were more susceptible, an integrated program might be feasible.

ACKNOWLEDGMENT The following individuals helped greatly with this study: Joe Balciunas, Al Cof- rancesco, Mary Davis, Dale Habeck, Bonnie Ross, Steve Hampton, and Russell The- 362 Florida Entomologist 79(3) September, 1996 riot. Personnel at Manatee Springs State Park, Florida Department of Natural Resources, allowed us to collect hydrilla. Plants were identiﬁed by Kenneth Langdon and Carlos Artaud, Florida Department of Agriculture and Consumer Services, Divi- sion of Plant Industry, Gainesville. The moths were identiﬁed by D.C. Ferguson, SEL, ARS/USDA. The Aquatic Plant Control Research Program, U.S. Army Corps Engi- neers, Waterways Experiment Station, Vicksburg, MS, provided partial funds for this project and the Division of Plant Industry, Florida Department of Agriculture and Consumer Services provided laboratory and quarantine facilities and support. Our sincere thanks to all of these organizations and individuals, especially Bonnie Ross. Mention of a proprietary product does not constitute an endorsement or a recommen- dation for its use by USDA.

REFERENCES CITED

AGASSIZ, D. 1978. Five introduced species, including one new to science, of China mark moths (Lepidoptera: Pyralidae) new to Britain. Entomol. Gaz. 29: 117- 127. AGASSIZ, D. 1981. Further introduced China mark moths (Lepidoptera: Pyralidae) new to Britain. Entomol. Gaz. 32: 21-26. BALCIUNAS, J. K., AND T. D. CENTER. 1981. Preliminary host speciﬁcity tests of a Pan- amanian Parapoynx as a potential biological control agent for hydrilla. Envi- ron. Entomol. 10: 462-467. BALCIUNAS, J. K., AND D. H. HABECK. 1981. Recent range extensions of a hydrilla- damaging moth, Parapoynx diminutalis (Lepidoptera: Pyralidae). Florida En- tomol. 64: 195-196. BALCIUNAS, J. K., AND M. C. MINNO. 1985. Insects damaging hydrilla in the U.S.A., J. Aquat. Plant Manage. 23: 77-83. BALOCH, G. M., AND SANA-ULLAH. 1974. Insects and other organisms associated with Hydrilla verticillata (L.f.) L.C. (Hydrocharitaceae) in Pakistan, pp. 61-66 in A. J. Wapshere [ed.], Proc. 3rd Int. Symp. Biol. Control Weeds, Montpellier, France, 1973. Commonw. Inst. Biol. Control Misc. Publ. No. 8. BUCKINGHAM, G. R., AND C. A. BENNETT. 1989. Laboratory host range of Parapoynx diminutalis (Lepidoptera: Pyralidae), an Asian aquatic moth adventive in Flor- ida and Panama on Hydrilla verticillata (Hydrocharitaceae). Environ. Ento- mol. 18: 526-530. CHANTARAPRAPHA, N., AND J. A. LITSINGER. 1986. Host range and biology of three rice caseworms. IRRN 11(5): 33-34. DEL FOSSE, E. S., B. D. PERKINS, AND K. K. STEWARD. 1976. A new record for Parapo- ynx diminutalis (Lepidoptera: Pyralidae), a possible biological agent for Hyd- rilla verticillata. Florida Entomol. 59: 19-20. LANGELAND, K. A. 1990. Hydrilla (Hydrilla verticillata (L. ﬁl.) Royle), a continuing problem in Florida waters. Univ. Florida Coop. Ext. Serv. Circ. 884. LEKIC, M. 1971. Ecology of the aquatic insect species Parapoynx stratiotata L. (Pyraustidae, Lepidoptera). J. Sci. Agric. Res. 23: 49-63. MUNROE, E. 1973. Pyraloidea. Pyralidae (Part), Fasc. 13.1C, pp. 251-304 in R. B. Do- minick et al., The Moths of America North of Mexico. E. W. Classey, Ltd., Lon- don. SPEIDEL, W. 1984. Revision der Acentropinae des palaearktischen Faunengebietes (Lepidoptera: Crambidae). Neue Entomol. Nachrichten 12: 1-157. VARGHESE, G., AND G. SINGH. 1976. Progress in the search for natural enemies of hydrilla in Malaysia, pp. 341-352 in C. K. Varshney and J. Rzoska [eds.], Aquatic Weeds in South East Asia. W. Junk, The Hague. VIRAKTAMATH, C. A., M. PUTTARUDRIAH, AND G. P. CHANNA-BASAVANNA. 1974. Stud- ies on the biology of rice case-worm, Nymphula depunctalis Guenee (Pyraus- tidae: Lepidoptera). Mysore J. Agric. Sci. 8: 234-241. Buckingham and Bennett: Biology of Parapoynx diminutalis 363

WILSON, L. T., AND W. W. BARNETT. 1983. Degree-days: an aid in crop and pest management. California Agric. 37: 4-7. YOSHIYASU, Y. 1985. A systematic study of the Nymphulinae and the Musotiminae of Japan (Lepidoptera: Pyralidae). Kyoto Prefectural University, Agriculture, Sci- entiﬁc Report 37: 1-162. YOSHIYASU, Y. 1992. Nymphulinae, pp. 79-80 in J. B. Heppner and H. Inoue [eds.], Lepidoptera of Taiwan, Vol. 1, Pt. 2: checklist. Scientiﬁc Publishers, Inc., Gainesville, Florida.

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Jansson: Infectivity and Reproduction of Nematodes 363

INFECTIVITY AND REPRODUCTION OF THREE HETERORHABDITID NEMATODES (RHABDITIDA: HETERORHABDITIDAE) IN TWO INSECT HOSTS

RICHARD K. JANSSON1 Merck Research Laboratories P.O. Box 450, Hillsborough Road Three Bridges, NJ 08887

1Previous address, University of Florida, Institute of Food and Agricultural Sciences, Tropical Research and Education Center, Homestead, FL 33031

ABSTRACT

The infectivity, incubation time, and reproduction of three heterorhabditid nematodes, Heterorhabditis sp. Bacardis and FL2122 strains and H. bacteriophora Poinar HP88 strain were studied in two insect hosts, an apionid weevil, Cylas formicarius (F.), and a pyralid moth, Galleria mellonella (L.). Two of the nematodes, Heterorhab- ditis sp. Bacardis and FL2122 strains, were of tropical or subtropical origin, whereas the third nematode, H. bacteriophora HP88 strain, was of temperate origin. Infectiv- ity did not differ among nematodes within each host; however, it did differ between hosts for the Bacardis strain. Cylas formicarius was more susceptible to this nematode than G. mellonella. Incubation times also did not differ among nematodes within hosts; however, incubation times were 3.2-4.3 d shorter in C. formicarius than in G. mellonella. Progeny production differed (although not signiﬁcantly consistent) among nematodes and was highest for Heterorhabditis sp. Bacardis followed by the Heter- orhabditis sp. FL2122 and H. bacteriophora HP88 in both hosts. Percentages of infected cadavers that produced progeny were consistently higher for the tropical and subtropical nematodes, Heterorhabditis sp. Bacardis and FL2122, than for the temperate nematode, H. bacteriophora HP88, in both hosts. Patterns of emergence from cadavers were consistent in G. mellonella; most progeny emerged by 23 d after inoculation and emergence lasted for up to 48 d after inoculation. Conversely, emergence patterns varied markedly in C. formicarius. Emergence lasted for up to 29 d after inoculation and peak emergence varied between 12 and 28 d after inoculation. Progeny production in C. formicarius was not related to the biomass of the host cadaver.

Key Words: Heterorhabditis spp., Cylas formicarius, Galleria mellonella, infectivity

364 Florida Entomologist 79(3) September, 1996

RESUMEN

Fueron estudiadas la infectividad, el tiempo de incubación y la reproducción de tres nemátodos heterorhabdítidos, las cepas Bacardis y FL2122 de Heterorhabditis sp., y la cepa HP88 de H. bacteriophora Poinar, en dos insectos hospedantes, un gor- gojo apiónido, Cylas formicarius (F.), y una polilla pirálida, Galleria mellonella (L.). Dos de los nemátodos, las cepas Bacardis y FL2122 de Heterorhabditis sp., fueron de origen tropical o subtropical, mientras que el tercer nemátodo, la cepa HP88 de H. bacteriophora Poinar, fue de origen templado. La infectividad no difirió entre los ne- mátodos dentro de cada hospedante; sin embargo, difirió entre los hospedantes para la cepa Bacardis. Cylas formicarius fue más susceptible a este nemátodo que G. mellonella. Los períodos de incubación tampoco difirieron entre los nemátodos dentro de los hospedantes; sin embargo, los tiempos de incubación fueron 3.2-4.3 días más cor- tos en C. formicarius que en G. mellonella. La producción de progenie difirió (aunque no con significación consistente) entre los nemátodos y fue más alta para Heterorha- bditis sp. Bacardis, seguida por Heterorhabditis sp. FL2122 y por H. bacteriophora HP88 en ambos hospedantes. Los porcentajes de cadáveres infectados que produjeron progenie fueron consistentemente más altos para los nemátodos tropicales y subtro- picales, Heterorhabditis sp. Bacardis y Heterorhabditis sp. FL2122, que para el templado, H. bactriophora HP88, en ambos hospedantes. Los patrones de emergencia de los cadáveres fueron consistentes en G. mellonella; la mayoría de la progenie emergió antes de los 23 días posteriores a la inoculación y la emergencia duró al menos 48 días después de la inoculación. Por el contrario, los patrones de emergencia variaron mar- cadamente entre los 12 y los 28 días después de la inoculación. La producción de progenie en C. formicarius no estuvo relacionada con la biomasa del cadáver del hospedante.

Two population parameters of entomopathogenic nematodes that affect their suitability as a biological control agent against specific target insects are their level of infectivity and reproductive capacity. Infectivity refers to the ability of nematodes to cause infection in a target insect (Tanada & Fuxa 1989) and has been shown to vary among nematodes within specific target hosts (Bedding et al. 1983, Molyneux et al. 1983, Morris et al. 1990, Mannion 1992) and among hosts for a given nematode species or strain (Bedding et al. 1983, Morris et al. 1990). The reproductive capacity of nematodes was also shown to differ among nematodes within target insects (Morris et al. 1990, Mannion & Jansson 1992), and among hosts within specific nematode species or strains (Morris et al. 1990). Nematodes with higher levels of infectivity and reproduction within a specific target host may be more effective at controlling a particular insect under field conditions. These two population parameters are also central to long-term persistence. Morris et al. (1990) noted that a high infection rate of soil insects followed by a high rate of reproduction is critical to ensure reinfestation of the habitat by nematode progeny. Recent studies conducted in the laboratory that used a variety of different bioassay systems showed that heterorhabditid nematodes, especially an undescribed nematode isolated in Florida, Heterorhabditis sp. FL2122 strain, were most suitable as biological control agents of the sweetpotato weevil, Cylas formicarius (F.) (Mannion

1992). She found that heterorhabditid nematodes had some of the lowest LC50 and

LC90 values, produced more progeny per cadaver, had higher levels of infectivity in sand, soil, and Petri plates, killed more hosts within sweet potato storage roots, and had a greater ability to exit infected weevil cadavers within storage roots and infect new hosts in the soil than steinernematid nematodes. Her data concur with previous

Jansson: Infectivity and Reproduction of Nematodes 365 reports (Jansson 1991, Jansson et al. 1990, 1992, 1993) that found heterorhabditid nematodes to be more efficacious against C. formicarius. Jansson et al. (1992, 1993) also found that heterorhabditid nematodes persisted longer than steinernematids in the field. A recent study that determined the potential for applying nematode-infected wax moth, Galleria mellonella (L.), cadavers for controlling C. formicarius in the field found that an undescribed nematode isolated from Puerto Rico, Heterorhabditis sp. Bacardis strain, produced more progeny per G. mellonella cadaver than H. bacteriophora Poinar HP88 strain (Jansson et al. 1993). The present studies were conducted to more fully compare the suitability of certain nematodes as biological control agents of C. formicarius and determined if fitness parameters of three heterorhabditid nematodes differed between two insect hosts, C. formicarius and G. mellonella.

MATERIALS AND METHODS

Two insect hosts were used in these experiments: late instar wax moth, G. mellonella, and third instar sweetpotato weevil, C. formicarius. Galleria mellonella larvae were obtained from a commercial supplier (JA-DA Bait, Antigo, Wisconsin). Cylas formicarius were reared in the laboratory using methods described previously (Man- nion & Jansson 1992). Three heterorhabditid nematodes were tested: H. bacteriophora HP88 strain, and two undescribed nematodes, Heterorhabditis sp. FL2122 and Bacardis. The two latter nematodes were of tropical or subtropical origin. The FL2122 isolate was found in central Florida and the Bacardis nematode was isolated near the San Juan harbor in Pu- erto Rico (R. K. J. et al., unpublished). These two undescribed isolates are presumably local variants of a previously unrecorded species of Heterorhabditis (J. Curran, personal communication) reported earlier as Heterorhabditis sp. B in Poinar (1990). The HP88 strain was of temperate origin (Poinar 1990). Nematodes were reared in vivo (Dutky et al. 1964) in G. mellonella larvae as previously described (Mannion & Jans- son 1992). Infective juveniles used in these tests were less than two weeks old at the time of the experiment.

Infectivity

The level of infectivity was determined in each host using a single nematode/single host bioassay system (Miller 1989). Single infective juveniles were removed from an aqueous suspension taken from laboratory cultures and pipetted (0.3 ml) onto a double layer of filter paper in individual wells (1.5 cm diam) of a Multiwell TM 24 well, flat bottom tissue culture plate (Falcon R, model 3047, Becton Dickinson and Co., Lincoln Park, New Jersey). One host larva was then placed on the filter paper in each well. Fil- ter paper treated with deionized water served as the control. Microsoap (Interna- tional Products Corp., Burlington, New Jersey) was added (1.25 ml/liter) to nematode suspensions to improve efficiency of nematode transfer. Tissue culture plates were covered, sealed with parafilm and stored in the dark at 25±2°C. Larvae were checked daily for nematode-induced mortality for four consecutive days. Two trials were conducted against G. mellonella larvae and five trials were conducted against C. formicarius larvae. A total of four (G. mellonella) and twelve (C. formicarius) tissue culture plates (96 and 288 larvae, respectively) per nematode treatment were used to determine infectivity of these nematodes. Efficiency of nematode transfer was determined by removing an aliquot (0.3 ml) with a single nematode from suspensions, dispensing each aliquot onto a Petri plate,

366 Florida Entomologist 79(3) September, 1996 and then counting the number of infective juveniles in each aliquot. A total of 80 aliquots were removed from each suspension on the day of the experiment. The efﬁciency of transferring single nematodes were 93.7±3.7, 97.5±1.4, and 95.0±2.9 for H. bacteriophora HP88, Heterorhabditis sp. FL2122, and Heterorhabditis sp. Bacardis, respectively. These efﬁciencies were then used to adjust infectivity data to estimate real infectivity of each nematode.

Incubation Time and Reproduction

All nematode-infected G. mellonella larvae and only those C. formicarius larvae infected in the first three trials were removed from the tissue culture plates and placed in individual, modified White traps (White 1924) and incubated in the dark at 25±2°C. Cadavers were inspected daily for nematode emergence. The time required for infective juveniles to emerge from each cadaver was recorded. The percentage of cadavers that produced infective juveniles was recorded in both trials with G. mellonella, but in only the first and third trials with C. formicarius. Once emergence began, infective juveniles were removed from the outer well of each White trap once per week and counted. The time that emergence ceased was also recorded at which time cadavers were dissected and the numbers of infective juveniles within each cadaver were recorded. In the first two trials with C. formicarius, the biomass of each larva was recorded before each trial to determine if progeny production was related to host biomass.

Data Analysis

Most data were analyzed by least squares analysis of variance or regression techniques, accordingly (Zar 1984). Percentage infectivity was compared among nematodes within hosts by chi-square analysis (Conover 1980). Mean incubation periods and numbers of progeny produced were compared among nematodes by the Waller- Duncan K-ratio t test (Waller & Duncan 1984). Numbers of progeny produced per C. formicarius cadaver in the ﬁrst two trials were pooled and regressed on biomass to determine if reproduction was dependent upon host biomass.

RESULTS

Infectivity

Infectivity to G. mellonella larvae did not differ (X2 = 1.71, df = 2, P > 0.05) among nematodes. Adjusted percentages of infective juveniles that invaded larvae were 15.4, 16.0, and 26.7% for Heterorhabditis sp. Bacardis, Heterorhabditis sp. FL2122, and H. bacteriophora HP88, respectively. Infectivity to C. formicarius also did not differ (X2 = 1.9, df = 2, P > 0.05) among nematodes. Adjusted percentages of infective juveniles that invaded C. formicarius larvae were 30.3, 25.9, and 20.4% for Heterorhabditis sp. Bacardis, Heterorhabditis sp. FL2122, and H. bacteriophora HP88, respectively. Infectivity of the Heterorhabditis sp. Bacardis nematode was affected by the insect host (X2 = 4.9, df = 1, P < 0.05). A higher percentage of infective juveniles invaded C. formicarius larvae than G. mellonella larvae. Infectivity of Heterorhabditis sp. FL2122 and H. bacteriophora HP88 did not differ between hosts (X2 ≤ 2.3, df = 1, P > 0.05).

Jansson: Infectivity and Reproduction of Nematodes 367

Incubation Time

The incubation times of nematodes within the two hosts also did not differ (F ≤ 1.1, df = 2,4, P > 0.05) among nematodes. In G. mellonella larvae, Heterorhabditis sp. Bac- ardis, Heterorhabditis sp. FL2122, and H. bacteriophora HP88 required 11.9±0.1, 12.1±0.1, and 12.0±0.1 d, respectively, to emerge from cadavers. In C. formicarius larvae, these nematodes required only 8.7±0.2, 7.8±0.2, and 8.1±0.2 d, respectively, to emerge from hosts. Incubation periods for each nematode were signiﬁcantly shorter in C. formicarius than in G. mellonella, which is probably related to differences in host size.

Reproduction In the first trial, percentages of G. mellonella cadavers that produced progeny differed among nematodes (X2 = 10.7, df = 2, P < 0.01). Higher percentages of G. mellonella infected with Heterorhabditis sp. Bacardis (66.7%; n = 6) and FL2122 (71.4%; n = 7) produced progeny than those infected with H. bacteriophora HP88 (38.5%; n = 13). In the second trial, percentages of cadavers that produced progeny did not differ among nematodes (X2 = 3.6, df = 2, P > 0.05). All nematode-infected cadavers produced progeny in high percentages and were 75% (n = 8), 100% (n = 8), and 91% (n = 11) for Bacardis, FL2122, and HP88, respectively. Patterns of progeny production were consistent for each nematode in G. mellonella larvae (Fig. 1). Most progeny emerged from cadavers infected with Heterorhabditis sp. Bacardis (67.7-79.3%), Heterorhabditis sp. FL2122 (69.0-81.3%) and H. bacteriophora HP88 (85.6-96.2%) within 23 d after inoculation. Emergence of progeny declined 23 d after inoculation for all nematodes and lasted for up to 48 d after inoculation. Total progeny production in G. mellonella cadavers did not differ (F = 2.3, df = 2,12, P > 0.05) among nematodes in the first trial, but did differ (F = 4.4, df = 2,12, P < 0.05) among nematodes in the second trial (Table 1). Trends in the data were consistent between the two trials. More progeny were consistently produced by Heterorhabditis sp. Bacardis followed in decreasing order by Heterorhabditis sp. FL2122 and H. bacteriophora HP88. Considerably more progeny were produced in the second trial than in the first trial, and the reasons for this are unclear. Percentages of C. formicarius cadavers that produced progeny differed among nematodes in the two trials (X2 ≥ 15.3, df = 2, P < 0.001). In the first trial, the highest percentages of C. formicarius cadavers produced progeny when infected with Heter- orhabditis sp. Bacardis (90%; n = 20) followed in decreasing order by those infected with Heterorhabditis sp. FL2122 (41.7%; n = 12) and H. bacteriophora HP88 (35.3%; n = 17). In the third trial, cadavers infected with Heterorhabditis sp. FL2122 had the highest percentage of nematode production (90%, n = 20) followed by Heterorhabditis sp. Bacardis (58.8%; n = 17) and H. bacteriophora HP88 (46.7%; n = 15). No consistent patterns of emergence of infective juveniles from C. formicarius cadavers were found in the three trials (Fig. 2). In the first trial, emergence of Heter- orhabditis sp. Bacardis peaked 19 d after inoculation, whereas those of H. bacteriophora HP88 and Heterorhabditis sp. FL2122 peaked 28 d after inoculation. In trial 2, emergence of H. bacteriophora HP88 peaked 12 d after inoculation, whereas those of Heterorhabditis sp. FL2122 and Bacardis peaked 19 d after inoculation. Few progeny emerged after 19 d. In the third trial, emergence of all three nematodes peaked 16 d after inoculation and few progeny emerged after 16 d. Total progeny production in C. formicarius cadavers did not differ (F ≤ 1.3, df = 2,24, P > 0.05) among nematodes in the first two trials, but did differ (F = 11.2, df = 2,32, P < 0.001) in the third trial (Table 1). As found in G. mellonella, more progeny 368 Florida Entomologist 79(3) September, 1996

Fig. 1. Emergence patterns of infective juveniles from G. mellonella cadavers infected with three different heterorhabditid nematodes: Heterorhabditis sp. Bacardis strain, Heterorhabditis sp. FL2122 strain, and H. bacteriophora HP88 strain. Num- bers counted on the last sample date of each trial are the sum of those that emerged plus any remaining infective juveniles within cadavers. Jansson: Infectivity and Reproduction of Nematodes 369

TABLE 1. PROGENY PRODUCTION OF THREE HETERORHABDITID NEMATODES IN TWO IN- SECT HOSTS, CYLAS FORMICARIUS AND GALLERIA MELONELLA.

Trial

Host × Nematode 123

C. formicarius Heterorhabditis sp. 6,522.7(1,844.0)a1 7,919.5(1,061.0)a 8,625.6(1,162.6)a Bacardis Heterorhabditis sp. 5,610.6(1,086.5)a 6,896.8(1,167.1)a 4,834.6(676.7)b FL2122 H. bacteriophora 1,880.8(469.0)a 3,387.0(259.0)a 2,071.6(388.7)c HP88

G. mellonella Heterorhabditis sp. 194,222.5(54,228.0)a 238,692.2(45,442.2)a Bacardis Heterorhabditis sp. 122,580.2(34,579.4)a 229,903.4(30,825.3)a FL2122 H. bacteriophora 74,992.5(30,940.0)a 137,228.8(13,072.1)b HP88

1Data are means ± sem. Means within a column followed by the same letter do not differ by the Waller-Dun- can K-ratio t test (Waller & Duncan,1969).

were consistently produced (although not consistently signiﬁcant) by Heterorhabditis sp. Bacardis followed in decreasing order by Heterorhabditis sp. FL2122 and H. bacteriophora HP88. The nematode host inﬂuenced progeny production. Approximately 28.1-, 30.5-, and 43.4-fold more progeny were produced in G. mellonella than in C. formicarius for Heterorhabditis sp. Bacardis, Heterorhabditis sp. FL2122, and H. bacteriophora HP88, respectively. Progeny production in C. formicarius was not related to host biomass for any of the three nematodes (Bacardis: Y = 1054 + 646713X, F = 1.3, df = 1,31, P > 0.05, r 2 = 0.04; FL2122: Y = 91 + 687518X, F = 1.2, df = 1,13, P > 0.05, r 2 = 0.09; HP88: Y = -1901 + 510346X, F = 2.4, df = 1,6, P > 0.05, r 2 = 0.29).

DISCUSSION

Few differences in infectivity were found among nematodes within insect hosts; however, infectivity differed between hosts for Heterorhabditis sp. Bacardis strain. This nematode was more infective against the weevil, C. formicarius, than against the lepidopteran, G. mellonella, despite the fact that these nematodes were all reared in vivo in G. mellonella before experiments. Bedding et al. (1983) showed that H. bacteriophora was least infective against the host from which it was isolated, Heliothis punctigera (Wallengren). They also found that Steinernema feltiae (Filipjev) (= Neoa- plectana bibionis) isolated from Lucilia cuprina (Wiedemann) and Otiorhynchus sulcatus (F.) was least infective to these insects. Kaya (1987) suggested that the host from which a nematode is isolated in soil probably has little, if any, affect on the suitability of the nematode/host encounter. Entomopathogenic nematodes attack a broad 370 Florida Entomologist 79(3) September, 1996

Fig. 2. Emergence patterns of infective juveniles from C. formicarius cadavers infected with three different heterorhabditid nematodes: Heterorhabditis sp. Bacardis strain, Heterorhabditis sp. FL2122 strain, and H. bacteriophora HP88 strain. Num- bers counted on the last sample date of each trial are the sum of those that emerged plus any remaining infective juveniles within cadavers.

spectrum of insects; thus, isolation from soil is, in part, due to a chance encounter between the isolation host and the nematode. Kaya (1987) also suggested that, in certain cases, continued association with the same insect species may reduce virulence rather than enhance it. Incubation times also did not differ among nematodes within each species; however, incubation times were considerably shorter for all nematodes in the weevil, C. formicarius, than in the lepidopteran, G. mellonella. It is well known that emergence of infective juveniles is related to depletion of food reserves and crowding within the host cadaver (Kaya 1985, 1987). Patterns in total reproduction of nematodes differed among the three nematodes in both hosts. Heterorhabditis sp. Bacardis consistently produced more (although not consistently signiﬁcant) progeny than the other two nematodes in both hosts. These data concur with previous studies (Jansson & Lecrone 1994, Jansson et al. 1993) which showed that Heterorhabditis sp. Bacardis produced more progeny per cadaver than H. bacteriophora HP88 in G. mellonella larvae. Progeny production of Bacardis was higher in the previous study (range, 272,576-396,598 per cadaver) than in the present study (range, 137,229-238,692 per cadaver). However, a dose of 20 infective juveniles per larva was used in the previous study compared with only 1 per larva in the present study. Progeny production of H. bacteriophora HP88 was comparable between the two studies despite large differences in dose (range of previous study, 76,260-219,181 per cadaver; range of present study, 74,992-194,222 per cadaver). Jansson: Infectivity and Reproduction of Nematodes 371

Patterns of emergence from cadavers were consistent in G. mellonella, but not in C. formicarius. In G. mellonella, most infective juveniles emerged within 23 d after inoculation. In C. formicarius, emergence patterns varied among trials. As noted earlier, emergence of infective juveniles is related to depletion of food reserves and crowding (Kaya 1985, 1987). These factors may have been less apparent to emerging infective juveniles from C. formicarius due to the smaller size of this host compared with G. mellonella. Total progeny production of heterorhabditid nematodes in C. formicarius concurred with a previous report (Mannion & Jansson 1992), although progeny production was higher for FL2122 and lower for HP88 in the present study. The dose used might have affected these data. Mannion & Jansson (1992) used a dose of 25 infective juveniles per larva compared with 1 per larva in the present study. It is recognized that a laboratory bioassay that predicts performance of entomopathogenic nematodes in the field is needed to facilitate selection of nematodes in biological control programs (Hominick 1990, Mannion 1992). Mannion (1992) conducted Petri dish, sand, soil, and simulated field bioassays to select suitable entomopathogenic nematodes for the biological control of C. formicarius and consistently found that heterorhabditids were superior to steinernematids in all bioassay systems tested. The present bioassay system may also have potential for selecting suitable entomopathogenic nematodes, especially heterorhabditid nematodes, for C. formicarius. Morris et al. (1990) noted that both infectivity and reproduction within hosts were important attributes of nematodes capable of reinfesting new hosts in the field. The present study demonstrated that Heterorhabditis sp. Bacardis was most infective and reproductive against C. formicarius, and, for this reason, it should be a suitable nematode for use in biological control of this weevil. Recent results from field studies confirm this belief. Heterorhabditis sp. Bacardis was shown to be very efficacious at controlling weevil damage to storage roots and persisted at levels higher than those of all other nematodes tested, including H. bacteriophora HP88, S. carpocapsae (Weiser) All and S20, and S. feltiae N27 (Jansson et al. 1993). Collectively, these data suggest that the use of this single nematode/single host bioassay may be an important tool for identifying potential candidate heterorhabditid nematodes in biological control programs of target insect pests. More work is needed, however, to confirm this belief. These data also suggest that certain Neotropical and subtropical nematodes, Het- erorhabditis sp. Bacardis and FL2122, may be more suitable as biological control agents against the pantropical sweetpotato weevil, C. formicarius, than the temperate nematode, H. bacteriophora HP88 strain. Recent studies by Lawrence (1994) also suggested that certain Neotropical nematodes may be more suitable as biological control agents of this weevil; however, not all tropical and subtropical isolates were superior to all temperate isolates.

ACKNOWLEDGMENTS

I thank K. Ericsson for assistance with data collection and R. Miller (Biosys Inc., Columbia, MD), R. Gaugler (Rutgers University, New Brunswick, NJ), and C. M. Mannion and R. J. Zimmerman (University of Florida, I.F.A.S., T.R.E.C., Homestead) for critically reviewing the manuscript. Thanks are also extended to R. Miller for technical assistance. I thank G. C. Smart, Jr. (University of Florida, Gainesville) for providing Heterorhabditis sp. FL2122. Heterorhabditis sp. Bacardis was collected near San Juan, Puerto Rico by W. Figueroa (University of Puerto Rico, Rio Piedras) as part of a study [supported by the U.S. Department of Agriculture under CSRS Special Grant No. 91-34135-6134 (to R.K.J.)] to survey the Caribbean for entomopathogenic 372 Florida Entomologist 79(3) September, 1996 nematodes. These nematodes are currently being held at the University of Florida, I.F.A.S., Entomology and Nematology Department, Gainesville, FL and at the Depart- ment of Entomology, Rutgers University. The present research was supported by the U.S. Department of Agriculture under CSRS Special Grant No. 91-34135-6134 (to R.K.J.) managed by the Caribbean Basin Administrative Group (CBAG). Research was conducted at the University of Florida, I.F.A.S., T.R.E.C., Homestead. The manuscript was prepared, in part, at Merck & Co., Inc.

REFERENCES

BEDDING, R. A., A. S. MOLYNEUX, AND R. J. AKHURST. 1983. Heterorhabditis spp., Neoaplectana spp., and Steinernema kraussei: interspecific and intraspecific differences in the infectivity for insect hosts. Exp. Parasitol. 55: 249-257. CONOVER, W. J. 1980. Practical nonparametric statistics, 2nd ed. J. Wiley, New York. DUTKY, S. R., J. V. THOMPSON, AND G. E. CANTWEL. 1964. A technique for the mass propagation of the DD-136 nematode. J. Insect Pathol. 6: 417-422. HOMINICK, W. M., AND A. P. REID. 1990. Perspectives in entomopathogenic nematology, pp. 327-345 in R. Gaugler and H. K. Kaya [eds.], Entomopathogenic nematodes in biological control. CRC Press, Boca Raton, FL. JANSSON, R. K. 1991. Biological control of Cylas spp., pp. 169-201 in R. K. Jansson, and K. V. Raman [eds.], Sweet potato pest management: a global perspective. Westview Press, Boulder and London. JANSSON, R. K., AND S. H. LECRONE. 1994. Application methods for entomopathogenic nematodes (Rhabditida: Heterorhabditidae): aqueous suspensions versus infected cadavers. Florida Entomol. 77: 281-284. JANSSON, R. K., S. H. LECRONE, AND R. GAUGLER. 1991. Comparison of single and multiple releases of Heterorhabditis bacteriophora Poinar (Nematoda: Heter- orhabditidae) for control of Cylas formicarius (F.) (Coleoptera: Apionidae). Biol. Control. 1: 320-328. JANSSON, R. K., S. H. LECRONE, AND R. GAUGLER. 1993. Field efficacy and persistence of entomopathogenic nematodes (Nematoda: Steinernematidae, Heterorhab- ditidae) for control of sweetpotato weevil (Coleoptera: Apionidae) in southern Florida. J. Econ. Entomol. 86: 1055-1063. JANSSON, R. K., S. H. LECRONE, R. R. GAUGLER, AND G. C. SMART, JR. 1990. Potential of entomopathogenic nematodes as biological control agents of sweetpotato weevil (Coleoptera: Curculionidae). J. Econ. Entomol. 83: 1818-1826. KAYA, H. K. 1985. Entomogenous nematodes for insect control in IPM systems, pp. 283-302 in M. A. Hoy and D. C. Herzog [eds.], Biological control in agricultural IPM systems. Academic, New York. KAYA, H. K. 1987. Diseases caused by nematodes, pp. 453-470 in J. R. Fuxa and Y. Tanada [eds.], Epizootiology of insect diseases. J. Wiley, New York. LAWRENCE, J. L. 1994. Potential of Neotropical and temperate Heterorhabditid nematodes as biological control agents for the sweetpotato weevil, Cylas formicarius (Fabricius) (Coleoptera: Apionidae). M.S. Thesis, University of Florida, Gainesville, FL. MANNION, C. M. 1992. Selection of suitable entomopathogenic nematodes for biological control of Cylas formicarius (Coleoptera: Apionidae). Ph.D. Diss., Univer- sity of Florida, Gainesville, FL. MANNION, C. M., AND R. K. JANSSON. 1992. Comparison of ten entomopathogenic nematodes for biological control of the sweetpotato weevil (Coleoptera: Apion- idae). J. Econ. Entomol. 85: 1642-1650. MILLER, R. W. 1989. Novel pathogenicity assessment technique for Steinernema and Heterorhabditis entomopathogenic nematodes. J. Nematol. 21: 574 (abstract). MOLYNEUX, A. S., R. A. BEDDING, AND R. J. AKHURST. 1983. Susceptibility of larvae of the sheep blowfly Lucilia cuprina to various Heterorhabditis spp., Neoaplec- tana spp., and an undescribed steinernematid (Nematoda). J. Invertebr. Pathol. 42: 1-7. Jansson: Infectivity and Reproduction of Nematodes 373

MORRIS, O. N., V. CONVERSE, AND J. HARDING. 1990. Virulence of entompathogenic nematode-bacteria complexes for larvae of noctuids, a geometrid, and a pyralid. Canadian Entomol. 122: 309-319. POINAR, G. O., JR. 1990. Taxonomy and biology of Steinernematidae and Heterorhab- ditidae, pp. 23-61 in R. Gaugler and H. K. Kaya [eds.], Entomopathogenic nematodes in biological control. CRC Press, Boca Raton. TANADA, Y., AND J. R. FUXA, J. R. 1987. The pathogen population, pp. 113-157 in J. R. Fuxa and Y. Tanada [eds.], Epizootiology of insect diseases. J. Wiley, New York. WALLER, R. A., AND D. B. DUNCAN. 1969. A Bayes rule for the symmetric multiple comparison problem. J. American Stat. Assoc. 64: 1484-1489. WHITE, G. F. 1924. A method for obtaining infective nematode larvae from cultures. Science 66: 302-303. ZAR, J. H. 1984. Biostatistical analysis. Prentice-Hall, Englewood Cliffs, NJ.

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Morse and Lindegren: Suppression of Fuller Rose Beetle 373

SUPPRESSION OF FULLER ROSE BEETLE (COLEOPTERA: CURCULIONIDAE) ON CITRUS WITH STEINERNEMA CARPOCAPSAE (RHABDITIDA: STEINERNEMATIDAE)

JOSEPH G. MORSE1 AND JAMES E. LINDEGREN2,3 1Department of Entomology University of California Riverside, CA 92521

2USDA ARS Rt. Horticultural Crops Research Laboratory Commodity Protection & Quarantine Insect Research Unit 2021 South Peach Avenue Fresno, CA 93727

3Current address: Kapow Consulting, 2826 E. Los Altos, Fresno, CA 93710

ABSTRACT

A laboratory bioassay with larvae and adults of the Fuller rose beetle, Asynony- chus godmani Crotch, and the Kapow strain of Steinernema carpocapsae (Weiser), resulted in 12, 44, and 67% mortality with rates of 50, 150, and 500 infective juveniles per 3-wk old larva, respectively; 100% mortality with 150 infective juveniles per 3-mo old larva; and 24, 48, and 83% mortality with adults. A ﬁeld trial was conducted on Va- lencia orange trees harboring high levels of Fuller rose beetle late instar larvae. A single application of either the Kapow or All strain of S. carpocapsae each applied at 3 rates (50, 150, and 500 infective juveniles per cm2) reduced the number of emerging adult Fuller rose beetles a combined 55 and 38% compared with the water control the year following treatment and 79 and 82%, respectively, the 2nd year. Because of high variability between treatments, however, it was difﬁcult to choose between the two nematode strains or the 3 rates of each strain. Infested fruit was reduced by a combined mean level of 62% one year after treatment. Based on nematode recovery at 6 months and the further reduction of Fuller rose beetle emergence in the second year

374 Florida Entomologist 79(3) September, 1996 after application, we suspect that nematodes persisted and recycled in the soil and provided added control in the second year of the trial. In order to conduct the laboratory bioassay, a method of rearing Fuller rose beetle from egg to adult was developed. A corn rootworm artiﬁcial diet was used but resulted in high larval mortality. After 6 months on the diet under laboratory conditions, 12 parthenogenetic females resulted from approximately 1,000 eggs and 8 of these females produced viable eggs masses.

Key Words: Asynonychus godmani, parasitic nematodes, Kapow strain, All strain, Biovector™, biological control

RESUMEN

Un bioensayo con larvas y adultos de Asynonychus godmani Crotch, y la cepa Ka- pow de Steinernema carpocapsae (Weiser), dió como resultado 12, 44, y 67% de mortalidad con dosis de 50, 150 y 500 juveniles infectivos por larva de 3 días de edad, repectivamente; 100% de mortalidad con 150 juveniles infectivos por larvas de 3 meses de edad, y 24, 48 y 83% de mortalidad con adultos. Un esayo de campo fue condu- cido en árboles de nanranjo Valencia, con altos niveles de instares tardios de A. godmani. Una sola aplicación de la cepas Kapow o All de S. carpocapsae aplicadas cada una a 3 dosis (50, 150, y 500 juveniles infectivos por cm2) redujo el número de A. godmani emergidos en un 55 y 38%, comparado con un testigo con agua al año si- guiente al tratamiento, y 79 y 82%, respectivamente, en el segundo año. Sin embargo, debido a la alta variabilidad entre los tratamientos, fue difícil de escoger entre las dos cepas del nemátodo o las tres dosis de cada cepa. Los frutos infestados fueron reduci- dos por un nivel de media combinada del 62% un año después del tratamiento. Basas- dos en la recuperación de nemátodos en el segundo año después de la aplicación, sospechamos que los nemátodos persistieron, se reciclaron en el suelo, y aportaron control adicional en el segundo año del ensayo. Para conducir los bioensayos de laboratorio, fue desarrollado un método de cría para A. godmani desde el huevo hasta el adulto. Fue usada una dieta artiﬁcial para gusanos de la raíz del maíz pero produjo una alta mortalidad larval. Luego de 6 meses en la dieta bajo condiciones de laboratorio, 12 hembras partenogenéticas se obtuvie- ron de aproximadamente 1,000 huevos y 8 de esas hembras produjeron masas viables de huevos.

The Fuller rose beetle (FRB), Asynonychus godmani Crotch (Pantomorus cervinus (Boheman)), is a ﬂightless, parthenogenetic beetle which was reported in California as early as 1879 (Chadwick 1965). Until recently, however, it was considered to be a relatively unimportant pest of citrus in the state. In 1985, viable egg masses were found under the calyx of fruit shipped to Japan. Japan lists FRB as a quarantine pest and loads of fruit found to contain a single fruit infested with a viable egg mass are fumigated with methyl bromide. Fumigation damages the fruit and is costly to the grower. The FRB is thought to have one generation per year in California with peak emergence of adults from the soil occurring August to October although a few adults emerge each month of the year (Morse et al. 1987). After emerging from a pupal chamber in the soil, adults feed on the leaves of citrus for several wks prior to laying egg masses in cracks and crevices in the tree and under the calyx of fruit. Eggs hatch after 3-4 wks, larvae drop to the ground, enter the soil, and feed on citrus roots for most of a year before building a pupation chamber several centimeters below the soil surface.

Morse and Lindegren: Suppression of Fuller Rose Beetle 375

Methods of reducing FRB infestation of citrus shipped to Japan include: (1) monitoring citrus groves or fruit lots in the packinghouse and prioritizing fruit with low levels of viable FRB egg masses for shipment to Japan, (2) skirt-pruning and trunk treatments to exclude adult beetles from ovipositing on fruit in the tree, (3) foliar chemical treatments to kill adults in the tree, and (4) holding lemons in cold storage long enough to allow eggs to hatch (Morse et al. 1987, 1988, Haney & Morse 1988, Lakin & Morse 1989). None of these options has yet proven to be totally satisfactory due to a combination of logistic and economic constraints consistent with the need for almost 100% control required by quarantine protocols (Morse et al. 1987). This research investigates an alternative control strategy—use of the entomopathogenic nematode, Steinernema carpocapsae (Weiser) (= S. feltiae Filipjev), to reduce FRB larval populations in the soil thereby reducing subsequent adult emergence and oviposition on the fruit.

MATERIALS AND METHODS

FRB Laboratory Rearing

Adult FRB for larval rearing were collected from the Fairview citrus grove in Hemet, CA, and approximately 25 were isolated in each of 4 ventilated plastic sweater boxes (11 cm × 26 cm × 36 cm) with Valencia orange leaves, Citrus sinensis (L.) Os- beck, provided as a food source, and 10 tightly folded pieces of wax paper as an oviposition substrate. Eggs were collected, allowed to hatch, and larvae were reared for 3 wks or 3 mo at room temperature (24.4 ± 1.7°C) on a corn rootworm artiﬁcial diet (Diet Premix No. 1675, Bio-Serv. Inc., Frenchtown, NJ) as suggested by Dr. W. J. Schroeder, USDA-ARS, Orlando, FL. The diet mixture was prepared by mixing 25 liters cold water, 750 g agar, 60 ml formaldehyde, and 10 kg diet premix. The mixture was heated to 93˚C in a mixing machine, poured while still hot into small 25-ml plastic diet cups, dried for 5 d, and refrigerated prior to use.

Laboratory Nematode Bioassay

Preliminary laboratory bioassays with the Kapow selection of S. carpocapsae were conducted on ﬁve dates with adult FRB, and on a single date each with 3-week old (1- 2 mm in length) FRB larvae, and a small number of 3-month old larvae (about 5 mm in length). Adult FRB bioassays were conducted using beetles collected from a commercial citrus grove in Hemet, CA. Larvae used in the bioassays were reared from eggs collected in the laboratory. Kapow selection infective juveniles (IJ), selected since 1984 from the Mexican strain for increased production and pathogenicity (Agudelo-Silva et al. 1987, Linde- gren 1990), were produced at the USDA-ARS, Horticultural Crops Research Labora- tory, Fresno, California using the in vivo method described by Lindegren et al. (1993). The laboratory bioassays were conducted with recently harvested Kapow infective juveniles (IJ) counted by pipetting 0.02-ml drops of IJ-water suspension onto the bottom of a 10-cm diam, 1.5-cm high, plastic petri dish. The drops were overlaid with 9-cm diam ﬁlter paper, and deionized water was added to give a total volume of 1 ml per dish. The dish was covered with a lid and allowed to equilibrate for 1 h, then 10 FRB adults or larvae were added to each petri dish. Each test consisted of 10 replications of 0, 5, 15, and 50 nematodes per FRB (i.e., 0, 50, 150, and 500 IJ per dish). Adult FRB were bioassayed on 5 dates, 3-wk old larvae were bioassayed once, and because only twenty 3-mo old larvae were available, a single replicate was bioassayed using a wa-

376 Florida Entomologist 79(3) September, 1996 ter control and 150 IJ/ larva. The dishes were incubated at 25°C and 45% RH. Mor- tality was evaluated after 72 h, and data were corrected for control mortality using Abbott’s formula (1925). All larval and adult mortalities were veriﬁed by dissection.

Field Trial

A mature Valencia orange grove (Block 8, Fairview Ranch) in Hemet, CA was chosen for the ﬁeld trial based on high levels of FRB adults observed the previous year. Pre-treatment counts of fruit calyx infestation with FRB egg masses (both hatched and unhatched eggs) were taken 7 June 1989 by removing and examining the calyx of 20 fruit randomly picked from the exterior canopy of each of 160 trees. Seventy trees with the highest infestation levels were chosen and seven single tree replicates were assigned to each of 10 blocks based on the percent of fruit infested (there were 10 replicates per treatment). One tree in each block was assigned randomly to each of the following seven treatments: water control, Kapow strain at 50, 150, and 500 nematodes per cm2, and All strain (Biovector™, Biosys, Columbia, MD) at 50, 150, and 500 per cm2 applied to the soil surface beneath the drip line of the tree. Data trees were separated by a minimum of one untreated buffer tree in each direction down the row and were spread uniformly over an area of 13 rows by 47 trees (611 trees total). Tree spacing was 6.1 m both down and across the row, trees were approximately 5.5 m high, and the tree canopy extended approximately 5.5 m in diam. Tree skirts were pruned to 1 m above ground level and, if necessary, between data trees and adjacent trees in a row, so that beetles could access data trees only via the trunk. Treatments were applied 26 June 1989 using a 189-liter diaphragm pump sprayer (Lindegren et al. 1987) to apply the nematodes in 1.9 liters of water, uniformly under the drip line of each tree. The grove was irrigated for 9 h before and for 15 h after nematode applications were made. The irrigation system consisted of fanjet J2 minisprinklers (Tee- jet, Wheaton, IL) positioned mid-way between each trunk down the row emitting 60.0 liters of water per h. FRB adult emergence from the soil was monitored every two wks July to December for two years following treatment using three 58.4-cm square emergence boxes placed under each tree. Emergence box frames were constructed of wood, covered with screen, and were pushed one cm into the soil to prevent beetle escape. Counted adults were released under the sampled data tree. Fruit FRB egg mass contamination was determined again 21 June 1990 by examining 100 fruit selected randomly from the exterior of each data tree. Persistence of nematodes in the soil was bioassayed on 0-, 0+ (several h before and after treatment, respectively), 9, 15, 23, and 193 d post-treatment using soil cores (5.0-cm diam, 7.0-cm deep) collected from 5 of the 10 replicates from each treatment using a circular aluminum cylinder with a volume of 137 cm3. On each date, 3 soil cores were collected 1.5 m from the trunk of each of the same 5 replicate trees (initially chosen randomly) and were shipped by 2-d mail to Fresno where bioassays were conducted one d later using wax moth larvae, Galleria mellonella (L.). The three samples from each tree were mixed and an aliquot of 88.4 cm3 of soil was spread evenly in individual 15-cm diam by 2.5-cm high, plastic petri dishes with 10 larvae. Mortality was evaluated after 72 h incubation at 25°C. Soil samples from +15 and +23 d were quite dry and 10 ml of water was mixed with the soil prior to bioassays being conducted. Statistical analysis was conducted using PROC GLM in SAS (SAS Institute 1985) and mean separation was determined using the REGWQ option.

Morse and Lindegren: Suppression of Fuller Rose Beetle 377

RESULTS

FRB Laboratory Rearing

The corn rootworm diet appears suitable for rearing FRB larvae although mortality rates were quite high especially during early larval instars. Diet moisture content may be important and some manipulation of this might lead to increased survivorship. Under laboratory conditions, 12 adults were obtained from approximately 1,000 neonate larvae placed on the diet with the larval and pupal stages lasting ﬁve and one months, respectively. Of the 12 adults obtained, 8 laid egg masses within 4 wks of pupal emergence. Egg hatch was comparable to that observed with ﬁeld collected adults and neonate larvae appeared to be normal.

Laboratory Nematode Bioassay

The laboratory bioassay with 3-week old FRB larvae and the Kapow strain of S. carpocapsae resulted in 7% control mortality and 12, 44, and 67% corrected mortality with rates of 50, 150, and 500 IJ per larva, respectively (Table 1). A limited number of large FRB larvae (3 mo old, about 5 mm in length) were available and in the single bioassay conducted, 10% control mortality was observed and 100% mortality with 150 IJ per larva. Five laboratory bioassays were conducted with adult FRB and results were variable (Table 1). On average, 7.6% control mortality was observed and 24.1, 47.6, and 83.3% corrected mortality with 50, 150, and 500 IJ per larva, respectively. Based on the above data, a ﬁeld trial was determined to be worthwhile.

Field Trial

The Valencia orange grove in Hemet chosen for the ﬁeld trial had as high a level of FRB infestation for the two years prior to the trial as we have seen in California since 1985. As a result of choosing the most heavily infested trees for the trial and blocking treatments based on fruit egg mass infestation levels, pre-trial levels were similar among treatments and varied from 24.0 to 25.0% of the outside fruit infested just prior to harvest in June, 1989 (Table 2). Petal-fall for these fruit was May, 1988 and thus, they were exposed to FRB oviposition for 13 mo prior to harvest with the highest levels of oviposition expected approximately August to December, 1988 (Morse et al. 1987). Nematodes were applied 26 June 1989. Morse et al. (1987) monitored FRB soil emergence in four groves in Riverside and San Bernardino counties. One of these groves was the same Block 8 used in this S. carpocapsae trial and a second was a nearby grapefruit grove. Based on data from all four groves, monthly adult emergence was 2.7, 6.8, 32.0, 34.9, 16.0, 6.0, and 1.5% of the yearly emergence during the months of June through December, respectively, and only 0.1% of the FRB adults emerged in the months of January through May. Thus, at the time of nematode application, we assume that the majority of the FRB were in the late larval or pupal instars. Soil laboratory wax moth bioassays indicated that nematodes initially persisted in the soil for at least 23 d and the All strain was recovered in the persistence bioassay 193 d later (Table 2). Soil samples from the +9 d sample overheated in transport and low bioassay mortalities for this date were suspect. Although not statistically different than laboratory wax moth mortality seen with soil from the water control, numerically higher mortality was observed with soil from the 150 and 500 nematodes per

378 Florida Entomologist 79(3) September, 1996 1 . Mortality Percent Adult Percent Mean Corrected TRIALS

LABORATORY

LARVAE

AND

ADULTS

= 19.69). Percentage data were transformed using arcsine square-root frac- Percentage = 19.69). 3,16 FRB

CARPOCAPSAE

4 22 30 44 48 24.1 bc Corrected Percent Adult FRB Mortality in Tests on 5 Dates Tests Adult FRB Mortality in Corrected Percent 27-Nov-88 10-Jan-89 15-Jan-89 31-Jan-89 7-Feb-89 EOAPLECTANA N

2 OF

Larvae with 3-mo Old Mean Corrected Percent Mortality Percent SELECTION

KAPOW

THE

Larvae OF

with 3-wk Old Mean Corrected Percent Mortality Percent MPACT 1. I Means followed by the same letter within a column are not significantly different (p = 0.05, REGWQ; F REGWQ; Means followed by the same letter within a column are not significantly different (p = 0.05, conducted with the control and 150 IJ/larva. only a single replicate was Due to an insufficient number of 3-mo old larvae, 1 2 50 12 — ABLE Nematode Rate (IJ/FRB) tional mortality prior to analysis; untransformed means are listed. tional mortality prior to analysis; 150500 44 67 100 — 20 58 40 84 78 98 60 96 56 84 47.6 b 83.3 a Control 7 10 2 4 14 10 8 7.6 c T

Morse and Lindegren: Suppression of Fuller Rose Beetle 379 Fruit Percent Percent INFESTA- Infested

Post-trial Post-trial 21-Jun-90 FRUIT

AND

July-Dec. EMERGENCE

EETLE FRB Adult Emergence per Tree, Tree, Adult Emergence per FRB B OSE R ULLER F Mean reduction [46.6] [80.4] [61.5] [61.5] ADULT

Date NEMATODES

Days Post-treatmentDays [% Reduction Compared to Control] IMPACT

AND

SOIL

0- 0+ 9 15 23 193 1989 1990 Both years Percent Mortality in Laboratory Soil Bioassays with Wax Moth Larvae Wax with Mortality in Laboratory Soil Bioassays Percent 26-Jun-89 26-Jun-89 5-Jul-89 11-Jul-89 19-Jul-89 5-Jan-90 NEMATODES

0.02 1.58 46.51 1.74 1.48 1.31 1.35 1.89 10.34 5.21 3.73 Fruit OF Percent Percent Infested Pre-trial

7-Jun-89 1 . Nematode Strain ERSISTENCE TION 2. P Means followed by the same letter within a column are not signiﬁcantly different (p = 0.05, REGWQ). Percentage data were transformed using arcsine square-root fractional infestation Percentage REGWQ). Means followed by the same letter within a column are not signiﬁcantly different (p = 0.05, Rate 1 6,63 —5050 Control Kapow All 25.0 a 25.0 a 4 a 24.5 a 0 a 4 c 0 a 96 ab 0 a 82 b 2 a 0 a 0 a 0 a 0 a 0 a 0 a 0 a 0 a 0 a 24.1—a 13.4[44]a 12 a 19.0—a 4.5[76]b 17.9[59]b 15.2[37]a 43.1—a 3.9[64]b 3.0[84]b 10.7—a 18.2[58]b 3.6[66]b ABLE Nematode or mortality prior to analysis; untransformed means are listed. or mortality prior to analysis; 150150500 Kapow500 All Kapow 24.0 a AllF 25.0 a 24.5 a 2 a 24.5 a 0 a 4 a 94 ab 0 a 100 a 98 a 2 a 100 a 2 a 24 a 4 a 16 a 18 a 20 a 0 a 32 a 4 a 6 a 0 a 10 a 0 a 10.6[56]a 9 a 4.4[77]b 0 a 12.2[49]a 8.2[66]a 15.0[65]b 2.0[90]b 17.6[27]a 3.1[84]b 3.3[69]b 14.2[67]b 11.3[74]b 5.4[72]b 4.4[59]b 23.0[47]b 4.3[60]b 5.2[51]b (nem/sq cm) T

380 Florida Entomologist 79(3) September, 1996 cm2 treatments of the All strain at 9, 15, and 23 d and with the 500 per cm2 treatment of the Kapow strain at 15 d. Because the life cycle of S. carpocapsae is approximately 2 to 3 wks, we assume that this indicates persistence and recycling of the nematodes for at least one generation on FRB or other insects present in the soil and the 193 d data suggest persistence for 6 months. Soil emergence of adult FRB followed the general pattern reported by Morse et al. (1987), with the highest levels of emergence occurring in August through October, independent of treatments. In the water control plots, total emergence over the six months July - December was 24.1 and 19.0 adults collected from the three emergence boxes per tree for 1989 and 1990, respectively. In 1989, treatments reduced emergence 27% (All 500) to 66% (Kapow 500) (mean of 47% over the 6 treatments) compared with the water control although data were not statistically significant due to high variability among replicates. In 1990, however, all nematode treatments resulted in statistically reduced emergence, 72% to 90% compared with the control, with an average reduction for all 6 treatments of 80%. Based on this further reduction of FRB emergence in 1990 over that seen in 1989, and the 193 d Galleria bioassay, we suspect that nematode populations persisted in the soil beyond 1989 and gave added control of FRB larvae and pupae in the second year of the trial. Fruit infestation by FRB egg masses one year after the trial began were significantly reduced 51 (All 500) to 69% (Kapow 150) with an average of 62% compared with levels in the water control. Note that these mature Valencia oranges were set May, 1989 and thus, the majority of egg masses observed resulted from adults emerging July to December 1989. We have no clear explanation for the decline in egg mass infestation in the water control trees from June, 1989 (25.0% infested) to June, 1990 (10.7%). Data trees were separated by at least one buffer tree down the row and a total of 60 of 611 trees in this part of the grove were treated with nematodes in this trial. It is possible that nematode applications in June, 1989, to selected heavily infested trees distributed throughout the grove, suppressed FRB levels in the entire block resulting in reduced oviposition in the control trees. Persistence and dispersal of entomopathogenic nematodes has been observed in other tests (Rick Miller, Biosys, Inc., personal communication). This possibility, however, remains unproven. Although results were not statistically separable among the six nematode treatments, there were numerical trends. With the Kapow strain, there was a consistent trend towards reduced emergence and reduced post-trial fruit infestation with higher rates of nematodes. With the All strain, however, despite the numerically higher levels of nematode soil persistence as indicated by the G. mellonella laboratory bioassays, FRB emergence and fruit infestation was numerically higher at the highest rate of 500 nematodes per cm2. Because of this unusual result, it is difficult to choose between the Kapow and All strains although the Kapow strain at 500 nematodes per cm2 gave the best results numerically.

DISCUSSION

Parasitic rhabditid nematodes have been shown to be effective against a number of insect pests (Poinar 1971, 1979, Kaya 1985, Morris 1985, Gaugler & Kaya 1990, Parkman et al. 1994), have been evaluated in a number of trials against root weevils (Diaz & Hernandez 1978, Bedding & Miller 1981, Montes et al. 1981, Simons 1981, Beavers 1984, Belair & Boivin 1985, Roman & Figueroa 1985, Rutherford et al. 1987, Schroeder 1987, 1990a, b, 1992, 1994, Boivin & Belair 1989, Downing et al. 1991, Mannion & Jansson 1992, Burlando et al. 1993, Mracek et al. 1993, Smith 1994, Dun- can & McCoy 1996) and are currently applied commercially for control of Diaprepes

Morse and Lindegren: Suppression of Fuller Rose Beetle 381 abbreviatus L. in Florida citrus. Results in different trials have been variable, however, and as might be expected, appear to be influenced by: application method; air, water suspension, and soil temperatures; soil type; soil salinity; soil moisture level as influenced by irrigation or natural rainfall; host species; vertical distribution of the host species; the level of alternate hosts available for nematode parasitization and persistence; and nematode species and quality. In this study, we evaluated soil persistence of nematodes using the following three parameters: (1) laboratory bioassays of field-collected soil using wax moth larvae as substitute hosts, (2) reduction in adult FRB emergence for two years after treatment, and (3) reduction in FRB egg masses deposited on fruit one year after treatment. With one nematode application (June 25, 1989), and three rates of two strains of S. carpocapsae, a combined average of 95% of the wax moth larvae were killed in soil collected 1 day after treatment. In addition, emergent adult and egg mass reductions of 46.6 (year 1), 80.4 (year 2), and 61.5% were observed, respectively. An exceedingly high level of FRB control is required to reduce egg mass contamination of fruit to the degree required to pass quarantine inspection in Japan (Morse et al. 1987). In citrus groves with moderate to high levels of FRB, it appears that repeated nematode applications, perhaps multiple applications per year over 2-3 successive years, might be required to approach this level of control. Cost is a factor, although treatment with 2 billion nematodes per acre (50 IJ/ cm2) is considered economically feasible (Smith 1994). Because large prepupal larvae are most susceptible, nematode applications should be optimally timed to impact this stage. Nevertheless, with a single treatment applied in June, 1989, the additional reduction of FRB emergence observed in 1990 beyond that seen in 1989 is encouraging. The obvious impli- cation is that nematodes persisted in the soil, providing additional control of FRB larvae in 1990. Application costs may be reduced by the use of more efficacious entomopathogenic nematodes. FRB susceptibility should be evaluated for S. riobravis Cabanillas, Poinar & Raulston, a more temperature tolerant nematode, indigenous to the Rio Grande Valley of Texas (Cabanillas et al. 1994). This commercially available nematode was the most efficacious of six species and strains tested with a substitute host - G. mellonella (Lindegren et al. 1993), persisted for 3 months in spring field tests in Phoenix, AZ (Lindegren et al. 1995), and was significantly more effective than S. carpocapsae in mid-summer small-scale field tests on pink bollworm, Pectinophora gossypiella (Saunders) in Phoenix (Lindegren et al. 1994). This study indicates nematode establishment and reduction of FRB populations into the second year after application. Because FRB infested trees can be readily identified by notched leaves, costs might also be reduced by applying nematodes to single previously identified and marked infested trees throughout the orchard, similar to the method used in this study.

ACKNOWLEDGMENTS

We thank Alan Urena, Karen Valero, Joyce Nishio-Wong, and Pamela Watkins for technical assistance; Harry Griffiths and Joe Barcinas of Entomological Services, Inc., John Gless of Gless Ranch, Inc., and Randy Noe for making available the field site at the Fairview Ranch; William Schroeder for advice on rearing FRB on the corn rootworm artificial diet; and Rick Miller of Biosys, Inc. for assistance with the field applications and for providing the Biovector™ strain of S. carpocapsae. This research was supported in part by grants from the California Citrus Research Board.

382 Florida Entomologist 79(3) September, 1996

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LINDEGREN, J. E., T. J. HENNEBERRY, P. V. VAIL, L. J. F. JECH, AND K. A. VALERO. 1994. Current status of pink bollworm control with entomopathogenic nematodes. Proc. Beltwide Cotton Prod. & Res. Conf. Vol. 2: 1242-1243. LINDEGREN, J. E., K. A. VALERO, AND L. J. F. JECH. 1993. Host searching response of various entomopathogenic nematodes: sand barrier bioassay. Proc. Beltwide Cotton Prod. & Res. Conf. Vol. 2: 1044-1045. LINDEGREN, J. E., K. A. VALERO, AND B. E. MACKEY. 1993. Simple in vivo production and storage methods of Steinernema carpocapsae infective juveniles. J. Nema- tol. 25: 193-197. MANNION, C. M., AND R. K. JANSSON. 1992. Comparison of ten entomopathogenic nematodes for control of sweetpotato weevil (Coleoptera: Apionidae). J. Econ. Entomol. 85: 1642-1650. MONTES, M., E. ARTAGA, AND R. BROCHE. 1981. Control of the citrus root weevil with the nematode, Neoaplectana P2M in citrus nurseries. Proc. Int. Soc. Citricul- ture 1981, Vol. 2: 670-672. MORRIS, O. N. 1985. Susceptibility of 31 species of agricultural insect pests to the entomogenous nematodes Steinernema feltiae and Heterorhabditis bacteriophora. Canadian Entomol. 117: 401-407. MORSE, J. G., D. A. DEMASON, M. L. ARPAIA, P. A. PHILLIPS, P. B. GOODELL, A. A. URENA, P. B. HANEY, AND D. J. SMITH. 1988. Options in controlling the Fuller rose beetle. Citrograph 73: 135-140. MORSE, J. G., P. A. PHILLIPS, P. B. GOODELL, D. L. FLAHERTY, C. J. ADAMS, AND S. I. FROMMER. 1987. Monitoring Fuller rose beetle populations in citrus groves and egg mass levels on fruit. The Pest Control Circular, Sunkist Growers, Inc., On- tario, CA. No. 547. 8 pp. MRACEK, Z., K. JISKRA, AND L. KAHOUNOVA. 1993. Efficacy of steinernematid nematodes (Nematoda: Steinernematidae) in controlling larvae of the black vine weevil, Otiorrhynchus sulcatus (Coleoptera: Curculionidae) in laboratory and field experiments. European J. Entomol. 90: 71-76. PARKMAN, J. P., J. H. FRANK, K. B. NGUYEN, AND G. C. SMART, JR. 1994. Inoculative release of Steinernema scapterisci (Rhabditida: Steinernematidae) to suppress pest mole crickets (Orthoptera: Gryllotalpidae) on golf courses. Environ. Ento- mol. 23: 1331-1337. POINAR, G. O., JR. 1971. Use of nematodes to control insects, pp. 181-203 in H. D. Burges and N. W. Hussey [eds.], Microbial Control of Insects and Mites. Aca- demic, London. POINAR, G. O., JR. 1979. Nematodes for Biological Control of Insects. CRC, Boca Ra- ton, FL. ROMAN, J., AND W. FIGUEROA. 1985. Control of the sugarcane rootstalk borer weevil, Diaprepres abbreviatus (L.) with entomogenous nematode Neoaplectana carpocapsae Weiser. J. Agric. Univ. Puerto Rico 69: 153-158. RUTHERFORD, T. A., D. TROTTER, AND J. M. WEBSTER. 1987. The potential of heterorhabditid nematodes as control agents of root weevils. Canadian Entomol. 199: 67-73. SAS INSTITUTE, INC. 1985. SAS User’s Guide: Statistics, Version 5 Edition. SAS Insti- tute, Inc., Cary, NC. 956 pp. SCHROEDER, W. J. 1987. Laboratory bioassays and field trials of entomogenous nematodes for control of Diaprepres abbreviatus (Coleoptera: Curculionidae) in citrus. Environ. 1 Entomol. 16: 987-989. SCHROEDER, W. J. 1990a. Water-absorbent starch polymer as a survival aid to entomogenous nematodes for control of Diaprepres abbreviatus (Coleoptera: Curcu- lionidae) in citrus. Florida. Entomol. 73: 129-132. SCHROEDER, W. J. 1990b. Suppression of Diaprepres abbreviatus (Coleoptera: Curcu- lionidae) adult emergence with soil application of entomopathogenic nematodes (Nematoda: Rhabditida). Florida Entomol. 73: 680-683. SCHROEDER, W. J. 1992. Entomopathogenic nematodes for control of root weevils of citrus. Florida Entomol. 75: 563-567. 384 Florida Entomologist 79(3) September, 1996

SCHROEDER, W. J. 1994. Comparison of two steinernematid species for control of the root weevil Diaprepes abbreviatus. J. Nematol. 26: 360-362. SIMONS, W. R. 1981. Biological control of Otiorrhynchus sulcatus with Heterorhab- ditid nematodes in the glasshouse. Netherland J. Pl. Path. 87: 149-158. SMITH, K. A. 1994. Control of insect pests with entomopathogenic nematodes. I. Con- trol of weevils with entomopathogenic nematodes. Food & Fertilizer Technology Center, Republic of China, Taiwan, Technical Bull. 139: 1-13.

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384 Florida Entomologist 79(3) September, 1996

GRAVIMETRIC METHOD FOR THE MEASUREMENT OF SUGAR CONSUMPTION BY ADULT VELVETBEAN CATERPILLAR (LEPIDOPTERA: NOCTUIDAE)

XIKUI WEI1 AND SETH J. JOHNSON Department of Entomology, Louisiana Agricultural Experiment Station, Louisiana State University Agricultural Center, Baton Rouge, LA 70803

1Current address: Texas A&M University, Research & Extension Center, 17360 Coit Road, Dallas, Texas 75252

ABSTRACT

Determination of food consumption by adult Lepidoptera, especially noctuid species, has not been well studied, possibly because of a lack of appropriate methodology. In order to measure sugar consumption by velvetbean caterpillar, Anticarsia gemmatalis Hübner, a gravimetric method was developed and is presented here together with results obtained using this method. After 24-h exposure to a moth, liquid consumption by the moth was determined by weighing the remaining solution on an electronic balance. The amount of solution consumed was calculated by converting the difference between the weight of the remaining solution and that of the unfed control to a volumetric value. Velvetbean caterpillar moths consumed a significantly greater volume of solution at lower sugar concentrations than at higher concentrations. Fe- male moths from larvae reared on soybean foliage in field cages consumed three times that of moths from larvae reared on artificial diet in the laboratory. This method was simple, accurate, and required minimum handling of test insects. It was suitable for the measurement of food consumption of velvetbean caterpillar moths and could also be suitable for other lepidopteran species.

Key Words: Anticarsia gemmatalis, adult feeding, sugar consumption, compensatory feeding

RESUMEN

La determinación del consumo de alimento por lepidópteros adultos, especial- mente por especies de noctuidos, no ha sido bien estudiada, posiblemente debido a la falta de una metodología adecuada. A ﬁn de medir el consumo de azúcar por la oruga del frijol de terciopelo, Anticarsia gemmatalis Hübner, fue desarrollado un método gravimétrico que es presentado aquí junto a los resultados obtenidos mediante su uso. Luego de 24 horas de exposición a la polilla, el consumo del líquido fue determinado

Wei & Johnson: Measurement of Sugar Consumption by a Moth 385 mediante el pesaje en una balanza electrónica de la solución remanente. La cantidad de solución consumida fue calculada mediante la conversión a un valor volumétrico de la diferencia entre el peso de la solución remanente y el del testigo donde las polillas no se alimentaron. Las polillas consumieron un volumen signiﬁcativamente mayor de las soluciones con baja concentración de azúcar. Las hembras de larvas criadas con fo- llaje de frijol de soya en jaulas de campo consumieron tres veces más que las polillas de larvas criadas en dieta artiﬁcial en el laboratorio. Este método fue simple, preciso, y requirió una mínima manipulación de los insectos. El método fue adecuado para la medida del consumo de alimento de A. gemmatalis y podría además ser adecuado para otras especies de lepidópteros.

The adult velvetbean caterpillar, Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae), like many other noctuid species, feeds on floral and extrafloral nectars (Lukefahr & Rhyne 1960). The moths have also been observed feeding on crushed grapes (Greene et al. 1973), cut apples (X. W., personal observation), whitefly honey- dew on hemp sesbania, Sesbania exaltata (Raf.) (Collins & Johnson 1985), and honey- dew on seed heads of a few species of grasses infected with ergot fungi including Bahiagrass, Paspalum notatum Flugge (Greene et al. 1973) and Dallisgrass, Paspalum dilatatum Poiretat (Collins & Johnson 1985, Wei & Johnson 1995). The importance of nectar sources to adult Lepidoptera has been demonstrated in studies that have shown increased numbers of eggs (Collins & Johnson 1985), increased egg-laying (Wales 1983, Adler 1989, Mason et al. 1989), increased population density (Lukefahr & Rhyne 1960, Collins 1984), insect pest outbreaks in certain agro- ecosystems (Burleigh 1972), and probably enhanced long-distance dispersal (Mason et al. 1989). However, comprehensive knowledge of adult food consumption is needed before we can understand how adult Lepidoptera utilize nectars in nature, and how important adult feeding is to reproduction and population dynamics, especially noctuids of economic importance. This knowledge could improve IPM programs or adult control technologies. However, adult feeding of most lepidopteran species, including noctuids, has been poorly studied (Adler 1989). One of the reasons appears to have been a lack of appropriate experimental methodology. In measuring liquid consumption by adult Lepidoptera, the following methods have been explored: an artificial flower consisting of graduated pipette and small funnel (Alm et al. 1990), hollow plastic stoppers covered with Parafilm® to serve as a reservoir for artificial nectar (Pivnick & McNeil 1985), weighing the test insect before and after feeding (Pivnick & McNeil 1985, Adler 1989), and using a 50 or 100 ul microcapillary to hold sugar solution to feed test insects (May 1985a, b). Adler (1989) adopted a microcapillary method combined with spring-action clothes pins to bind the wings of Helicoverpa zea (Boddie) moths to restrain them while they were feeding. While the above mentioned techniques were obviously acceptable for measuring consumption in the studies cited, each one has some problem or problems which would preclude its use in measuring daily or lifetime sugar consumption by a noctuid moth. Also, previous studies with Lepidoptera, except for Alm et al. (1990) with the white cabbage butterfly, have used uptake rate as the consumption variable rather than daily consumption. In uptake rate measurement, test insects were given food once or twice a day and the liquid ingested per unit time was then calculated (May 1985a, Piv- nick & McNeil 1985, Adler 1989). However, because daily consumption was not determined, the estimates to predict daily and total consumption were not suited to our objectives which were to determine the effects of sugar consumption on activity, feed-

386 Florida Entomologist 79(3) September, 1996 ing pattern, lipid accumulation, and fecundity of the velvetbean caterpillar. To reach this objective we needed a technique that would give us absolute values of sucrose consumed on a daily basis. In the present study, we developed a new method to measure the daily and lifetime total nectar consumption by velvetbean caterpillar adults.

MATERIALS AND METHODS

Insect

Two sources of test insects were used in this study, lab-reared and field-reared moths. Laboratory rearing procedures were a modification of the techniques of Greene et al. (1976) and King & Hartley (1985) which had been used for maintaining the velvetbean caterpillar colony in our laboratory from 1991 to 1994 (Wei 1995). Field- reared moths were obtained from larvae reared in field cages in a soybean field at the Ben Hur Experiment Farm of Louisiana State University Agricultural Center, Baton Rouge, LA. A 1.5-ha field was planted with a soybean variety, ‘Pioneer 9791’, which is susceptible to velvetbean caterpillar. During the summer of 1993, six cages, 1.8 × 1.8 × 1.8 m, were set-up in the field when the soybeans were mostly at developmental stage R2 (Fehr & Caviness 1979). For ease of collection and reduction of damage, the procedures of Wei & Johnson (1994) for rearing velvetbean caterpillar larvae and harvesting their pupae from field cages were followed. The ground inside the cages was covered with one layer of saran screen (32 × 32 mesh) over which a 2-cm layer of coarse vermiculite (STRONG-LITE® Products Corp., Seneca, IL) was evenly placed at time of cage set-up. Forty lab-reared adults (20 males and 20 females) were released in each cage and removed three days later. After the larvae emerged, densities were monitored to ensure they were below levels which would result in total defoliation of soybean foliage prior to pupation. After most larvae had pupated, pupae were collected and transported to the laboratory. Pupae from either field cages or laboratory sources were sexed, and placed in individual 155 ml plastic diet cups (SOLO® Cup Company, Urbana, IL). They were held in a walk-in environmental chamber at 27 ± 1°C, 85 ± 5% RH, and a photoperiod of 14:10 (L:D) until emergence. We performed three different experiments to examine sugar consumption by velvetbean caterpillar moths using the gravimetric method. First, we examined total liquid consumption for 10 female and 10 male field-reared moths over their entire adult life span when the insects were provided with both 30% sucrose solution and distilled water. Second, we measured daily consumption over 14 days for 10 lab-reared female moths and 10 field-reared female moths when the insects were provided with both 30% sucrose solution and distilled water. Third, we measured consumption of five concentrations of sucrose solutions (0%, 5%, 10%, 20% and 40%) in the 48 h after adult emergence. We tested 10 field-reared female moths at each of these concentrations. In each test, only moths that emerged within a 24-h period were randomly selected and transferred singly from the diet cups to 1.8 liter paper cartons (16 cm high × 13 cm in diam, Fonda® Group Inc., Union, NJ), covered with cheese cloth secured with a rubber band.

Experimental Conditions and Evaporation Monitoring

The experiments were conducted in a walk-in environmental chamber at 27 ± 1°C, 85 ± 5% RH, and a photoperiod of 14:10 (L:D). To monitor evaporation under such conditions, we measured the evaporation of distilled water and 30% sucrose solution over a 24-h period using the gravimetric method described in the present study. We also ex-

Wei & Johnson: Measurement of Sugar Consumption by a Moth 387 amined the effects of location (horizontal and vertical locations inside the walk-in environmental chamber) on evaporation.

Development of Methods for Measuring Sugar Consumption

When we were investigating methods to use for measuring sugar consumption by adult moths, we tested most of the available methods. We modified the method used by Pivnick & McNeil (1985) (using hollow plastic stoppers covered with Parafilm® ) by weighing the plastic stoppers instead of weighing the test insects before and after feeding. However, the moths could not easily locate the food source. Weighing test insects before and after feeding not only was too time-consuming, but some test insects were damaged. More importantly, weighing the insect before and after feeding would certainly not be suitable for the velvetbean caterpillar moth which has the ability to quickly excrete liquid from both proboscis and anus during or shortly after feeding (X. W., S. J. J., & Abner M. Hammond, unpublished data). We also tested the method of using a 100 ul microcapillary to hold the sugar solution to feed test insects (May 1985a, b). This method was not suitable for our experiment because threading the proboscis manually into the openings of the microcapillary was not practical when measuring food consumption for 24 h per day over an extended period of time and with multiple test insects involved. We did not use the method described by Adler (1989) (using a microcapillary to hold the artificial nectar and spring-action clothes pins to bind the wings of moths to restrain them while they were feeding) because we felt that refrigerating, weighing, and pinning the wings of moths each time they were fed would cause an unacceptable amount of damage to the moths. The method of Alm et al. (1990) (which involved the use of a small funnel connected to a graduated pipette though plastic tubing) was designed to measure the daily nectar consumption by white cabbage butterfly. However, this method was not sensitive enough to measure consumption by a velvetbean caterpillar moth. Using a similar apparatus, the difference in the liquid levels could not be detected before and after 200 ul of liquid were removed from a 5 ml funnel connected to a 0.1 ml pipette. Also, evaporation associated with this method would be problematic because of evaporation from the relatively large surface area of the funnel although no dimensions of the “funnel” or “graduated pipette” were given in their method. During the development of our method, we also experimented with several kinds of containers as a reservoir for the sugar solution, including clear plastic micro tubing, plastic cells cut from blister cards (used for packing medicine by pharmacies), hollow plastic stoppers, porcelain liquid holders, and polystyrene wells. However, none of them, except for the small polystyrene wells, allowed for easy and accurate measurement without the risk of spill. The polystyrene wells, which were chosen for our method, were big enough for moths to locate the food source easily but, at the same time, small enough to minimize evaporation.

Feeding Platform

Feeding solutions were held in the 360 ul individual polystyrene wells (10.7 mm high × 6.9 mm in top diam) cut from EIA/RIA 8-well strips (Costar Corporation, Cam- bridge, MA). Two polystyrene wells were embedded 2-cm apart in a piece of styrofoam (5 × 4 × 2 cm) which formed a feeding platform. In the ﬁrst and second tests, 300 ul of distilled water and 300 ul of 30% sucrose solution (wt:vol) were separately provided in the 2 wells in each feeding platform. A 30% sugar solution was selected based on experimental results that indicated it was an ap-

388 Florida Entomologist 79(3) September, 1996 propriate concentration for velvetbean caterpillar moths (X. W., S. J. J., & Abner M. Hammond, unpublished data). For the third test, only sucrose solution of the designated concentration was added to the 2 wells in a platform with 300 ul in each well. A feeding platform containing measured solution was then placed in each paper car- ton in the morning (0800, CST), and the moths were allowed to feed for 24 h. Each moth received new wells and solutions daily for the designated experimental period.

Weighing Procedure

At the end of each 24-h exposure period, wells from each feeding platform were transferred to a 32-cell weighing board made of a 10 × 15 × 2 cm piece of styrofoam with rows of holes punched about the size of the outside diameter of the well. The following steps were carried out: (1) the weighing board (with wells and residual liquid) was placed on an electronic balance (precision to 0.001 g), and the balance was zeroed; (2) the liquid was pipetted from one well and the inside of that well dried with a paper towel; (3) the negative number then displayed indicated the weight of the liquid left in the well by the moth after feeding. This three-step procedure was repeated for each well in succession until all wells in a board were weighed. One of the advantages of the weighing procedure was that handling of each individual well was unnecessary, reducing the risk of spill while weighing and also increasing handling efﬁciency. The residual weight was subtracted from the weight of the appropriate control (distilled water or sugar solution) included for monitoring evaporation. The volumetric estimate of sugar solution or distilled water ingested was calculated by converting the solution weight to volume.

Statistical Analysis

All statistical analyses were performed using SuperANOVA version 1.11. Tukey- Kramer’s test (Gagnon et al. 1989) was used for means separation.

RESULTS AND DISCUSSION

Evaporation Monitoring

The monitoring of evaporation was very important to the accuracy of the consumption measurement. We found: (1) there was no significant difference (F= 0.0514; df= 1, 46; P = 0.8217) between distilled water and 30% sucrose solutions in the volume evaporated [see Alm et al. (1990)]; (2) the environmental chamber was uniform in effects on evaporation when relative humidity was high (85 ± 5%) and air circulation was maintained as evidenced by the finding that there was no significant difference in evaporation among various locations (F= 0.1209; df= 1, 22; P = 0.7314); (3) the average daily evaporation from a well was 24.87 ± 1.7 ul (8.3% water evaporated). Sugar concentration increased about 3% due to water loss by evaporation during a 24-h period, which was not considered significant in altering the sucrose concentration [velvetbean caterpillar moths were observed to feed on an extremely wide range of sugar solutions, ranging from 1% concentration to even sugar crystals (X. W., S. J. J., & Abner M. Hammond, unpublished data)]. However, in order to monitor evaporation closely, we included 2 controls (same as treatment except with no test insect) for each treatment group (usually 10 cartons) during all experiments. The mean weight of the liquid of these unfed controls served as the post-evaporation reference from which we

Wei & Johnson: Measurement of Sugar Consumption by a Moth 389 subtracted the residual liquid weight obtained after feeding by a moth for 24 h. Volu- metric consumption data were then obtained by converting the resulting weight (the difference in weight between fed and unfed) to a volumetric value.

Consumption

Using this gravimetric method, we obtained the daily food consumption pattern of male and female velvetbean caterpillar moths. Both male and female moths consumed heavily during the first 2 days after emergence, with peak consumption occurring on the first day. Females consumed 314.1 ± 72.8 ul in the initial 2 days when provided with 30% sucrose solution and distilled water, which represents 37% of the total consumption over their adult lifetime (19.8 ± 8.0 days at 27°C). Males consumed 356.9 ± 69.8 ul in the initial 2 days representing 36.9% of their total food consumption. After day 2 they consumed a nearly constant daily volume of 25.6 ± 8.1 ul for females and 25.8 ± 5.1 ul for males. The differences in consumption between day 2 and day 3 were found significant for both male (F= 16.6798; df= 1, 18; P= 0.007) and female moths (F= 6.7824; df= 1, 18; P= 0.0179). On average, the total liquid consumption for the entire life span of a female moth was 849.3 ± 328.9 ul. Males consumed a total of 964.0 ± 189.6 ul of liquid during their entire life of 27.7 ± 6.4 d. There was no significant difference (F= 0.9128; df= 1, 18; P= 0.3520) in total liquid consumption between sexes. Even with the presence of 30% sucrose solution, distilled water was still taken up daily by moths until death, at an almost constant volume: 8.1 ± 4.0 ul for females and 7.6 ± 3.7 ul for males. A total of 150.4 ± 68.2 ul and 211.0 ± 57.4 ul distilled water was consumed per female or male moth, representing respectively, 17.6% and 21.9% of their total liquid consumption. Total sucrose consumed in dry weight was 0.21 ± 0.08 g for a female and 0.23 ± 0.05 g for a male moth when they were provided with both 30% sucrose solution and distilled water. The daily total liquid consumption of field- versus lab-reared velvetbean caterpillar female moths is compared in Fig. 1. Females from larvae reared on soybean foliage in field cages consumed 644.2 ± 176.3 ul liquid in the initial 2 weeks compared with 204.8 ± 93.1 ul for females from the laboratory. The consumption by field-reared moths was 3 times that of lab-reared moths, showing a highly significant difference (F = 48.58; df= 1, 18; P= 0.0001) in consumption between the 2 sources, although there was no statistical difference in pupal weights between these 2 groups (F = 1.767; df= 1, 398; P= 0.1844). The lab-reared moths consumed much less, even during the peak consumption period which occurred in the first 2 days after emergence. Furthermore, distilled water constituted 22.5% of the total liquid consumption by the lab-reared moths while distilled water represented only 16.8% of the total liquid consumption by field moths. These results suggest that laboratory insects may have more stored energy carried over from larval to the adult stage and thus do not depend as heavily on feeding for their reproductive needs. Mason et al. (1989) found much more adult lipid when larvae of soybean loopers, Pseudoplusia includens (Walker), were reared on artificial medium in the laboratory than in adults from larvae reared on plants in the field. Also, this finding suggests that laboratory insects may be inappropriate experimental subjects for food consumption studies because their consumption is not representative of the consumption of natural populations. This could be especially important in research on adult feeding stimulants. Moths consumed significantly more solution when they fed at low concentrations than at high concentrations (Fig. 2; F= 61.54; df = 4, 44; P= 0.0001). As the sugar concentration increased, the volume consumed decreased dramatically. In the initial 48- h feeding period after emergence, they consumed 657.5 ± 151.4; 526.4 ± 134.4; 346.6

390 Florida Entomologist 79(3) September, 1996

Fig. 1. Daily liquid consumption by 10 females from larvae reared on soybean foliage in ﬁeld cages and 10 females from larvae reared on artiﬁcial diet in the laboratory. Moths were provided with both 30% sucrose solution and distilled water.

Fig. 2. Consumption by female velvetbean caterpillar moths during the initial 48- h period after emergence when provided with various concentrations of sucrose solution. Ten ﬁeld-reared moths were tested at each concentration. Different letters shown by each solid and standard error bar indicates signiﬁcant difference (a=0.05, Tukey-Kramer’s test) in consumption.

Wei & Johnson: Measurement of Sugar Consumption by a Moth 391

± 88.3; and 193.2 ± 36.8 ul, respectively, when they were fed with 5, 10, 20, or 40% sucrose solutions. Only 35 ± 16.6 ul of distilled water was taken up during the same period when the insects were provided with only distilled water. All pairwise comparisons of consumption between concentrations were highly signiﬁcantly different from each other. When moths were fed on 5% sucrose, they consumed 3 times as much as they did on 40%. This is an example of compensatory feeding by which moths consumed a greater volume of solution at a lower sugar concentration than at a higher concentration.

ACKNOWLEDGMENTS

We are grateful to Zhuofei Song for contributions to the weighing procedure development and assistance throughout the study and to A. M. Hammond, R. N. Story, J. R. Fuxa (Louisiana State University), R. L. Crocker, W. T. Nailon (Texas A&M Univer- sity), J. D. Lopez, Jr. (USDA-ARS, Area-Wide Pest Manage. Res. Unit, College Station, TX), and unknown reviewers for critical reviews of the manuscript. We also thank Terrie R. Thomas for assistance in insect rearing. Approved for publication by the director, Louisiana Agricultural Experiment Station, as manuscript number 94-17- 8234.

REFERENCES CITED

ADLER, P. H. 1989. Sugar feeding of the adult corn earworm (Lepidoptera: Noctuidae) in the laboratory. J. Econ. Entomol. 82: 1344-1349. ALM, J., T. E. OHNMEISS, J. LANKA, AND L. VRIESENGA. 1990. Preference of cabbage white butterflies and honey bees for nectar that contains amino acids. Oecolo- gia 84: 53-57. BURLEIGH, J. G. 1972. Population dynamics and biotic control of the soybean looper in Louisiana. Environ. Entomol. 1: 290-294. COLLINS, F. L. 1984. Interactions of soybean arthropods and crop weeds: diversity, moth nutrition, and stability. Ph.D. dissertation, Louisiana State University, Baton Rouge, LA. COLLINS, F. L., AND S. J. JOHNSON. 1985. Reproductive response of caged adult velvetbean caterpillar and soybean looper to the presence of weeds. Agric. Ecosyst. Environ. 14: 139-149. FEHR, W. R., AND C. E. CAVINESS. 1979. Stages of soybean development. Special Re- port 80. Cooperative Extension Service, Iowa State University, Ames, IA. GAGNON, J., J. M. ROTH, M. CARROLL, R. HOFMANN, K. A. HAYCOCK, J. PLAMONDON, D. S. FELDMAN, JR., AND J. SIMPSON. 1989. SuperANOVA: accessible general linear modeling. Abacus Concepts, Inc., Berkeley, CA. GREENE, G. L., J. C. REID, V. N. BLOUNT, AND T. C. RIDDLE. 1973. Mating and oviposition behavior of the velvetbean caterpillar in soybeans. Environ. Entomol. 2: 1113-1115. GREENE, G. L., N. C. LEPPLA, AND W. A. DICKERSON. 1976. Velvetbean caterpillar: A rearing procedure and artificial medium. J. Econ. Entomol. 69: 487-488. KING, E. G., AND G. G. HARTLEY. 1985. Diatraea saccharalis, in P. Singh and R. F. Morre [eds.], Handbook of Insect Rearing, Vol. 2. Elsevier, Amsterdam. LUKEFAHR, M. J., AND C. RHYNE. 1960. Effects of nectariless cottons on populations of three lepidopterous insects. J. Econ. Entomol. 53: 242-244. MASON, L. J., S. J. JOHNSON, AND J. P. WOODRING. 1989. Influence of carbohydrate deprivation and tethered flight on stored lipid, fecundity, and survivorship of the soybean looper (Lepidoptera: Noctuidae). Environ. Entomol. 18: 1090-1094. MAY, P. G. 1985a. A simple method for measuring nectar extraction rates in butterflies. J. Lepid. Soc. 39: 53-55. 392 Florida Entomologist 79(3) September, 1996

MAY, P. G. 1985b. Nectar uptake rates and optimal nectar concentrations of two butterfly species. Oecologia 66: 381-386. PIVNICK, K. A., AND J. N. MCNEIL. 1985. Effects of nectar concentration on butterfly feeding: measured feeding rates for Thymelicus lineola (Lepidoptera: Hesperi- idae) and a general feeding model for adult Lepidoptera. Oecologia 66: 226-237. WALES, P. J. 1983. Activity of velvetbean caterpillar moths as recorded by a new ac- tograph. M.S. thesis, University of Florida, Gainesville, FL. WEI, X. 1995. Adult velvetbean caterpillar, Anticarsia gemmatalis, (Lepidoptera: Noc- tuidae), sugar feeding strategy and its effect on fecundity and whole body lipid accumulation. M.S. thesis, Louisiana State University, Baton Rouge, LA. WEI, X., AND S. J. JOHNSON. 1994. An efficient method to harvest velvetbean caterpillar pupae from field cages. Southwestern Entomol. 19: 411. WEI, X., AND S. J. JOHNSON. 1995. Velvetbean caterpillar: surviving freezing weather in Louisiana. Florida Entomol. 78: 186-189.

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392 Florida Entomologist 79(3) September, 1996

ATTRACTION OF MOTHS (LEPIDOPTERA: ARCTIIDAE, GEOMETRIDAE, NOCTUIDAE) TO ENANTIOMERS OF SEVERAL EPOXYDIENES

P. J. LANDOLT, M. TÓTH1, W. FRANCKE2 AND K. MORI3 United States Department of Agriculture, Agricultural Research Service, 5230 Konnowac Pass Rd., Wapato, WA 98951

1Plant Protection Institute, Hungarian Academy of Sciences, Budapest, Pf. 102, H-1525, Hungary

2Institut für Organische Chemie der Universität Hamburg, Martin-Luther-King-Platz 6, D-20146, Hamburg 13, Germany

3Department of Chemistry, Science University of Tokyo, Kagurazaka 1-3, Shinjuku-ku, Tokyo 162, Japan

ABSTRACT Males of four species of moths were captured in traps baited with enantiomers of straight chain epoxydienes in north Florida. The noctuid Renia salusalis was captured in traps baited with (3Z,6S,7R,9Z)-6,7-epoxy-3,9-nonadecadiene, the geometrid Probole alienaria in traps baited with a 1:1 mixture of enantiomers of cis-6,7-epoxy- 3,9-nonadecadiene, and the noctuid Zale lunifera as well as the arctiid Halysidota tessellaris in traps baited with (3Z,6Z,9R,10S)-9,10-epoxy-3,6-henicosadiene. These compounds may be part of the sex pheromone of females of these 4 species.

Key Words: Insecta, sex attractant, trap, bait, pheromone

RESUMEN Fueron capturados machos de cuatro especies de polillas en trampas cebadas con enaantiómeros de epoxydienos de cadena recta en el norte de la Florida. El noctuido

Landolt et al: Moth Attraction to Epoxydienes 393

Renia salusalis fue capturado en trampas cebadas con (3Z,6S,7R,9Z)-6,7-epoxy-3,9- nonadecadiene, el geométrido Probole alienaria en trampas cebadas con una mezcla 1:1 de enantiómeros de cis-6,7-epoxy-3,9-nonadecadiene, y el noctuido Zale lunifera así como el arctiido Halysidota tessellaris en trampas cebadas con (3Z,6Z,9R,10S)- 9,10-epoxy-3,6-henicosadiene. Estos compuestos pueden ser parte de la feromona sexual de las hembras de estas 4 especies.

A number of triene hydrocarbon compounds and their monoepoxide derivatives are female sex pheromones, and/or function as sex attractants, for a number of Geometridae, Noctuidae, and Arctiidae. Many species of moths are known to be attracted to these compounds through screening tests designed to document the capture of attracted moths in baited traps. For example, Wong et al. (1985) captured 17 species of Geometridae and 9 species of Noctuidae in traps baited with mixtures of C18 to C22 (3Z,6Z,9Z)-triene hydrocarbons and corresponding epoxydienes derived from these compounds. Subsequent studies by Millar et al. (1990a,b,c), which expanded on the studies of Wong et al. (1985), clearly demonstrated the importance of optical isomers on the sexual attractiveness of particular epoxydienes and extended the list of species responding to these compounds and the triene hydrocarbons to over 80 in central Can- ada alone. Efforts by others in Europe have revealed similar usage of triene hydrocarbons and their monoepoxydiene derivatives as moth sex attractants, primarily within the Geometridae (i.e., Tóth et al. 1991,1992, 1995). Undoubtedly, comprehensive testing of these compounds in other geographic areas and throughout the seasons of moth activity would reveal a more complete picture of the patterns of activity of these compounds as sex attractants. As a contribution towards this goal, we conducted a screening program to evaluate the attractiveness of a set of epoxydiene derivatives of C17, C19, and C21 triene hydrocarbons. We report here the capture of four species of moths in traps baited with these compounds in screening trials conducted in north Florida. Enantiomers and 1:1 combinations of enantiomers of 6 compounds were tested in a forest in north-central Florida from early spring to late autumn of 1994.

MATERIALS AND METHODS

The enantiomers of (3Z,9Z)-cis-6,7-epoxy-3,9-nonadecadiene were synthesized by Mori & Brevet (1991). Other chemicals were synthesized and puriﬁed as reported in Szöcs et al. (1993). All compounds were tested in dosages of 100 micrograms per enantiomer, applied in hexane to 0.5 cm long pieces of 1 cm wide rubber tubing (Taurus, Budapest, Hungary, No., MSZ 9691/6). Prior to usage tubing was extracted 3 times in boiling ethanol for 10 min, then also 3 times overnight in methylene chloride (Tóth et al. 1995). Racemates produced as 1:1 mixtures of enantiomers consisted of 200 ug total material per lure. Lures were wrapped in aluminum foil for shipping and storage and were kept at -10°C until used in ﬁeld tests.

Seasonal Survey

Lures were placed in Pherocon 1C traps, suspended by wire from the trap roof to within 3 cm of the sticky trap liner. Traps were hung from vines, shrubs and tree branches at about 2 m above ground. Two linear blocks of traps were set up in late

394 Florida Entomologist 79(3) September, 1996

February of 1994, with each block comprised of one trap for each of the nine treatments. These were 1) (3R,4S,6Z,9Z)-3,4-epoxy-6,9-heptadecadiene (Z6Z9-3R4S-epo- 17Hy), 2) (3S,4R,6Z,9Z)-3,4-epoxy-6,9-heptadecadiene (Z6Z9-3S4R-epo-17Hy), 3)a 1:1 mixture of enantiomers of (6Z,9Z)-cis-3,4-epoxy-6,9-heptadecadiene (Z6Z9-3,4- epo-17Hy) 4) (3Z,6R,7S,9Z)-6,7-epoxy-3,9-nonadecadiene (Z3Z9-6R7S-epo-19Hy), 5) 3Z,6S,7R,9Z)-6,7-epoxy-3,9-nonadecadiene (Z3Z9-6S7R-epo-19Hy) 6) a racemic mixture of enantiomers of (3Z,9Z)-cis-6,7-epoxy-3,9-nonadecadiene (Z3Z9-6,7-epo-19Hy), 7) (3Z,6Z,9R,10S)-9,10-epoxy-3,6-henicosadiene (Z3Z6-9R10S-epo-21Hy), 8) (3Z,6Z, 9S,10R)-9,10-epoxy-3,6-henicosadiene (Z3Z6-9S10R-epo-21Hy), and 9) a 1:1 mixture of enantiomers of (3Z,6Z-cis-9,10-epoxy-3,6-henicosadiene (Z3Z6-9,10-epo-21Hy). Within a block, traps were placed 50 m apart. The two trapping blocks were located in two forested locations in Alachua County, Florida. Plot No. 1 was in an area comprised primarily of second growth hardwood trees. The area of the second plot was primarily planted slash pine (Pinus elliottii Engelman). Traps were set up initially in late February and were maintained until November. Traps were checked twice per week and were rotated one position in the plot each week. Trap liners were changed when needed and lures were replaced monthly.

Follow-up Treatment Comparisons

Additional traps were set up at other locations to evaluate further the attractiveness of moths to 3 treatments that showed some activity in the seasonal survey. In these cases, Pherocon 1C traps baited with a treatment were directly compared with unbaited traps, for statistical purposes. Seven pairs of traps were set up in 1994 and 3 pairs of traps were set up in 1995 to further evaluate the attraction of Probole alienaria Herrich-Schaeffer to Z3Z9-6,7-epo-19Hy. These traps were maintained for 1 week during 1994 and for 3 weeks in 1995. Similarly, twelve pairs of traps were set up in 1994 and 3 pair of traps were set up in 1995 to assess the attraction of Renia salusalis Walker to Z6Z9-6S7R-epo-19Hy. These traps were maintained for 3 days in 1994 and 3 weeks in 1995. Traps in 1995 were checked every 3 days. Three pairs of traps were set up in 1994 and 3 pairs in 1995 to evaluate the attractiveness of (3Z,6Z,9R,10S)-9,10-epoxy-3,6-heneicosadiene to Halysidota tessellaris Smith. These traps were monitored for 3 days in 1994, and 3 weeks in 1995 and were checked for captured moths every 3 days. Treatment and control positions for traps were alter- nated every 3 days. Mean trap captures for direct comparisons between treatments and unbaited control traps were evaluated by Student's T-test to determine signiﬁcant differences between treatments. Voucher specimens of P. alienaria, R. salusalis, H. tessellaris, and Zale lunifera Hübner are in the collection of the ﬁrst author.

RESULTS

Four species of moths were captured in traps baited with the listed epoxydienes during 1994 and 1995. These were R. salusalis, P. alienaria, Z. lunifera, and H. tessellaris. Fifty-eight male R. salusalis were captured in Pherocon 1C traps baited with Z3Z9-6S7R-epo-19Hy during the 1994 season-long survey, primarily in March (Fig. 1). No R. salusalis were captured in traps baited with any other treatment. Fifty-four of the 58 captured were from the ﬁrst plot, dominated by hardwood trees, with 4 in the second plot dominated by pine trees. In a direct comparison of unbaited traps and traps baited with Z3Z9-6S7R-epo-19-Hy, 4.92 ± 0.98 (x ± SE) male R. salusalis were captured per baited trap per 3 days, and no moths were captured in unbaited traps Landolt et al: Moth Attraction to Epoxydienes 395

Fig. 1. Numbers of moths captured per week in blocks 1 & 2 combined throughout the 1993 season. Solid line with open circles is for Probole alienaria; broken line with plus signs is for Halysidota tessellaris; and the dotted line with closed circles is for Re- nia salusalis.

(t=5.0, df=46, p=0.9 x 10-5). Nine additional R. salusalis were captured in a Universal moth trap (Unitrap) baited with Z3Z9-6S7R-epo-19Hy placed near the ﬁrst plot during the third week of March in order to obtain voucher specimens. During the 1994 season-long survey, forty male P. alienaria were captured in traps baited with the racemic mixture of Z3Z9-6,7-epo-19Hy. Fourteen of these moths were captured in plot 1 and 26 were captured in plot 2. This species was captured intermit- tently throughout the season, with 3 possible generations or ﬂight periods (Fig. 1). None were captured in traps baited with any other treatment. In a test which directly compared numbers of P. alienaria in traps baited with a 1:1 mixture of enantiomers of Z3Z9- 6,7-epo-19Hy and unbaited traps, 2.5 ± 0.6 (x ± SE) males were captured per treated trap per week compared to 0 moths captured in unbaited traps (t=4.2, df=46, p=0.0001). Eleven H. tessellaris males were captured in traps during the 1994 season-long survey, all in traps baited with Z3Z6-9R10S-epo-21Hy, and all were captured in March (Fig. 1). During the subsequent 1995 test which compared captures of H. tessellaris in unbaited traps to traps baited with Z3Z6-9R10S-epo-21Hy, only 1 H. tessellaris male was caught. However, 12 male Z. lunifera were caught in this test, all in baited traps and all during March 1994 (t = 2.69, df = 18, p = 0.015). One male Z. lunifera was captured during the 1994 season-long survey. This was in March in a trap baited with Z3Z6-9R10S-epo-21Hy.

DISCUSSION

The capture of males of these four species of moths, P. alienaria, R. salusalis, Z. lunifera and H. tessellaris, in traps baited with a 1:1 mixture of enantiomers of Z3Z9- 396 Florida Entomologist 79(3) September, 1996

6,7-epo-19Hy, with Z3Z9-6S7R-epo-19Hy and Z3Z6-9R10S-epo-21Hy, respectively, indicates that these compounds may function as sex attractants for these species. This is further supported by the failure to capture moths in traps baited with other compounds. In the cases of R. salusalis, P. alienaria and Z. lunifera, sexual attraction was subsequently demonstrated with significant captures of males in baited traps compared to unbaited traps. These compounds may be female-produced sex pheromone components for these species, or they may be structurally related and may mimic the natural pheromones. There are no previous demonstrations of sexual attraction or reported sex pheromones for P. alienaria, which we captured in traps baited with a 1:1 mixture of enantiomers of (3Z,9Z)-cis-6,7-epoxy-3,9-nonadecadiene. A closely related species, Probole amicaria Herrich-Schaffer, is attracted to racemic (6Z,9Z)-cis-3,4-epoxy-6,9-nonadecadiene, which is enhanced in attracting P. amicaria by the addition of racemic (3Z,9Z)-cis-6,7-epoxy-3,9-nonadecadiene (Millar et al. 1990b). These two species may share at least this one component in their respective sex pheromones. It is not known if the 3,4-epoxynonadecadiene has any effect on sex attraction of P. alienaria, but P. amicaria did not respond to the 6,7-epoxynonadecadiene when presented alone in field tests (Millar et al. 1990b). The genus Probole is placed in the geometrid subfamily Ennominae. A number of other species in this group are attracted to combinations of (3Z,6Z,9Z)-nonadecatriene and structurally-related epoxynonadecadienes (i.e., Prochoerodes transversata (Drury) and Epirrhoe sperryi (Herbulot) Wong et al. 1985), Erannis defoliaria Clerck (Hansson et al. 1990), and Boarmia selenaria Schiffer- muller (Becker et al. 1983). Also, the female pheromone of Peribatodes rhomboidaria (Schiffermuller) is a combination of (Z,Z)-6,9-nonadecadien-3-one and (Z,Z,Z)-3,6,9- nonadecatriene (Buser et al. 1985). There are no pheromone identifications and no prior demonstrations of sexual attraction of species of the herminiine noctuid genus Renia, which responded to (3Z,6S,7R,9Z)-6,7-epoxy-3,9-nonadecadiene. However, other species in the Herminii- nae are attracted to similar compounds. Rivula propinquialis (Guenee) is attracted to the same enantiomer of Z3Z9-6,7-epo-19Hy as well as to the related triene (Millar et al. 1990). Bleptina caradrinalis (Guenee), Idia americalis (Guenee), and Idia aemula (Hübner) are attracted to (Z3,Z6,Z9)-heneicosatriene (Wong et al. 1985). This report is the second documentation of a species of Zale responding to an ep- oxide derivative of a triene hydrocarbon. While we trapped Z. lunifera with Z3Z6- 9R10S-epo-21Hy, Wong et al. (1985) reported the capture of Zale duplicata (Bethune) in traps baited with this same compound. Males of H. tessellaris were also caught in traps baited with Z3Z6-9R10S-21Hy, and other arctiids are known to be attracted to similar compounds. The salt marsh caterpillar female, Estigmene acrea (Drury) produces a sex pheromone comprised in part of (3Z,6Z,9S,10R)-9,10-epoxy-3,6-henicosadiene (Hill & Roelofs 1981). A mixture of epoxydienes and epoxytrienes have been identified as possible pheromone components in the glands of female fall webworm adults, Hyphantria cunea (Drury) (Tóth et al. 1989; Hill et al. 1982). Another genus of Arctiidae, Creatonotos, uses 21 carbon trienes and an epoxy derivative as a sex pheromone (Bell & Meinwald 1986).

ACKNOWLEDGMENTS

Technical assistance was provided by K. M. Davis, S. Lovvorn, and T. Macom. We thank M. S. Adams of West Shokan, N.Y. for identifying Renia salusalis. Landolt et al: Moth Attraction to Epoxydienes 397

REFERENCES

BECKER, D., T. KIMMEL, R. CYJON, I. MOORE, M. WYSOK, H. J. BESTMANN, H. PLATE, K. ROTH, AND O. VOSTROVSKY. 1983. (3Z,6Z,9Z)-3,6,9-nonadecatriene-a component of the sex pheromonal system of the giant looper, Boarmia (Ascotis) selenaria Schiffermuller. Tet. Lett. 224: 5505-5508. BELL, T. W., AND J. MEINWALD. 1986. Pheromones of two arctiid moths (Creatonotos transiens and C. ganges): Chiral components from both sexes and achiral female components. J. Chem. Ecol. 12: 385-409. BUSER, H. R., P. M. GUERIN, M. TÓTH, G. SZÖCS, A. SCHMID, W. FRANCKE, AND H. ARN. 1985. (Z,Z)-6,9-nonadecadien-3-one and (Z,Z,Z)-3,6,9-nonadecatriene: identification and synthesis of sex pheromone components of Peribatodes rhomboidaria. Tet. Lett. 26: 403-406. HANSSON, B. S., G. SZÖCS, F. SCHMIDT, W. FRANCKE, C. LÖFSTEDT, AND M. TÓTH. 1990. Electrophysiological and chemical analysis of sex pheromone communication system of the mottled umber, Erannis defoliaria (Lepidoptera: Geometridae). J. Chem. Ecol.16: 1887-1897. HILL, A. S., AND W. L. ROELOFS. 1981. Sex pheromone of the saltmarsh caterpillar moth, Estigmene acrea. J. Chem. Ecol. 7: 655-668. HILL, A. S., B. G. KOVALE, L. N. NIKOLAEVA, AND W. L. ROELOFS. 1982. Sex pheromone of the fall webworm moth, Hyphantria cunea. J. Chem. Ecol. 8: 383-396. MILLAR, J. G., M. GIBLIN, D. BARTON, AND E. W. UNDERHILL. 1990a. (3Z,6Z,9Z)-nonadecatriene and enantiomers of (3Z,9Z)-cis- 6,7-epoxy-nonadecadiene as sex attractants for two geometrid and one noctuid moth species. J. Chem. Ecol. 16: 2153-2166. MILLAR, J. G., M. GIBLIN, D. BARTON, AND E. W. UNDERHILL. 1990b. 3Z,6Z,9Z-trienes and unsaturated epoxides as sex attractants for geometrid moths. J. Chem. Ecol. 16: 2307-2316. MILLAR, J. G., M. GIBLIN, D. BARTON, A. MORRISON, AND E. W. UNDERHILL. 1990c. Synthesis and field testing of enantiomers of 6Z,9Z-cis-3,4-epoxydienes as sex attractants for geometrid moths: interactions of enantiomers and regioisomers. J. Chem. Ecol. 16: 2317-2340. MORI, K., AND J. -L. BREVET. 1991. Pheromone synthesis CXXXIII. Synthesis of both the enantiomers of (3Z,9Z)-cis-6,7-epoxy- 3,9-nonadecadiene, a pheromone component of Erannis defoliaria. Synthesis. 1125-1129. SZÖCS, G, M. TÓTH, W. FRANCKE, F. SCHMIDT, P. PHILIPP, W. A. KONIG, K. MORI, B. S. HANSSON, AND C. LÖFSTEDT. 1993. Species discrimination in five species of winter flying geometrids (Lepidoptera) based on chirality of semiochemicals and flight season. J. Chem. Ecol. 19: 2721-2735. TÓTH, M., H. R. BUSER, A. PEÑA, H. ARN, K. MORI, T. TAKEUCHI, L. N. NIKOLAEVA, AND B. G. KOVALEV. 1989. Identification of (3Z,6Z)-1,3,6-9,10-epoxyheneicosa- triene and (3Z,6Z)-1,3,6- 9,10-epoxyeicosatriene in the sex pheromone of Hy- phantria cunea. Tet. Lett. 30: 3405-3408. TÓTH, M., G. SZÖCS, C. LÖFSTEDT, B. S. HANSSON, F. SCHMIDT, AND W. FRANCKE. 1991. Epoxyheptadecadienes identified as sex pheromone components of Teph- rina arenacearia Hbn. (Lepidoptera: Geometridae). Z. Naturforsch. C. 46: 257- 263. TÓTH, M., H. R. BUSER, P. M. GUERIN, H. ARN, F. SCHMIDT, W. FRANCKE, AND G. SZÖCS. 1992. Abraxas grossulariata L. (Lepidoptera: Geometridae): Identifica- tion of (3Z,6Z,9Z)-3,6,9-heptadecatriene and (6Z,9Z)-6,9-cis-3,4-epoxyhepta- decadiene in the female sex pheromone. J. Chem. Ecol. 20: 13-25. TÓTH, M., G. SZÖCS, W. FRANCKE, F. SCHMIDT, C. LÖFSTEDT, B. S. HANSSON, AND A. FARAG. 1995. Pheromonal production and response to optically-active epoxydienes in some geometrid moths (Lepidoptera: Geometridae). Z. Naturforsch. 49c: 516-521. WONG, J. W., E. W. UNDERHILL, S. L. MACKENZIE, AND M. D. CHISHOLM. 1985. Sex attractants for geometrid and noctuid moths. J. Chem. Ecol. 11: 727-756.

398 Florida Entomologist 79(3) September, 1996

TEMPORAL AND SPATIAL VARIATION IN THE FORAGING BEHAVIOR OF HONEY BEES (HYMENOPTERA: APIDAE) AT CHINESE VIOLETS

ETHEL M. VILLALOBOS1 AND TODD E. SHELLY2 1Department of Biology, Chaminade University, Honolulu, Hawaii, 96816

2Hawaiian Evolutionary Biology Program, University of Hawaii, Honolulu, HI 96822

ABSTRACT

This study describes temporal and spatial variation in the foraging behavior of honey bees, Apis mellifera L., at the Chinese violet, Asystasia gangetica (L.) T. Ander- son, in Hawaii. Workers made 3 types of visits: “out” (O-) visits involving nectar robbing through corollar slits made by carpenter bees (Xylocopa sonorina (Smith), “in- upright” (IR-) visits involving upright entry along the base of the corolla, and “in-upside down” (ID-) visits involving spiral, upside-down entry along the top of the corolla. In general, individual workers displayed only 1 tactic over 10 successive flower visits and over successive days. Nectar-robbing workers visited more flowers per min but spent less time per flower than workers making IR- or ID-visits. Bees making O- or IR- visits carried similar nectar loads but only very small amounts of pollen, whereas the reverse was true for bees making ID-visits. O- and IR-visits were made throughout the day, but ID-visits were observed only in the morning. Based on inter-site comparisons, the incidence of nectar robbing was influenced by the local density of carpenter bees which made the perforations used by nectar robbing honey bee workers.

Key Words: Apis mellifera, nectar robbing, pollination, Hawaii

RESUMEN

Este estudio describe la variación temporal y espacial del comportamiento forra- jero de la abeja de miel, Apis mellifera L., en la violeta china, Asystasia gangetica (L.) T. Anderson, en Hawaii. Las obreras hicieron 3 tipos de visitas: Las visitas “por fuera” (F-) incluyeron el robo de néctar a través de las incisiones hechas previamente a la corola por las abejas carpinteras, Xylocopa sonorina (Smith), las visistas “dentro-por arriba” (DA-) incluyeron entradas por encima de la flor, a lo largo de la base de la corola, y las visitas “dentro-volteadas” (DV-) incluyeron entradas en espirtal, volteadas a lo largo del extremo de la corola. En general, las obreras individuales desarrollaron una sola táctica cada 10 visitas sucesivas a las flores, en 10 días sucesivos. Las obreras ladronas de néctar visitaron más flores por minuto pero pasaron menos tiempo por flor que las obreras que efectuaban visitas de los tipos DA- y DV-. Las abejas haciendo visitas de los tipos F- y DA- llevaron cargas de néctar similares pero sólo pequeñas can- tidades de polen, mientras que lo inverso ocurrió en abejas haciendo visitas DV-. Las visitas F- y DA- fueron hechas a lo largo de todo el día, pero las visitas DV- fueron observadas solamente en la mañana. Basada en comparaciones inter-sitios, la incidencia del robo de néctar fue influenciada por la densidad local de las abejas carpinteras, las cuales hacen las perforaciones usadas por las obreras ladronas de la abeja de miel.

Research on the foraging biology of the honey bee, Apis mellifera L., has focused primarily on colony performance, and comparatively little attention has been given to

Villalobos & Shelly: Honey Bee Behavior 399 the food-collecting behavior of individual workers (Schmid-Hempel 1991). Available data reveal that workers visiting the same plant species typically specialize on either nectar or pollen collection. In some instances, this distinction is absolute, and individual workers gather either nectar or pollen exclusively (McGregor et al. 1959; Free 1960a,b). Often, however, workers may collect both nectar and pollen, although they may concentrate primarily on one of these resources. For example, among honey bees visiting native cotton, Gossypium thurberi, 40% collected pollen only and 60% concentrated on nectar collection but gathered pollen passively as well (Buchmann & Ship- man 1990; see also Fell 1986; Weaver 1956). Nectar foragers may also display variable foraging modes, either obtaining nectar “legitimately” (thereby serving as pollinators) or via nectar theft or robbing (thus avoiding contact with pollen-bearing anthers, sensu Inouye 1980). Though reports of nectar theft and robbing are common for honey bees (Burrill 1925; Hawkins 1961; Helms 1970; Benedek et al. 1973; Barrows 1980), to our knowledge only 1 study presented information on worker specialization regarding legitimate vs. illegitimate nectar collection. In observing honey bees visit hairy vetch (Vicia villosa Roth.), Weaver (1956) found that workers reached the nectary by either entering the flower or insert- ing their tongue through the petals at the base of the corolla. Observations of individual workers over 2-10 consecutive floral visits revealed a high degree of specialization for 1 tactic or the other, and only 10 of the 300 individuals observed used a mixture of foraging tactics. The present study describes temporal and spatial variation in the foraging behavior of honey bees at the Chinese violet, Asystasia gangetica (L.) T. Anderson (Acan- thaceae), in Hawaii. Earlier observations (Barrows 1980; Gerling 1982) recorded Xylocopa sonorina (Smith) as a primary nectar robber (actually perforating flowers) and A. mellifera as a secondary nectar robber (using existing perforations) of this plant species (sensu Inouye 1980). Here, we describe diurnal variation in the incidence of 3 types of floral visits—2 involving corollar entry and 1 involving nectar robbing—and compare body sizes, nectar and pollen loads, rates of floral visitation, and floral handling times among honey bee workers displaying the different foraging tactics. Workers were also individually marked to assess between-day consistency in foraging behavior. Finally, we compared the incidence of nectar robbing at several sites differing in honey bee, carpenter bee, and floral densities.

MATERIALS AND METHODS

Study Site and Species

A. gangetica, a procumbent or scandent perennial herb, was introduced to Hawaii around 1925 and is now widespread in disturbed lowland areas (Wagner et al. 1990). Flowers are pentamerous, zygomorphic, and vary in color from pale blue to purple (or less commonly, white or yellowish). Corollas vary from approximately 25-35 mm in length (Barrows 1980). Both A. mellifera and X. sonorina were introduced to Hawaii in the late 1800s (Lieftinck 1956; Barrows 1980). Most of the ﬁeld work was conducted during May-June 1993 in a small untended lot (100 m2) on the campus of Chaminade University, Honolulu, Hawaii. All observations were made under sunny or only slightly overcast skies, and air temperatures ranged between 25-31°C at 15 cm above ground. A. gangetica was the most abundant ﬂower within the study area. Patches of A. gangetica covered approximately 1/2 the lot, and the rest of the area was covered by various grasses or bare rock surfaces. A. mellifera and X. sonorina were the only species of bees observed at the study site.

400 Florida Entomologist 79(3) September, 1996

Censuses of Foraging Bees

We recorded the numbers of bees foraging in 4 quadrats located within representative patches of A. gangetica during morning (0800-1000 hours) and afternoon (1400- 1600 hours) periods on 6 d. Each 1 m2 quadrat was separated by a minimum of 3 m. On a given census day, 2-3 counts were made during the morning and afternoon study periods, respectively, and successive counts were separated by a minimum of 25 min. Also, on each census day we recorded flower abundance in the 4 quadrats and collected 100 flowers haphazardly from the study site at 1000 hours and examined them in the laboratory for corollar slits or perforations. As described below, nearly all the honey bees that were observed displayed only 1 type of foraging tactic. Thus, in addition to the estimates of absolute density provided by the quadrat censuses, we recorded the relative density of the different types of foragers by walking a 10 m transect through the study area and noting the behavior (based on a single floral visit) of all honey bees within 1 m of the line. Three surveys were made during morning and afternoon periods, respectively, on 5 d, and successive surveys were separated by at least 30 min.

Behavioral Observations

The type of foraging behavior displayed by individual honey bees was noted during observations of consecutive floral visits. Initially, we included only data for bees observed to visit 25 or more flowers. However, because of the invariant nature of foraging behavior, additional observations were limited to 10 consecutive floral visits per individual honey bee. Morning (0800-1000 hours) and afternoon (1400-1600 hours) observations of foraging behavior were made on 10 d, and during any given morning or afternoon we observed only 10-15 sequences of floral visits to reduce the likelihood of observing the same individual more than once. Also, we attempted to select focal bees randomly by initiating observations on individuals in flight (i.e., whose foraging tactic was unknown to us). Though emphasis was on honey bees, behavioral observations were also made for carpenter bees (2-6 sequences of floral visits were observed during a given morning or afternoon period on 5 d). Three types of floral visits were distinguished: “out” (O) visits during which bees did not enter the corolla but probed the base of the corolla from the outside (i.e., nectar robbing), “in-upright” (IR) visits during which bees landed on the lower petals and entered the corolla in an upright position, and “in-upside down” (ID) visits during which bees landed on the lower petals in an upright position but then entered the flower while making a spiral path to the top of the corolla, thus assuming an upside-down position. [Perforations were absent on previously bagged flowers visited exclusively by honey bees, and we conclude (as did Barrows 1980) that honey bees used existing slits and did not perforate flowers themselves.] For approximately 1/2 the bees observed, we also recorded the duration of each floral visit (to the nearest 0.1 s with a stop- watch), the total duration of the observations to obtain rates of flower visitation. Also, for bees making either type of in-visit we recorded the number of flowers they approached (operationally defined as 1-3 s hovering immediately in front of a flower) but did not land on and the number of flowers they landed on but did not enter.

Measurement of Nectar and Pollen Loads

Nectar and pollen loads were estimated for a sample of honey bees observed displaying each of the different foraging tactics. Bees were observed over 10 consecutive

Villalobos & Shelly: Honey Bee Behavior 401

ﬂoral visits between 0800-1000 hours over 3 consecutive mornings, captured with an aerial net, and immediately transported to the laboratory for dissection. Nectar in the crop was drawn into a 10 ul glass capillary tube. Pollen was scraped off the hindlegs using a dissecting pin, dried at 45°C for 24 h, and weighed to the nearest 0.1 mg on a Mettler AE163 balance. Because the display of nectar robbing may be related to bee size (e.g., Barrow & Pickard 1984), we also measured forewings of these bees (specifically, the length of the medial cross vein) to the nearest 0.1 mm using a dissecting microscope equipped with a disc micrometer.

Between-day Consistency in Individual Behavior

To determine whether individual workers displayed the same foraging tactic over successive days, we uniquely marked individuals with different color combinations of enamel paint on their thorax. Bees were observed for 10 floral visits, captured with an aerial net, and cooled in an ice chest for marking. Following marking, bees were placed in full sunlight, and after 1-2 min of warming the bees took flight and often re- sumed foraging activity. A total of 70 bees (approximately equal numbers of O-, IR-, and ID-foragers) was marked between 0830-1100 hours in a single morning, and over the next 7 d we monitored the site between 0900-1000 hours and observed the foraging behavior of marked individuals for 10 consecutive floral visits. Additionally, we checked for within-day consistency in behavior by observing marked workers between 1400-1500 hours on the first 2 d following marking.

Inter-site Variation in Foraging Behavior

Honey bee foraging tactics were monitored concurrently at 3 other locations, all of which were infrequently mowed lawns within 5 km of the main study site. A. gangetica was the dominant flowering plant at each of these additional sites, and honey bees and carpenter bees were the only floral visitors observed. Following the above protocol, we recorded bee abundance in 4 quadrats (each 1 m2) at each of these sites. At a given site, 3 censuses were made between 0800-1000 hours on 4-5 d. In addition, we walked a transect line (10-15 m) at each site and recorded the number of honey bees making 0-, IR-, and ID-visits. At a given site, 4-6 transects were made during a given morning over 4-5 d. We also made 1 count of flowers in the quadrats per census day collected 100 flowers at 1000 hours and examined them in the laboratory for corollar slits or perforations.

RESULTS

Bee and Flower Abundance

Both carpenter bees and honey bees were more abundant in the morning than in the afternoon. Over a total of 60 censuses made in the 4 quadrats at the Chaminade site, the density of carpenter bees averaged 0.33 per m2 in the morning (SE=0.07; range:0-2) but only 0.08 per m2 in the afternoon (SE=0.04; range: 0-2). Similarly, the average density of honey bees was 2.08 per m2 in the morning (SE=0.14; range: 0-5) compared to 1.15 per m2 in the afternoon. The mean density of A. gangetica ﬂowers was 31.2 per m2 (SE=2.2; range=23-37; n=6 d × 4 plots=24 censuses). Flower density was relatively constant over the study period and did not differ signiﬁcantly over the different census days (χ2=3.9; P > 0.05; Kruskal-Wallis test with χ2 approximation).

402 Florida Entomologist 79(3) September, 1996

Foraging Behavior of Carpenter Bees Carpenter bees made O-visits exclusively (n=21 and 10 sequences of 10 floral visits for morning and afternoon periods, respectively). Individuals typically approached a flower frontally, landed on the petals, and then walked over the flower to the base of the corolla. Carpenter bees moved rapidly between flowers, spending little time at each. Averaged over all timed observations, carpenter bees visited 16 flowers per min (SE=0.9; range: 12-24) and spent 1.5 s at each flower (SE=0.1; range=0.3-3.4; n=150).

Temporal Variation in Honey Bee Foraging Tactics During almost all of the ﬂoral visitation sequences observed (215/224=96%), individual honey bees made only 1 type of ﬂoral visit. However, the incidence of the different types of foragers varied through the day. In the morning, we observed approximately equal numbers of O-foragers (n=60) and I-foragers (I=IR and ID combined; n=57), but in the afternoon nearly 2/3 (59/98) of the bees observed made O-visits. Even more striking, IR- and ID-foragers were equally common during the morning (29 and 28 observations, respectively), but all I-foragers observed in the afternoon were making IR-visits exclusively. The few bees (n=9) that displayed mixed foraging tactics were all observed in the morning and were making a combination of IR- and ID-visits. Data from the transects showed the same temporal pattern in foraging behavior. On average, we noted 20 honey bees per morning transect (SE=0.9; range: 16-25; n=15) of which 11 (55%), 5 (25%), and 4 (20%) were making O-, IR-, and ID-visits, respectively. Consistent with the census data from the quadrats, fewer honey bees were present in the afternoon transects (x=12; SE=0.7; range: 6-19; n=15). Of these, an average of 8 individuals (67%) were making O-visits, and 4 individuals were making IR- visits. No honey bees were seen to make ID-visits during any afternoon transect.

Behavioral Differences Among Honey Bee Foraging Tactics Rates of floral visitation differed significantly among the different foraging modes (H=54.1; P < 0.001; Kruskal-Wallis test; no significant difference was found between morning and afternoon visitation rates for bees making O- or IR-visits, and data for each of these tactics were pooled over the 2 periods; Table 1). Floral visits per min were similar between bees making IR- and ID-visits (P > 0.05), but these rates were significantly lower than that observed for O-foragers (P < 0.05 for both tests; Dunn’s rank sum multiple-comparison test following Daniel 1990). Though IR- and ID-foragers had similar floral entry rates, bees making IR-visits both landed on a higher proportion of flowers (after approaching) and entered a higher proportion of flowers (after landing) than bees making ID-visits (U=558 and 660, respectively; P < 0.001 in both cases; Mann-Whitney test; Table 1). Bees utilizing different foraging tactics also exhibited different handling times at individual flowers (H=50.1; P < 0.001; Kruskal-Wallis test; handling times did not differ between morning and afternoon for bees making O- or IR-visits, and data for each of these tactics were pooled over the 2 periods; Table 1). Handling times were similar between bees making IR-and ID-visits (P < 0.05), but these individuals spent nearly twice as much time at a given flower as O-foragers (P < 0.05 in both cases; Dunn’s rank sum multiple comparison test following Daniel 1990).

Nectar and Pollen Loads Both nectar (H=7.0) and pollen (H=24.1) loads varied with foraging tactic (P < 0.05 in both cases; Kruskal-Wallis test; Table 1). Bees making O-visits had signiﬁcantly

Villalobos & Shelly: Honey Bee Behavior 403

TABLE 1. COMPARISONS AMONG BEES MAKING O-, IR-, OR ID-VISITS, RESPECTIVELY. AVERAGE VALUES (SE) ARE PRESENTED. SAMPLE SIZES FOR BEHAVIORAL DATA WERE: O-54, IR-36, AND ID-15 INDIVIDUAL BEES OBSERVED OVER 10 CONSEC- UTIVE FLORAL VISITS. FOR NECTAR AND POLLEN LOADS AND WING LENGTH, 15 BEES FROM EACH FORAGING TACTIC WERE EXAMINED.

Foraging Tactic

OIRID

Flowers/Min 10.1 (0.7) 6.9 (0.4) 5.9 (0.4) % Landings 95 (1.6) 73 (3.9) % Entries 83 (1.5) 58 (3.5) Time/Flower (s) 4.3 (0.2) 7.6 (0.4) 7.5 (0.5) Medial Cross Vein (mm) 1.31 (0.01) 1.29 (0.02) 1.31 (0.01) Nectar Load (ul) 9.7 (1.6) 7.8 (1.3) 3.3 (0.6) Pollen Load (mg) 0.0 (0) 1.1 (0.5) 5.1 (0.7) larger nectar loads than either IR- or ID-foragers, and IR-foragers, in turn, carried more nectar than ID-foragers (P < 0.05 in all cases; Dunn’s rank sum multiple-comparison test following Daniel 1990). Approximately 1/2 of the O- and IR-foragers had at least 8 ul of nectar, whereas none of the ID-foragers carried more than 5 ul. Con- versely, ID-foragers had signiﬁcantly larger pollen loads than O- or IR-foragers (P < 0.05 in both cases; Dunn’s rank sum multiple-comparison test following Daniel 1990; Table 1). All ID-foragers had pollen loads exceeding 1 mg (dry weight), whereas none of the O-foragers and only 27% (4/15) of the IR-foragers carried measurable amounts of pollen. Bees displaying different foraging tactics did not differ signiﬁcantly in body size (H=0.9; P > 0.05; Kruskal-Wallis test; Table 1). Also, body size was unrelated to the volume of nectar loads (rs=0.21; data pooled over O- and IR-foragers) or to the dry weight of pollen loads (rs=0.11; ID-foragers only; P > 0.05 in both cases; Spearman’s rank correlation).

Between-Day Consistency in Individual Behavior Nearly all of the re-sighted individuals displayed a single foraging tactic across days. Of the 70 bees marked, 22 were seen on 1 or more mornings after marking (x=4.3 d; range=2-6); re-sighting probabilities were similar among the 3 foraging types (29%, 35%, and 34% for O-, IR-, and ID-foragers, respectively). Of the 22 re- sighted bees, only 2 (2/22=9%) were seen displaying 2 different foraging modes. In the ﬁrst case, the worker made ID-visits for 2 d and then O-visits over the next 4 d, and in the second, the worker made IR-visits on 3 d and then O-visits on 2 d. Over the 2 d when we made morning and afternoon observations, a total of 11 different individuals (all O- or IR-foragers) was seen during both periods of the same day, and all displayed the same foraging behavior at the 2 times.

Inter-site Variation in Foraging Behavior The relative occurrence of the different foraging tactics varied considerably among the 4 sites (Fig. 1). Most notably, O-visits represented over 50% of all visits at the Wilson

404 Florida Entomologist 79(3) September, 1996

Fig. 1. Average frequencies of the different foraging tactics at the 4 study sites. Av- erage numbers (SE) of honey bees per transect (n=15-21) were: Makiki 12.0 (0.6); Manoa 12.2 (0.4); Wilson 26.4 (1.1); Chaminade 20.0 (0.9). and Chaminade sites but were relatively rare at Manoa and completely absent at Mak- iki. Conversely, IR-visits were much more common at Makiki and Manoa than Wilson or Chaminade. The incidence of ID-visits varied only between 11%-21% among the sites. Though only a few sites were monitored, the incidence of O-visits appeared to be correlated with the abundance of carpenter bees and consequently the frequency of perforated flowers (Table 2). At Makiki, where O-visits were absent, carpenter bees were never recorded from the quadrats, and less than 10% of the flowers had holes or slits. In contrast, carpenter bees and perforated flowers were most common at Wilson and Chaminade, where high proportions of honey bees made O-visits. The density of honey bees was also higher at these latter sites, but since only carpenter bees (and not honey bees) perforated flowers it seems unlikely that the occurrence of O-visits was directly related to the abundance of conspecific foragers (Table 2). Flower density also seemed unimportant as the Makiki site had a flower density nearly 50% higher than the Chaminade site (Table 2).

DISCUSSION

As commonly reported for honey bees (Seeley 1985; Winston 1987), workers at our study sites showed specialization for nectar vs. pollen collection. IR- and ID-foragers gathered both resources, but the former concentrated on nectar and the latter on pollen; workers making O-visits collected nectar exclusively. Also, with respect to nectar harvesting, workers either collected it legitimately (IR- and ID-visits) or via robbing

Villalobos & Shelly: Honey Bee Behavior 405

TABLE 2. BEE AND FLOWER DENSITY AND FREQUENCY OF PERFORATED FLOWERS AT THE 4 STUDY SITES. AVERAGE VALUES (SE) ARE PROVIDED. SEE TEXT FOR SAM- PLING METHODS.

Number/m2 Perforated Site A. mell. X. son. A. gan. Flowers (%)

Makiki 0.8 (0.1) 0.0 42 (2.2) 8 (1.2) Manoa 0.6 (0.1) 0.1 (0.05) 25 (1.9) 65 (3.0) Wilson 2.6 (0.2) 0.4 (0.2) 56 (2.9) 86 (3.8) Chaminade 2.1 (0.1) 0.3 (0.1) 31 (2.2) 89 (2.2)

through corollar slits made by carpenter bees (O-visits). Consistency in nectar-collecting tactics was evident both within individual trips (see also Weaver 1956) and between trips on the same day or on different days (see Higashi et al. 1988 for similar data on bumble bees). Nectar robbing appeared to be an opportunistic strategy whose incidence was correlated with the abundance of carpenter bees and corollar perforations. However, it is not known why particular individuals specialized on 1 tactic over another. In the bumble bee B. pratorum L., smaller workers were nectar robbers exclusively, whereas larger workers both robbed nectar and collected pollen (Barrow & Pickard 1984; see also Brian 1952). In our study, however, foraging behavior was unrelated to worker size. It also seems unlikely that worker age was important as other studies (Ribbands 1952; Free 1963) found no consistent age-related pattern in honey bee foraging behavior. Based on the observations of Weaver (1956), learning was perhaps an important determinant of foraging specialization, and workers may have become “fixed” to a particular foraging mode that was initially successful. Alternatively, recent studies (Cal- derone & Page 1988; Robinson & Page 1989) have demonstrated a genetic component to task specialization in A. mellifera, and it is possible that inter-individual variation in foraging behavior reflects underlying genetic differences. In addition to inter-individual variation in behavior, several behavioral differences were evident among the 3 foraging tactics. First, though abundance of all foragers declined in the afternoon, ID-workers were completely absent. Whether ID-foragers remained in the hive or visited another food source is unknown, but based on morning and afternoon sightings of the same individuals, it is unlikely that ID-workers switch to O- or IR-visits at A. gangetica in the afternoon. Second, rates of floral visitation varied with foraging mode: O-foragers visited more flowers per min and spent less time per flower than IR- or ID-foragers, which had similar movement rates and handling times. Travel distances between successive floral visits may have also varied with foraging tactic (Zimmerman 1982a), but this hypothesis remains untested. Finally, the degree of floral discrimination appeared to differ between I-tactics. Based on lower probabilities of landing after approach and floral entry after landing, ID-foragers were apparently more selective than IR-foragers. This observation suggests that visual signals associated with pollen availability (Zimmerman 1982b) were more con- spicuous and/or reliable than possible visual (Thorp et al. 1975) or odor (Heinrich 1979) cues associated with nectar levels. It is unlikely that scent-marking by previous honey bee visitors was responsible (Wetherwax 1986; Giurfa & Nunez 1992), because unless IR- and ID-foragers leave different odors, a worker would be unable to assess which type of floral visits was made previously.

406 Florida Entomologist 79(3) September, 1996

Description of the different foraging modes raises many questions regarding the economics of food collection. For example, how does the availability of nectar and pollen change through the day? How does the rate of nectar intake compare between workers making O- and IR-visits, respectively? How does this rate vary through the day for these 2 tactics? How does the rate of pollen collection compare between workers making IR- and ID-visits, respectively? Is ﬂoral rejection by IR- and ID-workers actually indicative of depleted resources? We intend to address these and related questions in future studies.

ACKNOWLEDGMENTS

We thank Nani Wong for identifying the plant and the students of Biology 110, Spring 1993, Chaminade University, for their enthusiastic assistance in the ﬁeld. Caryn Ihori, Emma Shelly, and Meredith Whitney assisted with marking and measuring honey bees.

REFERENCES CITED

BARROW, D. A., AND R. S. PICKARD. 1984. Size-related selection of food plants by bumblebees. Ecol. Entomol. 9: 369-373. BARROWS, E. M. 1980. Robbing of exotic plants by introduced carpenter and honey bees in Hawaii, with comparative notes. Biotropica 12: 23-29. BENEDEK, P., L. BANK, AND J. KOMLODI. 1973. Behaviour of wild bees (Hymenoptera: Apoidea) on hairy vetch (Vicia villosa Roth.) flowers. Z. Ang. Entomol. 74: 80- 85. BRIAN, A. D. 1952. Division of labour and foraging in Bombus agrorum Fabricius. J. Anim. Ecol. 21: 223-240. BUCHMANN, S. L., AND C. W. SHIPMAN. 1990. Pollen harvesting rates for Apis mellifera L. on Gossypium (Malvaceae) flowers. J. Kansas Entomol. Soc. 63: 92-100. BURRILL, A. C. 1925. Honey bees follow wood bees for nectar. Science 62: 134. CALDERONE, N. W., AND R. E. PAGE. 1988. Genotypic variability in age polyethism and task specialization in the honey bee, Apis mellifera (Hymenoptera: Apidae). Be- hav. Ecol. Sociobiol. 22: 17-25. DANIEL, W. W. 1990. Applied Nonparametric Statistics. PWS-Kent, Boston, Mass. FELL, R. D. 1986. Foraging behaviors of Apis mellifera L. and Bombus spp. on oilseed sunflower (Helianthus annuus L.). J. Kansas Entomol. Soc. 59: 72-81. FREE, J. B. 1960a. The pollination of fruit trees. Bee World 41: 141-151, 169-186. FREE, J. B. 1960b. The behaviour of honeybees visiting flowers of fruit trees. J. Anim. Ecol. 29: 385-395. FREE, J. B. 1963. The flower constancy of honeybees. J. Anim. Ecol. 32: 119-131. GERLING, D. 1982. Nesting biology and flower relationships of Xylocopa sonorina Smith in Hawaii (Hymenoptera: Anthophoridae). Pan-Pacific. Entomol. 58: 336-351. GIURFA, M., AND J. A. NUNEZ. 1992. Honeybees mark with scent and reject recently visited flowers. Oecologia 89: 113-117. HAWKINS, R. P. 1961. Observations on the pollination of red clover by bees. I. The yield of seed in relation to the numbers and kinds of pollinators. Ann. Appl. Biol. 49: 55-65. HEINRICH, B. 1979. Resource heterogeneity and patterns of movement in foraging bumblebees. Oecologia 40: 235-246. HELMS, C. W. 1970. Bees and blueberries. Canadian J. Zool. 48: 185. HIGASHI, S., M. OHARA, H. ARAI, AND K. MATSUO. 1988. Robber-like pollinators: over- wintered queen bumblebees foraging on Corydalis ambigua. Ecol. Entomol. 13: 411-418. INOUYE, D. W. 1980. The terminology of floral larceny. Ecology 61: 1251-1253. Villalobos & Shelly: Honey Bee Behavior 407

LIEFTINCK, M. A. 1956. Revision of the carpenter bees (Xylocopa Latreille) of the Moluccan Islands, with notes on other Indo-Australian species. Tijdschr. Ento- mol. 99: 55-73. MCGREGOR, S. E., S. M. ALCORN, E. B. KURTZ, AND G. D. BUTLER. 1959. Bee visitors to saguaro flowers. J. Econ. Entomol. 52: 1002-1004. RIBBANDS, C. R. 1952. Division of labour in the honeybee community. Proc. Roy. Soc. London (B) 140: 132-143. ROBINSON, G. E., AND R. E. PAGE. 1989. Genetic determination of nectar foraging, pollen foraging, and nest-site scouting in honey bee colonies. Behav. Ecol. Socio- biol. 24: 317-323. SCHMID-HEMPEL, P. 1991. The ergonomics of worker behavior in social Hymenoptera. Adv. Stud. Behav. 20: 87-134. SEELEY, T. D. 1985. Honeybee ecology. Princeton Univ. Press, Princeton, NJ. THORP, R. N., D. L. BRIGGS, J. R. ESTES, AND E. H. ERIKSON. 1975. Nectar fluorescence under ultraviolet irradiation. Science 189: 476- 478. WAGNER, W. L., D. R. HERBST, AND S. H. SOHMER. 1990. Manual of the flowering plants of Hawaii. Bishop Museum, Honolulu, Hawaii. WEAVER, N. 1956. The foraging behavior of honeybees on hairy vetch. I. Foraging methods and learning to forage. Insectes Soc. 3: 537-549. WETHERWAX, P. B. 1986. Why do honeybees reject certain flowers? Oecologia 69: 567- 570. WINSTON, M. L. 1987. The biology of the honey bee. Harvard University Press, Cam- bridge, MA. ZIMMERMAN, M. 1982a. The effect of nectar production on neighborhood size. Oecolo- gia 52: 104-108. ZIMMERMAN, M. 1982b. Optimal foraging: random movement by pollen collecting bumblebees. Oecologia 53: 394-398.

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Gould: Hot Air Quarantine Treatment for Papayas 407

MORTALITY OF TOXOTRYPANA CURVICAUDA (DIPTERA: TEPHRITIDAE) IN PAPAYAS EXPOSED TO FORCED HOT AIR

WALTER P. GOULD USDA-ARS, Subtropical Horticulture Research Station, Miami, FL

ABSTRACT

Papaya fruit fly, Toxotrypana curvicauda Gerstaecker, is a quarantined pest of papayas. Papayas with naturally occurring infestations of the papaya fruit fly were exposed to forced hot air at 48°C for 30 to 210 m. Forced hot air at 48°C provided 97% mortality of Papaya fruit fly immature stages in papayas treated for 60 min. Probit 9 mortality predicted that at least 167 min of treatment was needed, but probably would require that the center temperature of the coldest fruit reach 46°C. This is the first quarantine treatment tested against the papaya fruit fly which is a major pest of papayas in Florida.

Key Words: Papaya fruit ﬂy, Toxotrypana curvicauda, commodity treatment, forced hot air

408 Florida Entomologist 79(3) September, 1996

RESUMEN

La mosca de la papaya, Toxotrypana curvicauda Gerstaecker, es una plaga cuaren- tenada de la papaya. Papayas naturalmente infestadas con T. curvicauda fueron ex- puestas a aire caliente forzado a 48°C durante 30-210 minutos. El aire caliente forzado a 48°C produjo 97% de mortalidad de los estados inmaduros de las moscas en papayas tratadas durante 60 minutos. La mortalidad probit 9 predijo que al menos se necesitan 167 minutos de tratamiento, pero probablemente se requiera que la tempe- ratura del centro de la fruta más fría alcance los 46°C. Este es el primer tratamiento cuarentenario probado contra esta impotante plaga de la papaya en la Florida.

Papaya (Carica papaya L.) is grown throughout the tropical regions of the world (Morton 1987). This plant is a member of the Caricaceae and probably originated in the Neotropics. The plant is grown for its melon shaped fruit which has an orange or red flesh, and is eaten fresh, dried, or is made into juice. The papaya plant and fruit have several pest problems ranging from viruses to insects. Fruit flies will use the papaya as a host, but the latex laden sap probably prevents most species of flies from successfully ovipositing until the fruit is very ripe. The papaya fruit fly, Toxotrypana curvicauda Gerstaecker, is a specialist on papayas (Knab & Yothers 1914). Only a few other hosts are known for this species (Baker et al. 1944, Butcher 1952, Castrejon-Ayala 1987, Castrejon-Ayala & Camino-Lavin 1991, Landolt 1994). The papaya fruit fly is found from Mexico south to Brazil, on some Caribbean islands, and in southern Florida. It is one of the larger fruit flies. Fe- males have long ovipositors and eggs are deposited in the central cavity of the papaya fruit (Knab & Yothers 1914). The fruit fly prefers to oviposit in immature green fruit, the early instar larvae feed on the coating and endosperm of developing papaya seeds (Pena et al. 1986). As the larvae mature, they switch to feeding on fruit pulp (Pena et al. 1986). Papaya fruit flies are a major barrier to growing papaya fruits in Florida. A large percentage (up to 30 to 50%) of the papayas in a field is often infested. There are no postharvest quarantine treatments available for Florida papayas, which are also considered to be hosts of Caribbean fruit fly, Anastrepha suspensa (Loew). A postharvest quarantine treatment would greatly expand the export market for Florida papayas. Hot air, hot water, and fumigation with ethylene dibromide (EDB) have been studied in Hawaii as commodity treatments for papayas infested with a number of fruit fly species (Armstrong et al. 1989, Armstrong et al. 1995, Couey & Hayes 1986, Couey et al. 1985). The fumigant, ethylene dibromide, has been banned as a carcinogen (Ruckelshaus 1984), and hot air has been found to damage the fruit less than hot water immersion (Armstrong et al. 1989, 1995, Chan et al. 1981, Jones 1939). This study was conducted to determine if hot air would be feasible as a commodity treatment for papayas infested with immature stages of the papaya fruit fly.

MATERIALS AND METHODS

Initial attempts were unsuccessful to raise papaya fruit ﬂies for the experiment by rearing larvae in commercial papayas from the packinghouse. Not enough larvae were found after commercial fruits were exposed to captive ﬂies in cages to conduct the experiments, probably because the commercial papayas were too ripe. Therefore, wild papayas with naturally occurring infestations were collected on the United States Department of Agriculture—Agricultural Research Service (USDA-ARS) Sub-

Gould: Hot Air Quarantine Treatment for Papayas 409 tropical Horticulture Research Station in Miami, and from a mixed grove of papayas (cvs. Red Lady and Known-You-1) and guavas in western Dade county in both 1993 and 1994. Papayas were collected from Oct.-Dec. 1993 (Rep. 1-5), and July-Aug. 1994 (Rep. 6-12). There were not enough papayas available to divide them into different weight classes, thus the papayas were sorted into groups of 10 (Reps. 2-6), 11 (Reps. 1 & 8), or 12 (Reps. 7, 9-12) so that each group had equal numbers of similar sized papayas. The groups were then randomly assigned to treatments or control. All fruits were weighed and their diameter was measured. Three control groups were held to estimate the fly population present for each replication. One group of papayas was used for each duration of treatment time. A forced hot air machine described by Sharp et al. (1994) was used to treat the papayas. Treatments consisted of 30, 60, 90, 120, 150, 180 and 210 min of forced hot air at 48°C. For each replication, one group of papayas was treated separately for each treatment time. Three papayas in each group had 36 gauge type t (copper-constantan) thermocouples placed beneath the surface of the fruit skin. Five of the largest papayas had thermocouples placed at the center of each fruit. Fruit temperatures, air temperature and relative humidity were monitored and recorded by a personal computer. Humidity was measured with a General Eastern humidity meter and sampling head (model M1/SIM-12H, General Eastern Instruments, Woburn MA). The computer was programmed to maintain heat and humidity levels. The humidity was kept below the dew point using the papaya surface temperatures so that the papayas remained dry. Previous research indicated that papayas are very sensitive to surface moisture which may interfere with fruit respiration during heat treatments (Chan et al. 1981, Jones 1939). After treatment all treated fruit were allowed to cool at ambient air temperatures (≈25°C) and then placed in holding cages. Larvae emerging from fruits were collected and counted for several weeks until the fruits had completely decomposed and all larvae had left the fruit. The experiment was repeated 12 times. Statistical analysis was performed using probit analysis (SAS Institute 1988) and also by fitting linear and non-linear models using TableCurve 2D (Jandel Scientific 1994).

RESULTS AND DISCUSSION An average of 5.3 ±0.9 larvae emerged per papaya from the 10 to 12 nontreated papayas in each replicate (range of 0.1 to 24.4 larvae per papaya). The estimated treated population totaled an average of 697.3 over the 12 replications (Table 1, mean of 3 controls). The treatment caused 45% mortality in the first 30 min (Fig. 1). In two replications, larvae survived 60 min of treatment and in one replication 18 larvae survived 150 min of treatment (Table 1). The center temperatures of the heated papayas reached 46°C after about 90 min of treatment (Fig. 2, 557 g papaya). Previous research with heat has shown that other fruit flies are killed when center temperatures of heated commodities reach 46°C (Sharp 1994). There are two possible reasons as to why papaya fruit fly larvae survived 150 min in replicate 4. Either there was an extremely high number of larvae in that particular treatment and/or there was a very large fruit containing the larvae which did not reach the treatment temperature. Due to the limited availability of infested papayas, all sizes were used in the experiment ranging from 37 to 3059g (mean = 543.8g, st. dev. = 412.0). There was one papaya in treatment replicate 4 that weighed 2,605 g. which may have had larvae in it. Papayas were not held individually because of logistical constraints so the individual papaya from which the larvae came could not be determined.

410 Florida Entomologist 79(3) September, 1996 otal . AIR

HOT

WITH

TREATMENT

AFTER

PAPAYAS

FROM

RECOVERED

123456789101112T LARVAE

OTAL 1. T 306090 000 00000000 0 0 48 0 0 0 2 0 42 9 132 11 22 0 0 0 0 0 6 43 0 89 0 10 0 383 0 31 ABLE Time(Min) Replicate 5 010000018 000 00000000 0 0 000180000000 000 00000000 0 0 120 000 00000000 0 0 150 180 210 ControlControlControl 25 7 40 0 22 1 11 0 0 72 154 293 81 59 147 80 66 27 10 22 29 26 30 28 166 124 142 28 99 37 39 5 30 45 132 15 613 876 603 T

Gould: Hot Air Quarantine Treatment for Papayas 411

Fig. 1. Mean number of larvae recovered from hot air treated papayas for replications 1-12.

Using different models on the mortality data gives different predictions of probit 9 (99.9968% mortality). Probit (Normal) gave a prediction of 167.5 min, Logit 87.7 min, Gompertz 267.9 min, Gaussian Cumulative 120.6 min, and the best fitting linear equation (y=a+blnx/x2) 123 min. Ignoring the one data point from replicate 4 would give 100% mortality at about 90 min, but the true figure is probably close to 167.5 min. The treatment should probably be extended until the core temperature of the coldest fruit reaches 46°C and remains there for at least 10 min. Mangan & Ingle (1992, 1994) found that heating mangoes and grapefruit with forced hot air until the core reached 48°C (157 to 200+ min) gave probit 9 mortality of West Indian fruit fly, Anastrepha obliqua (Macquart), and Mexican fruit fly, Anas- trepha ludens (Loew), respectively. Sharp et al. (1991, 1994) found that heating man- gos and grapefruit with forced hot air from 101 to 214 min achieved probit 9 mortality of A. suspensa. Armstrong et al. (1995) found that heating papayas with forced hot air achieved probit 9 in 210 ± 15 min for Mediterranean fruit fly, Ceratitis capitata (Wiedemann), melon fly, Bactrocera curcubitae (Coquillett), and oriental fruit fly, Bac- trocera dorsalis (Hendel). This indicates that the papaya fruit fly is similar in its tol- erance to heat to the other tephritid species that have been tested.

ACKNOWLEDGMENT

I thank W. Montgomery, USDA-ARS, for his assistance. I thank M. Trunk, Brooks Tropicals, Homestead, FL, and M. Hennessey, USDA-ARS, for their critical review of this manuscript, and E. Schnell, USDA-ARS, for translation of the abstract to Span- ish.

412 Florida Entomologist 79(3) September, 1996

Fig. 2. Core temperature of average sized (557g) hot air treated papaya.

REFERENCES CITED

ARMSTRONG, J. W., J. D. HANSEN, B. K. S. HU, AND S. A. BROWN. 1989. High-temperature forced-air quarantine treatment for papayas infested with tephritid fruit flies (Diptera: Tephritidae). J. Econ. Entomol. 82: 1667-1674. ARMSTRONG, J. W., B. K. S. HU, AND S. A. BROWN. 1995. Single-temperature forced hot-air quarantine treatment to control fruit flies (Diptera: Tephritidae) in papaya. J. Econ. Entomol. 88: 678-682. BAKER, A. C., W. E. STONE, C. C. PLUMMER, AND M. MCPHAIL. 1944. A review of studies on the Mexican fruitfly and related Mexican species. USDA Misc. Pub. 531, 155 pp. BUTCHER, F. G. 1952. The occurrence of papaya fruit fly in mango. Proc. Florida State Hort. 65: 196. CASTREJON-AYALA, F. 1987. Aspectos de la biología y hábitos de Toxotrypana curvicauda Gerst. (Diptera: Tephritidae) en condiciones de laboratorio y su distribu- ción en una plantación de Carica papaya L. en Yautepec, Mor. B.S. Thesis, Instituto Politécnico Nacional, México D.F., México. 88 pp. CASTREJON-AYALA, F., AND M. CAMINO-LAVIN. 1991. New host plant record for Toxot- rypana curvicauda (Diptera: Tephritidae). Florida Entomol. 74: 466. CHAN, H. T. JR., S. Y. T. TAM, AND S. T. SEO. 1981. Papaya polygalacturonase and its role in thermally injured ripening fruit. J. Food. Sci. 46: 190-191, 197. COUEY, H. M., J. W. ARMSTRONG, J. W. HYLIN, W. THORNBURG, A. N. NAKAMURA, E. S. LINSE, J. OGATA, AND R. VETRO. 1985. Quarantine procedure for Hawaiian papaya, using a hot-water treatment and high-temperature, low-dose ethylene dibromide fumigation. J. Econ. Entomol. 78: 879-884. COUEY, H. M., AND C. F. HAYES. 1986. Quarantine procedure for Hawaiian papaya using fruit selection and a two-stage hot-water immersion. J. Econ. Entomol. 79: 1307-1314.

Gould: Hot Air Quarantine Treatment for Papayas 413

JANDEL SCIENTIFIC. 1994. TableCurve 2D Windows v2.0 User’s Manual. Jandel Scien- tific, San Rafael, California. 404 pp. JONES, W. W. 1939. The influence of relative humidity on the respiration of papaya at high temperatures. Proc. American Soc. Hort. Sci. 37: 119-124. KNAB, F., AND W. W. YOTHERS. 1914. The papaya fruit fly. J. Agric. Res. 2: 447-453. LANDOLT, P. J. 1994. Fruit of Morrenia odorata (Ascepiadaceae) as a host for the papaya fruit fly, Toxotrypana curvicauda (Diptera: Tephritidae). Florida Entomol. 77: 287-288. MANGAN, R. L., AND S. J. INGLE. 1992. Forced hot-air quarantine treatment for mangoes infested with West Indian fruit fly (Diptera: Tephritidae). J. Econ. Ento- mol. 85: 1859-1864. MANGAN, R. L., AND S. J. INGLE. 1994. Forced hot air quarantine treatment for grapefruit infested with Mexican fruit fly (Diptera: Tephritidae). J. Econ. Entomol. 87: 1574-1579. MORTON, J. F. 1987. Fruits of warm climates. J. F. Morton, Miami, Florida. 505 pp. PENA, J. E., D. F. HOWARD, AND R. E. LITZ. 1986. Feeding behavior of Toxotrypana curvicauda (Diptera: Tephritidae) on young papaya seeds. Florida Entomol. 69: 427-428. RUCKELSHAUS, W. D. 1984. Ethylene dibromide, amendment of notice of intent to can- cel registration of pesticide products containing ethylene dibromide. Fed. Reg- ist. 49: 14182-14185. SAS INSTITUTE. 1988. SAS/STAT User’s Guide. Release 6.03 Ed. SAS Institute, Cary, NC. SHARP, J. L. 1994. Hot water immersion, pp. 133-147 in J. L. Sharp and G. J. Hallman [eds.], Quarantine treatments for pests of food plants. Westview Press, Boulder, Colorado. 290 pp. SHARP, J. L., J. J. GAFFNEY, J. I. MOSS, AND W. P. GOULD. 1991. Hot-air treatment device for quarantine research. J. Econ. Entomol. 84: 520-527.

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Seal et al.: Euxesta stigmatis Frequency and Reproduction 413

ABUNDANCE AND REPRODUCTION OF EUXESTA STIGMATIS (DIPTERA: OTITIDAE) ON SWEET CORN IN DIFFERENT ENVIRONMENTAL CONDITIONS

D. R. SEAL, R. K. JANSSON AND K. BONDARI1 University of Florida, Institute of Food and Agricultural Sciences, Tropical Research and Education Center, Homestead, Florida 33031

1Statistical and Computer Services, Coastal Plain Experiment Station, The University of Georgia, Tifton, Ga. 31793-0748

ABSTRACT

The abundance of a picture-winged ﬂy, Euxesta stigmatis Loew (Diptera: Otitidae), was studied on sweet corn in different environmental conditions. Adults were more abundant on plants in the sun than on plants in the shade. More adults were observed on tall plants than on medium and short plants. Females found on corn tassels had smaller ovaries with fewer mature eggs than those found on other corn plant parts (i.e., stem, ear, or leaf). Females with many eggs per ovary were observed at 0900 hours EST, but the number of eggs per ovary decreased as the day progressed. The

414 Florida Entomologist 79(3) September, 1996 hosts of E. stigmatis in southern Florida included sweet corn, ﬁeld corn, sorghum, sugarcane, guava, banana, atemoya, orchid, and potato. No larvae were found on orange.

Key Words: Abundance, habitat, distribution, Otitidae, maize

RESUMEN

Fue estudiada la abundancia de la mosca Euxesta stigmatis Loew (Diptera: Otiti- dae) en maíz dulce en diferentes condiciones ambientales. Los adultos fueron más abundantes en plantas al sol que en plantas a la sombra. Más adultos fueron obser- vados en plantas altas que en plantas medianas y pequeñas. Las hembras encontradas en el penacho de la mazorca tuvieron los ovarios más pequeños con menos huevos maduros que las encontradas en otras partes de la planta. Fueron observadas hembras con varios huevos por ovario a las 0900 horas (hora estándar del este), pero el nú- mero de huevos por ovario disminuyó en la medida en que el día progresó. Los hospedantes de E. stigmatis en el Sur de la Florida inculyen maíz dulce, maíz de campo, sorgo, caña de azúcar, guayaba, banano, atemoya, orquídea y papa. No fueron encontradas larvas en naranja.

Sweet corn, Zea mays var. rugosa Bonaf, is an important winter crop in southern Florida and is valued at over $5.7 million annually (Anonymous 1987). Euxesta stigmatis Loew (Diptera: Otitidae) is a serious pest of sweet corn in southern Florida (Seal & Jansson 1989) and in various Caribbean countries. Surveys conducted in 1989 (D.R.S., unpublished data) showed this insect to cause approximately 95% loss of sweet corn in nontreated fields. In some instances, economic loss has been reported even after the application of recommended insecticides. The E. stigmatis female ovi- posits inside the silks at the tip of corn ears. Larvae hatch 2-3 days after oviposition (Seal & Jansson 1989). Larval development takes place inside corn ears. Foliar application of insecticides has minimal effect on eggs and immature stages (Seal & Jansson 1995). During the late growing season optimum pesticide coverage is difficult to achieve because of the thick plant canopy. Few insecticides can be applied at the time of ear maturation because of days to harvest limitations. Nevertheless, several attempts have been made to manage this pest using insecticides (Bailey 1940, Hayslip 1951, Seal & Jansson 1989). Proper pest management practice for E. stigmatis may be developed based on appropriate knowledge of its biology and behavior; however there is little published information on the biology and life history of E. stigmatis. App (1938) presented brief observations on its biology. Seal & Jansson (1989) elaborated on some additional aspects of the biology and management of this fly in Florida, and Seal & Jansson (1993) presented some information on its oviposition and development. Recently, Seal & Jan- sson (1995) studied the diel pattern of the sex ratio of E. stigmatis on different sweet corn plant parts. The objectives of the present study were to provide additional information on the biology and the ecology of this pest. Specifically, we studied the habitat and host plants, weather effect, within plant distribution of females and diel pattern of egg supply of E. stigmatis.

Seal et al.: Euxesta stigmatis Frequency and Reproduction 415

MATERIALS AND METHODS

Habitat and Host Plants

Different habitats of E. stigmatis were surveyed for the presence of adults, immatures, and eggs in April, May and June, 1991. Habitats sampled included various fruits and vegetables and wooded and weedy areas. Qualitative and quantitative as- sessments were made by observing E. stigmatis adults 2 to 5 times in each habitat type at an interval of at least 7 days. We collected 5 to 30 specimens on each sampling date from each host, and the life stage of each specimen was recorded. The identity of pre-imaginal stages was conﬁrmed by rearing them to the adult stage.

Weather

The response of E. stigmatis adults to sunny, cloudy, and rainy conditions was studied in a 0.4 ha sweet corn ﬁeld (10-week-old) located in Homestead, FL on different dates in April, May and June, 1991. On a sunny day, the air temperatures ranged from 25-35°C. On a cloudy day, air temperatures ranged from 23-28°C, dewpoint 13- 21.4°C, and barometric pressures 28-33 in Hg. A rainy day was characterized as a day when rain continued for 1 h or more. We surveyed the ﬁeld on 4 dates for each type of environmental condition. On each date, 10 plants were randomly selected and checked for E. stigmatis adults.

Effects of Sunlight and Plant Size on the Abundance of E. stigmatis Adults

The abundance of E. stigmatis on sweet corn plants in sunny and shaded conditions was determined in a nontreated 0.4-ha sweet corn field in which the mean ± SE height of plants was 102 ± 5.1 cm (n = 62) and most of the plants (about 85%) were tasseling. In the morning at 1000 hours EST, sweet corn plants on the east side of the field received direct sunlight and the plants on the west side were in natural shade. The reverse was observed in the afternoon at 1400 hours EST. Eight sun-lit and 8 shaded plants were sampled randomly at 1400 hours EST and the number of E. stigmatis adults found on each plant was recorded. Light intensity for each plant was measured by stationing a light meter (Luna Pro F, Gossen, Germany) at a height of 70 cm along the stem above the ground. Sampling of plants under each environmental condition was replicated 5 times. We also studied the abundance of E. stigmatis adults on sweet corn plants of 3 different heights, [short (≤ 70 cm), medium (71-119 cm), and large (120-165 cm], after tassels emerged. The field was planted to ‘Silver Queen’ sweet corn on 14 January 1991. Size differences among the plants were mainly due to the differences in the rate of plant growth. The field was divided into 5 uniform sections (about 0.1 ha each). As E. stigmatis adults settled on sweet corn plants at dawn (between 0600 and 0700 hours EST) and dusk (between 1800 and 1900 hours EST), 3 plants from each height category were randomly selected and the number of adults on each plant was recorded. Observations were made in each plot at these 2 times (dawn and dusk) 2 to 3 days a week for 4 consecutive weeks, between 25 March and 22 April, after tasseling.

Within Plant Distribution of Females and Diel Pattern of Egg Supply

This study was conducted using a 0.4 ha sweet corn ﬁeld planted on 4 January 1991 and was not treated with insecticides. Field management was as discussed ear-

416 Florida Entomologist 79(3) September, 1996 lier. E. stigmatis females (n = 25) were collected from 4 different locations on sweet corn plants (tassel, ear, stem, and leaf) at 0900, 1300, and 1700 hours EST each day for a 5 day period between 20 and 25 March. Collected females were preserved in 70% ethyl alcohol for later study. We recorded the length (posterior end of post abdominal segment to tip of head) and width (widest part of abdomen) of females. Females were then dissected in 0.7% saline solution under a stereo microscope (25×) to expose the ovaries. The lengths and widths of the ovaries, and the number of egg tubes in each ovary were recorded by using an ocular micrometer. Eggs were counted by severing the abdomen with iris scissors, cutting the sternum lengthwise, and transferring the ovaries to a dish containing 70% ethyl alcohol. We differentiated mature eggs [n = 85, 0.85 ± 0.004, mean ± SE, mm long, and 0.16 ± 0.001 (mean ± SE) mm wide] by size. Immature eggs were exposed by puncturing the epithelial layer of the egg tubes. The number of mature eggs found in each ovary was recorded.

Statistical Analysis Data on habitat preference, abundance of adults on sweet corn plants of different heights, within plant distribution of females with different levels of reproductive maturity, and number of eggs in ovaries were transformed to square root of (x+1) prior to analysis of variance. The transformed data were analyzed by least squares analysis of variance (PROC GLM, SAS 1989), but means of the original data are presented in all tables and ﬁgures for ease of interpretation. The pairwise t-test option PDIFF of PROC GLM (SAS 1989) was used to separate the treatment means where signiﬁcant statistical differences occurred. The Chi-square test (PROC FREQ, SAS 1989) was used to test the independence of the number of adults present on various plant heights and the number of weeks after tasseling had occurred.

RESULTS AND DISCUSSION

Habitat and Host Plants

Most adults were found on various tropical fruit and vegetable crops, but occasionally they were also found in wooded and weedy areas. Larvae and adults were by far the most abundant on sweet corn, followed by ﬁeld corn and with very small numbers on other crops (Table 1). Both larval and adult stages were recorded from all crops, except that no larvae were found in orange groves. Sweet corn, ﬁeld corn and sorghum became infested with E. stigmatis immediately upon appearance of the silks or seed heads. Other plants were infested only when they were decaying.

Weather

On cloudy days, abundance of E. stigmatis adults was 0.3 ± 0.1 (mean ± SE) (n = 40) per sweet corn plant at the pretassel stage. On sunny days, adult abundance was greater (1.3 ± 0.1, mean ± SE) than on cloudy days 0.2 (t = 7.37, df = 79, P ≤ 0.01). In general, adults exhibited decreased activity on rainy days and on sunny days when the wind velocity was >24 km per h.

Effect of Sunlight and Plant Size on the Abundance of E. stigmatis Adults

Adults occurred more frequently (t = 6.2, df = 79, P ≤ 0.01) on the sunlit sweet corn plants [1.3 ± 0.2 (mean ± SE) per plant] than on shaded ones (0.2 ± 0.1, mean ± SE).

Seal et al.: Euxesta stigmatis Frequency and Reproduction 417 0.1c 0.1c 0.1c 0.1c 0.1c 0.1c 0.0c 0.0c 0.1b 0.2a ± ± ± ± ± ± ± ± ± ± Number of 1 Mean 3.0b 6.0a 0.6 1.3 0.0d 0.1d 0.3d 0.3d 0.0d 0.5d 0.2 0.4d 0.2 0.2d 0.2 0.2 0.1 0.2 0.1 0.1 ± ± ± ± ± ± ± ± ± ± . Larvae Adults PLANTS

HOST

Plants < 0.05). Square root (x + 1) transformation was applied prior to the analysis of variance, Square root (x + 1) transformation was < 0.05). DIFFERENT P Number of

ADULTS Miller 21 0.8

AND

LARVAE L. 24 0.2

A. cherimolia A. × L. 63 0.3 L. L. L. 30 0.0 L. 20 0.7 Moench 60 0.2 STIGMATIS spp. 100 0.7 E.

L. ‘Pioneer 305’ L. ‘Silver Queen’ L. 54 64 11.1 57.8 OF spp. 20 0.3 SE ±

Zea mays Zea mays Sorghum bicolor ofﬁcinarum Saccharum Psidium guajava Musa Citrus aurantium Annona squamosa Dendrobium Solanum tuberosum NUMBER

EAN 1. M Means within a column followed by different lowercase letters are signiﬁcantly ( 1 ABLE Sugarcane Guava Banana Orange Atemoya Orchid Potato Sweet corn Sorghum but means are detransformed to the original scale. Host plants corn Field T

418 Florida Entomologist 79(3) September, 1996

In a small field (0.2 ha), the number of E. stigmatis adults were greater (2.7 ± 0.6, mean ± SE) on the first row of the east side of the field than on the first row (0.3 ± 0.1, mean ± SE) of the west side of the same field in the morning. In the late afternoon (1600-1800 hours EST), adults per plant were more abundant on the west side of the field (n = 30, 3.3 ± 1.2, mean ± SE) where light intensity was approximately 7,000 foot- candles than on the east side of the field (n = 30, 0.2 ± 0.1, mean ± SE) where light intensity was approximately 5,000 foot candles. At 1830 hour, light intensity below the canopy was approximately 3,000 to 3,500 foot-candles, when adults were observed to move to the top of plants where the light was somewhat brighter (approximately 4,000 foot-candles). Light intensity appears to play an important role in the determination of feeding and ovipositional activities and in the synchronization of mating behavior of this fly. Such influences of light on feeding, oviposition and mating of tephritid fruit flies was reported by several authors (Tzanakakis & Economopoulos 1967, Boller 1965, Flitters 1964, Barton Browne 1956, Myers 1952). Results of the Chi-square tests (Table 2) indicated that the abundance of adult flies on corn plants 1-4 weeks after tasseling depended on the size of the corn plants. E. stigmatis adults were more abundant on tall plants both in the morning (82% of the total adult population) and evening (86% of the total adult population). Fly abundance did not differ significantly between medium and short plants either in the morning (11% vs. 7% adults, respectively) or evening (9% vs. 5% adults, respectively) samples. Tall plants were exposed to more intense light that may have attracted adults in larger numbers than shorter plants. Irrespective of plant size, abundance of adults increased with increasing age of corn plants up to 3 weeks after anthesis (Table 2). Abundance increased linearly up to the third week after tasseling, but a significant decrease was observed thereafter.

Within-Plant Distribution of Adult Females

The length of females, irrespective of their locations on a sweet corn plant, did not differ significantly (Table 3). Females with a wider abdomen occurred on stems, but no significant differences were observed among the females collected from the other 3 parts of the sweet corn plants. Irrespective of the locations, each ovary contained 48 to 56 egg tubes, each tube containing one mature egg. The lengths and widths of ovaries possessed by females found on stems were significantly greater than those of the females found on leaves (Table 3). Accordingly, the number of unlaid eggs per female collected from stems was the greatest, followed in decreasing order by those collected from ears, leaves, and tassels (Table 3). The comparatively smaller ovary lengths of adult females found on tassels was probably the result of the presence of fewer eggs than in females collected on other plant parts. We found that after oviposition, most of the adult females moved to tassels for subsequent mating during the day time. Adults often moved to the tassels at the end of the day (light intensity, <3,000 foot-candles) possibly for mating and shelter. Most of these females had no eggs in their ovaries. Correlations between the length and width of the adult females (r = 0.68) and between length and width of their ovaries (r = 0.74) were significant (P < 0.05), indicating that an increase in the body length or ovarian length is associated with an increase in corresponding widths of the body or ovary. Also, correlations between the width of an adult and the length (r = 0.54) or width (r = 0.61) of the ovary were positive and statistically significant (P ≤ 0.05) indicating that the wider the body size of an adult, the wider and longer the ovarian size. Adult body width, ovarian length, and ovarian width were positively corre- Seal et al.: Euxesta stigmatis Frequency and Reproduction 419

TABLE 2. NUMBERS OF E. STIGMATIS ADULTS ON SWEET CORN PLANTS OF DIFFERENT SIZES AT DIFFERENT WEEKS AFTER TASSELING.

Plant Size2 Week After Plant Percentage Tasseling1 (n) Large Medium Small of Total Total

Morning 1481021131 2 72 321 28 17 366 28 3 72 519 82 58 759 58 4 72 123 29 15 167 13 All weeks Total 264 1073 141 91 1305 — % Total — 82 11 7 100 — Chi-square with 6 df = 17.5, P = 0.008

Evening 1 48720 91 2 72 428 34 21 483 34 3 72 696 69 47 812 57 4 728818111178 All weeks Total 264 1219 123 79 1421 — % Total — 86 9 5 100 — Chi-square with 6 df = 16.5, P = 0.011

1Samples taken on 2 (week 1 only) or 3 different dates from 24 plants per plant size each. 2Plant size categories: Small (< 70 cm), Medium (71-119 cm), Large (120-165 cm). lated with the number of matured eggs per female. These results indicate a link between the adult female’s shape, size, and reproductive performance. Variation in diel pattern of mating behavior has been observed in some tephritid fruit flies, including some pest species, although most of these flies mate at dusk under low light intensities of less than 1,000 lux (Fletcher 1987). In contrast, Dacus oleae (Gmelin) flies prefer to mate in the afternoon at high light intensities (Arakaki et al. 1984, Lee et al. 1983, Suzuki & Koyama 1981, Qureshi et al. 1974, Tychsen & Fletcher 1971).

Diel Pattern of Egg Supply in the Ovaries

The greatest numbers of eggs in the ovaries of females were recorded at 0900 hours EST for females collected on all 4 plant parts (Fig. 1). Numbers of eggs decreased per female signiﬁcantly at 1300 hours EST, especially for those females found on tassels or leaves, suggesting that the optimum egg laying period of E. stigmatis was between 0900 and 1300 hours EST. The present ﬁnding is in agreement with Seal & Jansson (1993) who reported that E. stigmatis oviposited the highest numbers of eggs between 0900 and 1300 hours EST. It was evident from the condition of the ovaries that egg-laying females were abundant on ears, leaves and stems of corn. Very few egg-laying females were found on tassels. 420 Florida Entomologist 79(3) September, 1996

TABLE 3. MEANS1 OF ADULT SIZE AND REPRODUCTIVE TRAITS OF E. STIGMATIS FEMALES COLLECTED FROM DIFFERENT LOCATIONS ON SWEET CORN PLANTS.

Adult Size2 Ovary Size2 Location on Adults Number of Plant (n) LW LWEggs/Female

Tassel 21 4.9a 1.0b 0.8b 0.6a 3.0c Ear 13 4.9a 0.9b 1.1a 0.5b 29.2ab Stem 15 5.1a 1.2a 1.1a 0.6a 38.6a Leaf 19 4.9a 1.0b 0.9b 0.5b 21.3b SE of mean 0.09 0.03 0.04 0.03 5.6

1Means within a column followed by different lowercase letters differ (P < 0.05). 2L, length (mm); W, width (mm).

Females located on the tassels had few eggs at 1300 hours EST. By contrast, at 0900 hours EST, some females collected from tassels had a full complement of eggs in their ovaries. Upon dissection, the ovaries of some females collected from different locations on the plant at different times (0900, 1300 and 1700 hours EST) contained between zero and a full complement of eggs. These results indicated that egg maturation in E. stigmatis proceeds continuously throughout the day and the night to reach a maximum in the morning. In conclusion, our study showed that E. stigmatis was much more abundant on sweet corn than on other host crops and that the adults were more active in sun than in shade. Females with greater numbers of mature eggs were more abundant on stems than on other plant parts. We observed adults moving to tassels at the end of the day. Because of the variable abundance of adults in different locations on corn plants at different times of the day, selective, prudent and timely use of insecticide appears to be an important management strategy for E. stigmatis. This information may be useful in the quest for a combination of biorational chemical treatments with biological and cultural measures to achieve environmentally sound integrated pest management of E. stigmatis.

ACKNOWLEDGMENTS

We thank P. R. Espinosa and J. M. Hazel for their technical assistance; Drs. R. E. Foster, Department of Entomology, Purdue University, and G. S. Nuessly, Univ. of Florida, I.F.A.S., Everglades Research and Education Center, Belle Glade, FL, for reviewing the manuscript. We thank J. Alger for providing commercial ﬁelds, sweet corn seed, and other miscellaneous supplies. The research was supported in part by a grant from Alger Farms, Homestead, FL. This is Florida Agricultural Experiment Station Journal Series No. R-05310.

REFERENCES CITED

ANONYMOUS. 1987. Dade County Agricultural Statistics. APP, B. A. 1938. Euxesta stigmatis Loew, an otitid ﬂy infesting ear corn in Puerto Rico. J. Agric. Univ. Puerto Rico. 22: 181-8. Seal et al.: Euxesta stigmatis Frequency and Reproduction 421

Fig. 1. Diel pattern of egg supply of E. stigmatis females on different parts of sweet corn plants. Rovary, right ovary; Lovary, left ovary; EST, eastern standard time (n = 25 adults).

ARAKAKI, N., H. KUBA, AND H. SOEMORI. 1984. Mating behavior of the oriental fruit fly, Dacus dorsalis Hendel (Diptera: Tephritidae). Appl. Entomol. Zool. 19: 42- 51. BAILEY, W. K. 1940. Insecticidal control of corn-silk fly. Puerto Rico Exp. St. Circ. Vol. 23. BARTON BROWNE, L. 1956. The effect of light on the fecundity of the Queensland fruit fly, Strumeta tryoni. Australian J. Zool. 5: 145-158. BOLLER, E. F. 1965. Beitrag zur Kenntnis der Eiablage und Fertilitat der Kirschen- fliege Rhagoletis cerasi. Mitt. Schweiz, Entomol. Ges. 38: 194-202. FLETCHER, B. S. 1987. The biology of dacine fruit flies. Annu. Rev. Entomol. 32: 115- 144. FLITTERS, N. E. 1964. The effect of photoperiod, light intensity, and temperature on copulation, oviposition, and fertility of the Mexican fruit fly. J. Econ. Entomol. 57: 811-813. HAYSLIP, N. C. 1951. Corn-silk fly control on sweet corn. Florida Cooperative Exten- sion Circular S-41: 1-5. LEE, L. W. Y., T. H. CHANG, AND C. K. TSANG. 1983. Sexual selection and mating behavior of normal and irradiated oriental fruit flies. CEC/IOBC Symp., Athens, 1982. Rotterdam, Balkema. p. 439-444. 422 Florida Entomologist 79(3) September, 1996

MYERS, K. 1952. Oviposition and mating behavior of the Queensland fruit fly (Dacus (Strumeta) tryoni) and the solanum fruit fly (Dacus (Strumeta) cacuminatus). Australian J. Sci. Res. Ser. B. 5: 264-281. QURESHI, Z. A., M. ASHRAF, A. R. BUGHIO, AND S. HUSSAIN. 1974. Rearing, reproductive behavior and gamma sterilization of the fruit fly, Dacus zonatus (Diptera: Tephritidae). Entomol. Exp. Appl. 17: 504-510. SAS INSTITUTE. 1989. SAS user’s guide: statistics. SAS Institute, Cary, NC. SEAL, D. R., AND R. K. JANSSON. 1989. Biology and management of corn silk fly, Eux- esta stigmatis Loew (Diptera: Otitidae), on sweet corn in southern Florida. Proc. Florida State Hort. Soc. 102: 370-373. SEAL, D. R., AND R. K. JANSSON. 1993. Oviposition and development of Euxesta stigmatis (Diptera: Otitidae). Environ. Entomol. 22: 88-92. SEAL, D. R., AND R. K. JANSSON. 1995. Bionomics of Euxesta stigmatis Loew. (Diptera: Otitidae). Environ. Entomol. 24: 917-922. SUZUKI, Y., AND J. KOYAMA. 1981. Courtship behavior of the melon fly, Dacus cucurb- itae Coquillett (Diptera: Tephritidae). Appl. Entomol. Zool. 16: 164-166. TYCHSEN, P. H., AND B. S. FLETCHER. 1971. Studies on the rhythm of mating in the Queensland fruit fly, Dacus tryoni. J. Insect Physiol. 17: 2139-2156. TZANAKAKIS, M. E., AND A. P. ECONOMOPOULOS. 1967. Two efficient larval diets for continuous rearing of the olive fruit fly. J. Econ. Entomol. 60: 660-663.

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422 Florida Entomologist 79(3) September, 1996

POST-ALIGHTING BEHAVIOR OF CERATITIS CAPITATA (DIPTERA: TEPHRITIDAE) ON ODOR-BAITED TRAPS

RONALD J. PROKOPY1, SOTERO S. RESILVA2 AND ROGER I. VARGAS3 1Department of Entomology, University of Massachusetts, Amherst, MA 01003

2Philippine Nuclear Research Institute, Diliman, Quezon City, Philippines

3Tropical Fruit and Vegetable Research Laboratory, USDA-ARS, Honolulu, HI 96804

ABSTRACT

The influence of physiological state on the behavior of released laboratory-cultured Ceratitis capitata (Wiedemann) flies was evaluated after their arrival on four commonly-used odor-baited traps hung in a potted host tree in a field cage. Three-day-old and 12-day-old protein-deprived females and males were significantly more inclined than 3-day-old and 12-day-old protein-fed females and males to enter protein-odor- baited McPhail and Heath-Epsky traps. There was little or no influence of physiological state on propensity of flies to enter trimedlure-baited Jackson or Nadel-Harris traps. Across all physiological states combined, 74, 64, 0 and 0% of arriving females entered McPhail, Heath-Epsky, Jackson and Nadel-Harris traps, respectively. Corre- sponding values for arriving males were 60, 59, 86 and 82%. We suggest that future research on protein-odor-baited traps should be aimed at enticing a greater proportion of alighting C. capitata flies to enter the trap.

Key Words: Odor response, Mediterranean fruit ﬂies, physiological state

Prokopy et al.: Medﬂy Behavior 423

RESUMEN

Fue evaluada la influencia del estado fisiológico en el comportamiento de Ceratitis capitata (Wiedemann), criadas en el laboratorio, al llegar a cuatro trampas con cebos odoríferos. Las trampas fueron colgadas de un árbol hospedante plantado en una ma- ceta dentro de una jaula. Las hembras y machos de 3 y 12 días de edad privados de proteína se inclinaron significativamente más a entrar en las trampas de McPhail y Heath-Epsky cebadas con proteína odorífera que las hembras y machos de la misma edad alimentados con proteína. Hubo poca o ninguna influencia del estado fisiológico en la propención de las moscas a entrar en las trampas de Jackson o Nadel-Harris cebadas con trimedlure. A través de todos los estados fisiólogicos combinados, 74, 64, 0, y 0% de las hembras que llegaban entraron a las trampas de McPhail, Heath-Epsky, Jackson y Nadel-Harris, respectivamente. Los valores correspondientes a los machos que llegaban a las trampas fueron 60, 59, 86 y 82%. Sugerimos que en el futuro debe investigarse sobre trampas cebadas con proteína odorífera que permitan atraer hacia su interior a una mayor proporción de C. capitata que se posen sobre ellas.

The Mediterranean fruit fly, Ceratitis capitata (Wiedemann), is a major pest of fruit and vegetables on several continents. Numerous studies have been conducted evaluating the effectiveness of a wide variety of traps for detecting C. capitata adults (Cunningham 1989; Economopoulos 1989; Economopoulos & Haniotakis 1994; Kat- soyannos 1994; Millar 1995). The most commonly used traps employ either trimedlure (a synthetic parapheromone) as a male attractant or protein hydrolysate (a food- type lure) as an attractant for both sexes (Liquido et al. 1993). Typically, C. capitata response to trap stimuli is evaluated on the basis of total number of flies captured per trap. Only a few studies have distinguished, through direct behavioral observation or otherwise, between the number of C. capitata arriving nearby or upon a trap and the proportion of arrivers actually captured (Prokopy & Economopoulos 1975; Villeda et al. 1988; Hendrichs et al. 1989; Liquido et al. 1993). Observed variation in proportion of attracted flies caught could have been due to variation in trap characteristics, the structure of the habitat in the immediate vicinity of a trap, weather conditions or the physiological state of responding flies. Here, in a potted host tree in a field cage, we evaluated the influence of fly physiological state on the proportion of C. capitata alighting on a trap that eventually was captured. Specifically, we examined four physiological states of released females and males (immature or mature, protein-deprived or protein-fed) and four types of protein-baited or trimedlure-baited traps.

MATERIALS AND METHODS

C. capitata used in all trials originated from a laboratory colony in culture at the USDA Tropical Fruit and Vegetable Research Laboratory in Honolulu for about 100 generations. From eclosion until time of release, females and males were held together in 30 × 30 × 30 cm screened cages supplied with sucrose and water, with or without protein (enzymatic yeast hydrolysate). Holding conditions were about 25˚C, 50% RH and 13 h natural daylength. Flies were tested when 3 days old (females immature and males of unknown maturity) or 12 days old (females and males mature). Immature females lacked fully developed eggs whereas mature females carried an average of 38 or 125 fully developed eggs per female according to whether they did not or did receive protein. Being immature, 3-day-old females were presumed to be un-

424 Florida Entomologist 79(3) September, 1996 mated; being mature, 12-day-old females were presumed to be mated. Females and males of the same class were released into the lower center of a non-fruiting potted guava tree whose canopy was about 1 m diam. The field cage containing the tree was located on the grounds of the USDA laboratory in Honolulu and was protected from direct sunlight and rainfall by an opaque tarpaulin above the ceiling. For each release event, we collected about 10 female and about 10 male flies of the same class in glass vials and allowed the flies to crawl from the tip of a vial onto the surface of leaves. Im- mediately following release of a batch of about 20 flies, we hung a trap in the upper third of the tree canopy. Batches of about 20 flies were released until a total of 25 flies of both sexes combined (=one replicate) alighted on or in a trap, at which time a trap of a different type was tested. Trap types evaluated were: (a) a 16-cm-diam clear glass invaginated bell-shaped McPhail trap baited with a 200 ml liquid solution of 9% Nulure (Miller Chemical Co., Hanover, PA) (a proteinaceous food-type attractant), 5% sodium borate and 86% water (depicted in Katsoyannos 1994); (b) a Phase-2 Heath-Epsky green-colored plastic cylindrical (10 cm diam × 15 cm tall) dry trap baited on the interior with single dispens- ers of ammonium acetate and putrescine and three evenly-spaced 22-mm-diam entry holes midway between the top and bottom of the trap (flies that entered were killed by contact with squares of methomyl-impregnated waxed cardboard affixed to the top and bottom interior of the trap) (Heath & Epsky 1995); (c) a white cardboard Jackson dry trap (delta shaped) with a white sticky basal insert, baited with a trimedlure impregnated polymeric plug (AgriSense Ltd., Fresno, CA) and placed in a basket hung at the top center of the trap interior (depicted in Katsoyannos 1994); and (d) a clear plastic Nadel-Harris lid-covered bucket trap baited with a trimedlure-impregnated plug (as in c) placed in a basket hung at the top center of the trap interior with three evenly-spaced 22-mm holes entry holes at the upper third of the trap wall (entering flies were killed by the fumigant action of a naled dispenser on the floor of the trap) (depicted in Katsoyannos 1994). Exterior surfaces of all traps were washed before use. Each fly that alighted on the exterior of a trap or flew directly to the trap interior was observed. Observations continued until the fly either was captured or killed (by drowning, sticky, or insecticide) or left the trap without being captured or killed. For each type of trap we carried out a total of four replicates (total of 100 alighting flies) for each of the four physiological states tested. Trap types and fly types were evaluated in random order. All flies that remained in the cage after completion of a replicate of a treatment were removed before the next treatment commenced.

RESULTS

Across all four fly physiological-state classes combined, 74, 64, 0 and 0% of arriving females entered McPhail, Heath-Epsky, Jackson and Nadel-Harris traps, respectively. Corresponding values for arriving males were 60, 59, 86 and 82%. Across the four physiological-state classes of flies arriving on McPhail traps, 68- 80% were females and 20-32% were males (Table 1). Comparable values for flies arriving on Heath-Epsky traps were 56-64% females and 36-44% males. In contrast, across the four physiological states of flies arriving on Jackson and Nadel-Harris traps, 0-6% were females and 94-100% were males. Because we released approximately, not precisely, 10 females and 10 males at each release event, statistical comparison among trap types of the proportion of individuals of each sex released that arrived on a trap was considered invalid. With respect to females, a significantly greater proportion of arrivers that were protein-deprived than protein-fed entered both McPhail and Heath-Epsky traps (Ta-

Prokopy et al.: Medﬂy Behavior 425 . 1 TRAP

THE

ENTERED

THAT

TRAP

ARRIVING

FLIES

CAPITATA C.

Mean No. Arriving Flies and Mean % Arrivers that Entered Traps Arrivers that Entered Arriving Flies and Mean % Mean No. RELEASED

McPhail Trap Heath-Epsky Trap Trap Jackson Nadel-Harris Trap No. ArriveNo. % Enter Arrive No. % Enter Arrive No. % Enter Arrive No. % Enter CULTURED - LABORATORY

3 Fed3 20.0 Fed 72b(a) 5.0 13.8 53b(b) 56c(b) 11.3 0.8 48b(b) 0a(c) 24.3 1.5 93a(a) 0a(c) 23.5 86a(a) 1212 Deprived Fed 19.51212 Deprived 85a(a) 16.8 Fed 5.5 14.3 51c(a) 68a(b) 81a(a) 8.3 15.8 10.8 48b(b) 0.3 48c(a) 70a(b) 9.3 0a(b) 0.0 24.8 46b(b) 0a(b) 0.5 83ab(a) 25.0 0a(b) 0.3 24.5 81b(a) 84a(a) 0a(b) 24.8 79a(a) (days) Protein Fly age ROPORTION 1. P For each sex, values in the same column not followed by the same letter without parentheses, or values in the same row not followed by the same letter within parentheses, are sig- or values in the same row not followed by letter within parentheses, values in the same column not followed by letter without parentheses, sex, each For 1 ABLE nificantly different from one another according to one-way ANOVA (following arc-sine transformation) and the least significant difference test criterion at the 0.05 level. For each sex, val- sex, each For (following arc-sine transformation) and the least significant difference test criterion at 0.05 level. ANOVA nificantly different from one another according to one-way There were four replicates per treatment. consisted of the proportion arriving flies that entered trap. ANOVA cell for ues in each Sex Female 3Male Deprived 17.8 3 87a(a) Deprived 16.0 7.3 69b(b) 72a(b) 1.0 9.0 0a(c) 73a(b) 24.0 1.0 87ab(a) 0a(c) 24.0 77a(ab) T

426 Florida Entomologist 79(3) September, 1996 ble 1). This was true for both ages of females tested. Among all four classes of females tested, arriving 12-day-old protein-fed individuals were the least inclined to enter McPhail or Heath-Epsky traps. No arriving females entered a Jackson or Nadel-Har- ris trap. A significantly greater proportion of arriving 3-day-old protein-deprived and protein-fed females entered McPhail traps than Heath-Epsky traps, but there were no significant differences between these two traps in proportion of arriving 12-day-old females that entered a trap (Table 1). With respect to males, again, a significantly greater proportion of protein-deprived than protein-fed flies entered both McPhail and Heath-Epsky traps (Table 1). This was true for both age classes of flies tested. Again, among all four classes of males tested, arriving 12-day-old protein-fed individuals were the least inclined to enter McPhail or Heath-Epsky traps. The proportion of arrivers that entered a Jackson or Nadel-Harris trap varied little according to class of males tested (Table 1). Almost without exception, a significantly greater proportion of males arriving on Jackson or Nadel-Harris traps than on McPhail or Heath-Epsky traps entered the trap (Table 1). No female or male that arrived at a McPhail, Heath-Epsky or Nadel-Harris trap entered the interior of the trap without first landing on the exterior and then walking into the interior. In contrast, 53-59% of arriving males flew directly into the interior of Jackson traps. Of flies that entered the interior of traps, percentages that were captured (did not escape) were as follows for McPhail, Heath-Epsky, Jackson and Nadel-Harris traps, respectively: 96-98, 89-93, 86-92 and 95-99.

DISCUSSION

Our findings indicate that for all four classes of released laboratory-cultured C. capitata flies combined, about 74, 60, 2, and 3% of flies attracted to McPhail, Heath- Epsky, Jackson and Nadel-Harris traps, respectively, were females (we released approximately equal numbers of females and males). If the total number of female and male arrivers on a given trap type is multiplied by the proportion of each sex arriving that entered the trap (again for all four fly classes combined), then the percentage of all female flies released that entered a trap and was female turns out to be 78, 62, 0 and 0, respectively, for the four types of traps. These values parallel quite closely the proportions of all natural-population C. capitata captured that were females in field studies with Nulure-baited McPhail traps (71%) (Katsoyannos 1994), ammonium acetate/putrescine-baited Heath-Epsky traps (53%) (Heath & Epsky 1995), trimedlure- baited Jackson traps (0%) (Katsoyannos 1994), and trimedlure-baited Nadel-Harris traps (0%) (Katsoyannos 1994). Thus, even though we were unable to obtain and test wild-origin C. capitata, our combined findings across four physiological states of laboratory-cultured C. capitata tested under field cage conditions are similar (with respect to sex ratio of responding flies) to findings for wild C. capitata obtained under field conditions. Our findings revealed a significant effect of protein diet on proportions of arriving flies that entered McPhail and Heath-Epsky traps. Indeed, for all four comparisons involving 3-day or 12-day-old protein-deprived versus protein-fed females or males, the response of arrivers to protein-type odor emanating from the interior of McPhail traps was always significantly greater on the part of protein-deprived flies. The same was true for Heath-Epsky traps. On the other hand, in only one of the four comparisons for both the McPhail and Heath-Epsky traps was the response different between 3-day and 12-day-old flies, suggesting that the protein diet rather than fly age had a greater effect on propensity of alighting flies to enter traps containing protein-type

Prokopy et al.: Medfly Behavior 427 lures. Effects of female age per se may have coincided with effects of mating status on female response patterns in that 3-day-old females were presumed to be unmated and 12-day-old females were presumed to be mated. Our study, however, was not designed to distinguished between effects of age and mating status. Earlier, it was found that protein-deprivation was more important than fly age on degree of attraction of wild- origin C. capitata to sources of protein in a field-caged tree (Prokopy et al. 1992). Katsoyannos (1994) observed that significantly more natural-population C. capitata females were captured in protein-baited McPhail traps than in trimedlure-baited Jackson or Nadel-Harris traps, whereas the reverse was true for male medflies. Heath & Epsky (1995) found that on average across several experiments, numerically (but not always significantly) more natural-population C. capitata of both sexes combined were captured in McPhail traps than in Heath-Epsky traps. Here, a significantly greater proportion of arriving 3-day-old protein-deprived and protein-fed females entered McPhail traps than traps of any other types, while a significantly greater proportion of arriving males of all types (except 3-day-old protein-deprived ones) entered Jackson and Nadel-Harris traps than McPhail or Heath-Epsky traps. Perhaps difference in medfly captures among trap types tested by Katsoyannos (1994) and Heath & Epsky (1995) stemmed in part from differential attraction towards, and differential post-alighting behavior at, respective sources of trap odor according to the physiological state of responding flies. In conclusion, we have shown that the physiological state of C. capitata, with respect to amount of protein consumed in life and to a lesser extent fly age, can have a significant impact on the propensity of flies attracted to a protein-odor-baited trap to enter the trap. Perhaps certain characteristics or practices associated with current protein-odor baited traps could be adjusted to facilitate entry of flies of all physiological states into traps following their arrival on or near the trap.

ACKNOWLEDGMENTS

We thank Dale Kanehisa for maintaining the ﬂies prior to testing, Alan Reynolds for assistance with data analyses, Bob Heath and Nancy Epsky for providing Heath- Epsky traps, and R. Cunningham, J. J. Duan, E. Harris, R. Heath and T. Nishida for reviewing an earlier version of this manuscript.

REFERENCES CITED

CUNNINGHAM, R. T. 1989. Population detection, pp. 169-173 in A. S. Robinson and G. Hooper [eds.], Fruit flies: their biology, natural enemies and control. Elsevier Science, Amsterdam. ECONOMOPOULOS, A. P. 1989. Use of traps based on color and/or shape, pp. 315-327 in A. S. Robinson and G. Hooper [eds.], Fruit flies: their biology, natural enemies and control. Elsevier Science, Amsterdam. ECONOMOPOULOS, A. P., AND G. E. HANIOTAKIS. 1994. Advances in attractant and trapping technologies for tephritids, pp. 57-66 in C. O. Calkins, W. Klassen and P. Liedo [eds.], Fruit flies and the sterile insect technique. CRC Press, Boca Ra- ton, Florida. HEATH, R. R., AND N. D. EPSKY. 1995. Mediterranean fruit fly trap methodology, pp. 145-159 in J. G. Morse, R. L. Metcalf, J. R. Carey and R. V. Dowell [eds.], The Mediterranean fruit fly in California: defining critical research. College of Nat- ural and Agricultural Sciences, University of California, Davis. HENDRICHS, J., J. REYES, AND M. ALUJA. 1989. Behavior of female and male Mediter- ranean fruit flies in and around Jackson traps placed on fruiting host trees. In- sect. Sci. Applic. 10: 285-294.

428 Florida Entomologist 79(3) September, 1996

KATSOYANNOS, B. I. 1994. Evaluation of Mediterranean fruit fly traps for use in sterile-insect-technique programs. J. Appl. Entomol. 118: 442-452. LIQUIDO, N., R. TERANISHI, AND S. KINT. 1993. Increasing the efficiency of catching Mediterranean fruit fly males in trimedlure-baited traps with ammonia. J. Econ. Entomol. 86: 1700-1705. MILLAR, J. G. 1995. An overview of attractants for the Mediterranean fruit fly, pp. 123-143 in J. G. Morse, R. L. Metcalf, J. R. Carey and R. V. Dowell [eds.], The Mediterranean fruit fly in California: defining critical research. College of Nat- ural and Agricultural Science, University of California, Riverside. PROKOPY, R. J., AND A. P. ECONOMOPOULOS. 1975. Attraction of laboratory-cultured and wild Dacus oleae flies to sticky-coated McPhail traps of different colors and odors. Environ. Entomol. 4: 187-192. PROKOPY, R. J., D. R. PAPAJ, J. HENDRICHS, AND T. T. Y. WONG. 1992. Behavioral responses of Ceratitis capitata flies to bait spray droplets and natural food. Ento- mol. Exp. Appl. 64: 247-257. VILLEDA, M. P., J. HENDRICHS, M. ALUJA, AND J. REYES. 1988. Mediterranean fruit fly behavior in nature in relation to different Jackson traps. Florida Entomol. 71: 154-162.

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428 Florida Entomologist 79(3) September, 1996

A NEW SPECIES OF GALL MIDGE (DIPTERA: CECIDOMYIIDAE) FROM SUBTERRANEAN STEM GALLS OF LICANIA MICHAUXII (CHRYSOBALANACEAE) IN FLORIDA

RAYMOND J. GAGNÉ1 AND KENNETH L. HIBBARD2 1Systematic Entomology Laboratory, PSI, ARS, USDA c/o National Museum of Natural History MRC-168 Washington, D. C. 20560

2Division of Plant Industry Florida Department of Agriculture and Consumer Services Fort Pierce, FL 34945-3045

ABSTRACT A new species of gall midge is described from subterranean stem galls on Licania michauxii Prance from Florida. The gall former is a new species of Lopesia and the ﬁrst record of this genus in North America. The limits of Lopesia, a genus previously recorded only from South America and Africa, are enlarged to accept the new species. The species is described and illustrated. The pupa of this species is unique in Cecid- omyiidae for its large, robust, dorsal abdominal spines that may be used in pushing through sandy soil after leaving the gall.

Key Words: Gall midge, Nearctic Region, new species, gopher apple

RESUMEN

Una nueva especie de mosca gallícola es descrita de agallas subterráneas de Lica- nia michauxii Prance en la Florida. El insecto que produce la agalla es una nueva es-

Gagné & Hibbard: Gall Midge on Licania 429 pecie de Lopesia y representa el primer registro de este género en América del Norte. Los límites de Lopesia, un género previamente registrado sólo de América del Sur y Africa, han sido ampliados para acomodar la nueva especie. La especie es descrita e ilustrada. La pupa de esta especie es única en la familia Cecidomyidae por las espinas grandes y robustas del dorso del abdomen que deben ser usadas para empujar a tra- vés del suelo arenoso después de dejar la agalla.

Licania michauxii Prance, or gopher apple, is a low shrub that grows to 3-4 deci- meters high and spreads partly by means of long, horizontal, subterranean stems that grow 1-10 centimeters below the soil surface (Bell & Taylor 1982, Taylor 1992). The plant belongs to the family Chrysobalanaceae, a chieﬂy Neotropical family related to Rosaceae. This plant is found throughout most of Florida and on the coastal plains to Mississippi and the Carolinas. The fruit, 2-3 cm long, is made up of a large seed sur- rounded by white, juicy pulp. It is favored by wildlife, including tortoises, thus the common name, gopher apple, after the Florida gopher tortoise. In this paper we report for the ﬁrst time the occurrence of galls on the subterranean stems of L. michauxii in Florida and that these woody, spherical outgrowths are formed by the larvae of a new species of gall midge. The cecidomyiid responsible for the gall is a new species of Lopesia, a genus heretofore known only from South Amer- ica and Africa (Gagné 1994).

MATERIALS AND METHODS Specimens used for this study were taken from or reared from galls collected from subterranean stems of gopher apple. Adults were reared from galls kept indoors in plastic bags containing some paper towels to absorb excess moisture. Fresh specimens were killed and preserved in 70% ethanol. Specimens were slide mounted for identi- ﬁcation and scientiﬁc study using the method outlined in Gagné (1989, 1994). Termi- nology for adult morphology follows usage in McAlpine et al. (1981); that for larval morphology follows Gagné (1989). The new species is to be credited to Gagné.

Lopesia licaniae Gagné, new species

Adult.—Head (Fig. 6): Eyes about 9 facets long at vertex, connate; facets circular, closely adjacent. Vertex of occiput with wide, dorsal protuberance bearing several long setae. Frons with 2-5 setae per side. Labella short, acutely triangular in frontal view, each with several lateral setae. Palpus 4 segmented. Antenna with 12 flagellomeres. Male flagellomeres (Fig. 7): each with three circumfila, their loops not attaining the following circumfilum; the distal node of each flagellomere constricted near middle. Female flagellomeres (Fig. 8): becoming successively shorter in length from base to apex; last flagellomere with narrow apical elongation; necks setulose. Thorax: Mesanepisternum with 5-12 scales dorsally. Mesepimeron with 8-13 setae. Wing (Fig. 10) length: (, 2.4-2.7 mm (n=5); &, 2.4-3.1 mm (n=3); R5 curved api- cally, joining C posterior to wing apex; Rs weak, oblique, situated slightly distad of mid distance between arculus and R1 apex; M3+4 present as a fold. Tarsal claws (Fig. 9) bent near basal third, without teeth; empodia not reaching bend in claws. Male abdomen: First through sixth tergites entire, rectangular, with mostly single, continuous, posterior row of setae, several lateral setae on each side near midlength, scales covering most of sclerites, and pair of trichoid sensilla on anterior margin. Sev-

430 Florida Entomologist 79(3) September, 1996

Figs. 1-2. Galls on uncovered subterranean branches of Licania michauxii. 1) Galls on several branches. 2) Gall cut open to reveal larva. enth tergite unsclerotized posteriorly, with 1-2 lateral setae, scattered scales, and anterior pair of trichoid sensilla. Eighth tergite sclerotized only anteriorly, with scattered scales and anterior pair of trichoid sensilla. Genitalia (Fig. 11): Cerci broadly rounded, with several posterior setae. Hypoproct divided caudally into 2 long, narrow lobes, each with several setae. Aedeagus as long as splayed gonocoxite, grad- ually tapered to narrowly rounded apex. Gonocoxite strongly bowed at midlength,

Gagné & Hibbard: Gall Midge on Licania 431

Figs. 3-5. Pupa of Licania michauxii. 3) Anterior half only, ventral. 4) Dorsal. 5) Lateral. with right-angled mesobasal lobe. Gonostylus elongate, cylindrical, barely tapered from base to apex, setulose on basal fourth, asetulose and ridged beyond. Female abdomen (Figs. 12-13): First through seventh tergites as for male ﬁrst through sixth. Eighth tergite shorter, with scattered setae and anterior pair of trichoid sensilla. Ninth sternum evenly covered with setae. Tenth tergum without setae. Ovipositor abruptly tapered, barely protrusible; cerci elongate-ovoid, completely setulose and covered with setae, with several long, posteroventral, thick walled, sensory setae; hypoproct elongate, undivided. Pupa.—(Figs. 3-5) Antennal bases ﬂattened apicoventrally to form a horizontal ridge. Cephalic sclerite with short, inconspicuous seta set on each of two lateral con- vexities. Face smooth, without horns. Prothoracic spiracles elongate, tapering gradu- ally from base to apex. Abdominal second to eighth tergites with prominent, pigmented anterodorsal spines. Larva.—Third instar: Integument rugose. Antenna less than twice as long as wide. Spatula (Fig. 15) robust, with two triangular lobes anteriorly. Full complement of papillae basic for supertribe present (Gagné 1989); terminal segment (Fig. 14) with 8 short, setose papillae; setae of one pair thin and pointed, about 3 times as long as wide, setae of remaining pairs as wide as long, obtuse. Holotype.—(, from Licania michauxii, Indrio, St. Lucie Co., Florida, 6-VII-1983, K. L. Hibbard; deposited in the Florida State Collection of Arthropods, Gainesville. Paratypes (all from Licania michauxii).—2(, 2&&, 3 pupal exuviae, and larva, same data as holotype; Ft. Pierce, St. Lucie Co., Florida, 18-II-1987, K.L. Hibbard; 2((, 1&, and 5 larvae, Melbourne, Brevard Co., Florida, USA, 4-IX-1986, F. Smith and K.L. Hibbard. One male, 1 female, and 2 pupal paratypes will be deposited in the Florida State Collection of Arthropods, the remainder in the National Collection of In- sects, Washington, D.C. Etymology.—The name licaniae is the genitive of the host genus. Remarks on taxonomy.—This species is placed in Lopesia because it has the following characters: the R5 wing vein is closer to the end of R1 than to the arculus; tarsal

432 Florida Entomologist 79(3) September, 1996

Figs. 6-15 Licania michauxii. 6) Head. 7) Male third antennal ﬂagellomere. 8) Fe- male third antennal ﬂagellomere. 9) Tarsal claw and empodium. 10) Wing. 11) Male genitalia (dorsal). 12) Female postabdomen (lateral). 13) Detail of female tenth segment and cerci (lateral). 14) Eighth and terminal larval segments. 15) Larval spatula and lateral papillae of one side.

Gagné & Hibbard: Gall Midge on Licania 433 claws are bowed near the basal third and the empodia do not quite reach the bend in the claws; male antennal circumfila have short loops that do not reach the bases of the next distal circumfilum; male gonocoxites are bowed at midlength; the ovipositor is barely protrusible and its cerci have several apicoventral, thin-walled setae that are not shorter than surrounding setae; and larval terminal papillae have short, more or less corniform setae (Gagné & Marohasy 1993, Gagné 1994). This species differs from other known Lopesia in having simple instead of toothed tarsal claws. Several genera of Cecidomyiinae have representatives with toothed or simple tarsal claws, so it appears the tooth is easily lost. This species is the first cecidomyiid known to feed on Chrysobalanaceae, a chiefly Neotropical family. Four other species of Lopesia are known: Lopesia brasiliensis Rübsaamen from a Melastomataceae in Brazil; L. pari- narii Tavares from a Rosaceae in Mozambique; and L. armata Gagné and L. niloticae Gagné from Mimosaceae in Kenya (Gagné & Marohasy 1993). Several other, undescribed species from the Neotropical Region are represented in the USNM, which indicates to us that the diversity and host range of this genus will not be appreciated until the Neotropical fauna is better studied. In the key to Neotropical genera in Gagné (1994), the new species will not key to Lopesia because of the absence of teeth on the tarsal claws. Pupae of many plant-feeding Cecidomyiidae have prominent dorsal abdominal spines that are used by the pupa when pushing through plant tissue to the exterior (RJG, personal observation), but we know of no other species that has such prominent spines or, in the case of the spines of the second abdominal segment, where they are placed almost perpendicular to the body axis. The size and shape of the spines may be adaptations for pushing through the several centimeters of soil between the galls on the subterranean branches and the soil surface. Biological observations.—Woody, spheroidal outgrowths can be found on the subterranean stems (Figs. 1-2). These are 3-4 mm in diam and sometimes occur in large aggregates up to 1 cm in diam. Galls with the gallmaker inside are an off-white color. Old galls are a dark brown and usually have an exit hole. Fresh galls were present in the summer, fall, and winter months. A single cecidomyiid larva is found inside each gall. The full-grown larva is curled into a u-shape and completely fills the cavity (Fig. 2). Pupation occurs in the gall. When the adult is fully formed inside the pupa, the pupa breaks through the gall and crawls through the soil to the surface. A break in the pupal thorax then develops through which the adult escapes.

ACKNOWLEDGMENTS

We are grateful to Nit Malikul for preparing the microscopic slides and to E. E. Grissell, K. M. Harris, A. L. Norrbom, A. M. Young, and an anonymous reviewer for their comments on drafts of the manuscript.

REFERENCES CITED

BELL, C. R., AND B. J. TAYLOR. 1982. Florida Wild Flowers and Roadside Plants. Lau- rel Hill Press, Chapel Hill, NC. 308 pp. GAGNÉ, R. J. 1989. The Plant-Feeding Gall Midges of North America. Cornell Univer- sity Press, Ithaca, NY. xiii and 355 pp and 4 pls. GAGNÉ, R. J. 1994. The Gall Midges of the Neotropical Region. Cornell University Press, Ithaca, NY. xv and 352 pp. GAGNÉ, R. J., AND J. MAROHASY. 1993. The gall midges (Diptera: Cecidomyiidae) of Acacia spp. (Mimosaceae) in Kenya. Insecta Mundi 7: 77-124.

434 Florida Entomologist 79(3) September, 1996

MCALPINE, J. F., B. V. PETERSON, G. E. SHEWELL, H. J. TESKEY, J. R. VOCKEROTH, AND D. M. WOOD, eds. 1981. Manual of Nearctic Diptera, Vol. 1. Research Branch, Agriculture Canada, Monograph 27. vi + 674 pp. TAYLOR, W. K. 1992. The Guide to Florida Wildﬂowers. Taylor Publishing Co., Dallas, TX. 320 pp.

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434 Florida Entomologist 79(3) September, 1996

EFFICACY OF SOLID FORMULATIONS OF EMAMECTIN BENZOATE AT CONTROLLING LEPIDOPTEROUS PESTS

RICHARD K. JANSSON, ROBERT F. PETERSON, W. ROSS HALLIDAY1, PRADIP K. MOOKERJEE AND RICHARD A. DYBAS2 Merck Research Laboratories, Agricultural Research and Development, P.O. Box 450, Hillsborough Road, Three Bridges, NJ 08887-0450

1Ricerca, Inc., 7528 Auburn Road, P.O. Box 1000, Painesville, OH 44077

2Merck Research Laboratories, P.O. Box 2000, Rahway, NJ 07065-0900

ABSTRACT

Six solid formulations of emamectin benzoate (three impregnated powder blends, two dry powder blends, and one soluble granule) were compared with an emulsifiable concentrate (EC) formulation for their residual efficacy at killing tobacco budworm, Heliothis virescens (F.) (= Helicoverpa virescens (F.)), and beet armyworm, Spodoptera exigua (Hübner), in glasshouse tests. Two trials were conducted. Emamectin benzoate was applied to plants at two rates in each trial (8.4 and 0.084 g ai/ha in the first trial; and 8.4 and 0.84 g ai/ha in the second trial). The first trial was conducted in a glasshouse; in the second trial, plants were treated in the glasshouse and moved outdoors for the duration of the study. In the first trial, all three impregnated powder blends, one dry powder blend, and the EC formulation were comparable in their effectiveness at controlling both targets when applied at the high rate (8.4 g ai/ha). At the low rate, efficacy at controlling H. virescens did not differ among formulations, whereas the two powder formulations provided the longest residual efficacy against S. exigua. In the second trial, one impregnated powder blend, two dry powder blends, a soluble granule, and the EC formulation were comparable in their effectiveness at killing both species up to 10 days after application when applied at the high rate (8.4 g ai/ha). At the low rate (0.84 g ai/ ha), one powder formulation was consistently more effective at controlling S. exigua, whereas no formulation consistently outperformed all others at controlling H. virescens. Two field studies demonstrated that two dry powder blend formulations were very effective and comparable to the EC formulation at controlling Helicoverpa zea (Boddie), Keiferia lycopersicella (Walsingham), and S. exigua on tomato. These data demonstrate that solid formulations of emamectin benzoate have potential for control of Lepidoptera. The importance of a solid formulation for emamectin benzoate is discussed.

Key Words: Avermectin, emamectin benzoate, formulation, residual efﬁcacy

Jansson et al.: Efﬁcacy of Emamectin Benzoate Formulations 435

RESUMEN

La eficacia residual sobre el gusano cogollero del tabaco, Heliothis virescens (F.) (=Helicoverpa virescens (F.)) y el gusano de la remolacha, Spodoptera exigua Hübner, de seis formulaciones sólidas de benzoato de emamectin (tres mezclas de polvos impregnados, dos mezclas de polvo seco y un granulado sólido) fue comparada con la de un concentrado emulsionable (CE) en ensayos de invernadero. Dos ensayos fueron realizados. El benzoato de emamectin fue aplicado a las plantas a dos dosis en cada ensayo (8.4 y 0.084 g ai/ha en el primer ensayo, y 8.4 y 0.84 g ai/ha, en el segundo). El primer ensayo fue llevado a cabo en un invernadero; en el segundo ensayo las plantas fueron tratadas en el invernadero y se mantuvieron fuera durante el resto del estudio. En el primer ensayo, las efectividades de las tres mezclas de polvos impregnados, una mezcla de polvo seco y la EC fueron comparables en su efectividad para controlar a los dos sujetos cuando se aplicaron a alta dosis (8.4 g ai/ha). A la dosis baja, la eficacia para controlar H. virescens no difirió entre las formulaciones, mientras que las dos formulaciones de polvo mostraron la más larga eficacia residual contra S. exigua. En el segundo ensayo, una mezcla de polvo impregnado, dos mezclas de polvos secos, un granulado soluble y la formulación del CE tuvieron efectividades comparables contra ambas especies hasta los 10 días después de la aplicación, cuando se aplicaron a la dosis alta (8.4 g/ai/ha). A la dosis baja (0.84 g ai/ha), una formulación de polvo fue consistentemente más efectiva en el control de S. exigua, y ninguna formulación fue más efectiva que las otras controlando H. virescens. Dos estudios de campo demostraron que dos mezclas de polvo seco fueron muy efectivas y comparables con la formulación de CE en el control de Helicoverpa zea (Boddie), Keiferia lycopersicella (Walshingham) y S. exigua en el tomate. Estos datos demuestran que las formulaciones sólidas de benzoato de emamectin tienen potencial para el control de Lepidoptera. Se discute la importancia de una formulación para el benzoato de emamectin.

Avermectins are a family of 16-membered lactone natural product compounds produced by the soil microorganism, Streptomyces avermitilis MA-4680 (NRRL 8165), which was isolated in culture at the Kitasato Institute from a soil sample taken from Kawana Ito City, Shizuoka Prefecture, Japan. The major component of the fermenta- tion is avermectin B1, a mixture of B1a (80%) and B1b (20%) (Dybas et al. 1989). The discovery, structures, environmental fate, spectrum of activity, and applications for control of arthopods have been reviewed (Campbell et al. 1984, Dybas 1989, Lasota &

Dybas 1991). Abamectin, the common name assigned to avermectin B1, is currently sold commercially for control of mites and certain insect pests on a range of ornamental and horticultural crops in about 50 countries worldwide.

Emamectin [4”-epi-methylamino-4”-deoxyavermectin B1 hydrochloride salt (MK- 0243)] is a semisynthetic derivative of abamectin (Dybas 1988). This second generation avermectin was shown to be up to 1,500-fold more potent against armyworm species, e.g., beet armyworm, Spodoptera exigua (Hübner), and 105- and 43-fold more toxic to tomato fruitworm, Helicoverpa zea (Boddie), and tobacco budworm, Heliothis virescens (Guenee), larvae, respectively, than abamectin (Dybas & Babu 1988, Dybas et al. 1989, Mrozik et al. 1989, Trumble et al. 1987). This compound was also 1,720-, 884-, and 268-fold more potent against S. eridania (Cramer) than methomyl, thiodicarb, and fenvalerate, respectively (Dybas & Babu 1988). The benzoate salt of this compound (MK-0244; PROCLAIMR), was subsequently found to be more stable than the hydrochloride salt and is currently being developed for control of Lepidoptera on a variety of horticultural crops worldwide. Excellent efﬁcacy at unprecedented use rates (8.4 g ai/ha) has been demonstrated against numerous Lepidoptera on a variety

436 Florida Entomologist 79(3) September, 1996 of crops in the field (Jansson & Dybas 1996, Jansson & Lecrone 1991, Lasota & Dybas 1991, Leibee et al. 1995). Minor changes in the constituents of formulations may have marked effects on product efficacy (Hartley & Graham-Bryce 1980). Edwards et al. (1994) showed that formulation type may affect the behavior of arthropods and concomitantly affect product efficacy. Historically, solid formulations of avermectins have been less effective at controlling arthropods than liquid formulations. For this reason, most studies on emamectin benzoate conducted to date used an emulsifiable concentrate (EC) formulation of this semisynthetic derivative of avermectin. No studies have been conducted to compare efficacy of alternative formulations of emamectin benzoate at controlling arthropods. In addition, choice of formulation type and ingredients may significantly affect the acute toxicity of a formulation (Hudson & Tarwater 1988). Because of the scrutiny placed on emulsifiable concentrate formulations by the Environmental Protection Agency, many agrichemical companies have developed solid formulations for use in water soluble packaging. Such formulations result in lower worker exposure during the mixing and loading of pesticides into spray equipment. This paper presents data from glasshouse and field studies on the efficacy of novel solid formulations of emamectin benzoate against several economically important lepidopterous pests.

MATERIALS AND METHODS

Formulations Tested

Formulations tested were of four types: impregnated powder blends, dry powder blends, a soluble granule, and the EC formulation (Table 1). Impregnated powder blends (formulations 77, 78, and 79) were prepared by dissolving technical grade material of emamectin benzoate (Merck & Co., Rahway, NJ) in the solvent and spraying the solution onto a heated carrier, followed by the addition of other ingredients, and then blending until homogenous. These three formulations utilized different solvents and carriers. Dry powder blends (formulations 80 and 81) were prepared by combining all ingredients and then blending until homogenous. These two formulations differed in the diluents and surfactants. The soluble granule (formulation 82) was prepared by dissolving emamectin benzoate and a surfactant in a molten matrix. This mixture was blended until homogenous, cooled, and comminuted in a Waring blender; ﬂakes passing through a 10-mesh screen were tested. The EC formulation (formulation 49) was prepared by combining all ingredients and stirring until all solids had dissolved. All formulations, including the EC formulation (0.16 EC), contained 2.0- 2.2% w/w of emamectin benzoate.

Solubility and pH Tests

An experiment was conducted to determine the percentage of emamectin benzoate in each formulation that dissolved in water. A subsample (1.0 g) of each solid formulation was added to deionized water (50 ml). Two aliquots (1 ml each) were removed from the solution after about 0 min and 1 h. The solution was not agitated between the two sample periods. Aliquots were transferred to volumetric ﬂasks (25 ml), diluted with methanol (24 ml), and sonicated. Aliquots were drawn from this solution and centrifuged. The supernatant (10 ul samples) was then analyzed by HPLC with the following speciﬁcations: Zorbax C18 column (15 cm × 3.2 mm) run at 1 ml/min at 30°C with a wavelength of 245 nm and a 80:20:0.8 mobile phase (acetonitrile:water: etha-

Jansson et al.: Efﬁcacy of Emamectin Benzoate Formulations 437 GLASS-

Avail. ai., % ai., Avail. TESTED 1 (MK-0244) BENZOATE

EMAMECTIN

FORMULATIONS

THE

OF AI AVAILABLE

. PERCENTAGE

TRIALS

AND

FIELD

AND

OMPOSITION HOUSE 1. C GH, Glasshouse; F, Field; numbers correspond to trial number (e.g., GH 1,2, trials 1 and 2 in the glasshouse). GH 1,2, numbers correspond to trial number (e.g., Field; F, Glasshouse; GH, 1 ABLE MK-0244-77MK-0244-78MK-0244-79 Impregnated powder blendMK-0244-80 Impregnated powder blend surfactants carrier, solvents, A.I., MK-0244-81 Impregnated powder blend surfactants carrier, solvents, A.I., MK-0244-82 Dry powder blend surfactants carrier, solvents, A.I., MK-0244-49 Dry powder blend Soluble granule 10.3 Emulsiﬁable concentrate 5.1 GH 1 5.8 surfactants diluents, A.I., surfactants solvents, A.I., surfactants diluents, A.I., GH 1 GH 1,2 surfactants matrix A.I., 95 10.0 93 94 6.6 F 1,2 GH 1,2; 6.0 F 1,2 GH 2; F 1,2 GH 1,2; 79 GH 2 100 47 99 Treatment type Formulation Composition pH Trials T

438 Florida Entomologist 79(3) September, 1996 nolamine, respectively). The chemical availability of emamectin benzoate was then calculated by the following:

× % available = (Q1/Q0) 100

where Q0 and Q1 are the amount of emamectin benzoate recorded after 0 min and 1 h, respectively. pH was determined from a 1:50 suspension of each solid formulation in deionized water using a pH meter.

Glasshouse Tests

Two trials were conducted to compare the residual efficacy of the six solid formulations of emamectin benzoate with the EC formulation. The first trial was conducted in the glasshouse. In the second trial, plants were treated with the different formulations in the laboratory and then moved between the glasshouse and the outdoors daily for the duration of the experiment. Plants were kept outdoors on clear days, but were held indoors on cloudy and rainy days and were held indoors each evening to minimize the effects of rain splash and dew on foliar residues. The formulations that were tested in the two trials are listed in Table 1. The residual efficacy of these formulations was evaluated using two Lepidopteran targets, the tobacco budworm, H. virescens, and the beet armyworm, S. exigua. Heliothis virescens was tested on two-week old chickpea (Cicer arietinum cv. Burpee Garbanzo 5024) plants and S. exigua was tested on five-week old pepper (Capsicum annuum L. cv. Pi- mento) plants. In the first trial, the recommended field use rate (8.4 g ai/ha) and 1% of this rate (0.084 g ai/ha) of emamectin benzoate were applied to plants for each of four formulations (77, 78, 79, and 80) and the EC formulation (49). In the second trial, formulation batches 79 and 80 were retested along with two additional formulations (81 and 82), and the EC formulation. Rates tested were the recommended field use rate (8.4 g ai/ ha) and 10% of this rate (0.84 g ai/ha); the lower rate was increased (compared with trial 1) because of the increased ultraviolet radiation outdoors. In both trials, nontreated control plants were sprayed with water only. In both trials, 100 plants for each species were treated with two rates of each formulation using a CO2 track-sprayer system equipped with two disc-cone ceramic al- buz red drop nozzles (Teejet R cone type, model D2-25) that delivered approximately 153.2 liters/ha at a pressure of 128 kg/cm2 and a speed of 3.5 km/h. Plants were arranged beneath the track sprayer to ensure thorough coverage of spray materials. Plants were air-dried after applications were made and then moved to the glasshouse. All plants were bottom watered to minimize wash off of emamectin benzoate from foliage. Ten plants were randomly selected from each treatment on days 0, 1, 4, 7, 10, 14, and 21. One representative leaf was randomly excised from each plant and placed on the surface of water agar in petri dishes (9 cm diam). Ten neonate larvae were placed on each leaf on each sample date (n = 100 larvae per treatment per sample date); mortality was recorded after 96 hours.

Field Tests

Two ﬁeld trials were conducted on tomato in 1994 with formulations 80 and 81. Formulations were compared with the EC formulation and chemical standards at recommended ﬁeld rates. Emamectin benzoate treatments were applied at 7- and 14-day

Jansson et al.: Efficacy of Emamectin Benzoate Formulations 439 intervals, whereas the chemical standards were only applied at 7-day intervals. One trial was conducted in San Quintin, Baja California, Mexico in a commercial tomato, Lycopersicon esculentum Mill. var. UC-82, field. The second trial was conducted in Apex, North Carolina in a ‘Better Boy’ tomato field. In Mexico, tomatoes were direct seeded in two rows on 1.5 m raised beds (two rows per bed) that were drip-irrigated and covered with plastic mulch. Treatments were arranged in a randomized complete block design with four replications. Each plot was 3.1 m across by 9.3 m long. A 1 m buffer of nontreated plants separated replicates. Treatments were applied on either 3 (14-day intervals) or 6 dates (7 day intervals) in

July and August, 1994. Applications were made using a CO2 backpack sprayer with a boom width (1.5 m) that ranged across each bed and equipped with four equally- spaced hollow cone disk/core nozzles (D-7/25). The sprayer delivered approximately 750 liter/ha at a pressure of 1.7 kg/cm2. Lambda cyhalothrin (Karate 1 EC, Zeneca Ag Products, Wilmington, DE) was included (44 g ai/ha) as the chemical standard. Beet armyworm, S. exigua, and tomato pinworm, Keiferia lycopersicella (Walsing- ham), damage was assessed after the fourth and sixth applications. On the first evaluation date, 100 fruit from the center two rows per plot were randomly selected and examined for surface damage (armyworm) and small tunnels (pinworm). At final harvest, all fruit in the center 6.2 m section per plot were picked and evaluated for damage and marketability. The percentage of fruit damaged by each pest was recorded. In North Carolina, tomatoes were transplanted in single lines on raised beds (1.5 m across). Treatments were arranged in a randomized complete block design with four replications. Each plot was 1.5 m across by 6.2 m long. A single row of ‘Silver Queen’ sweet corn was planted midway between rows as a buffer to separate treatments. Treatments were applied on either 3 (14-day intervals) or 6 dates (7 day intervals) in July and August, 1994. Applications were made using a CO2 backpack sprayer with a boom width (1.5 m) that ranged across each bed, and equipped with three equally-spaced hollow cone disk/core nozzles (D-3/25). The sprayer delivered 560 liters/ha at a pressure of 2.9 kg/cm2. Methomyl (500 g ai/ha) (Lannate L, DuPont Agri- cultural Products., Wilmington, DE) and permethrin (112 g ai/ha) (Ambush 2E, Zeneca Ag Products, Wilmington, DE) were included as chemical standards. As in Mexico, the incidence of fruit damage was determined in each plot, except that damage was caused primarily by tomato fruitworm, Helicoverpa zea (Boddie). Damage was estimated on August 19, when fruit first reached maturity, and again on August 27 by picking and visually inspecting all ripe fruit. On September 2, all remaining fruit were harvested from the middle 4.6 m of each plot. Each harvested fruit was visually inspected and classified as either damaged by fruitworms or marketable. All damaged fruit were cut open and inspected to identify the pest responsible for damage. Percentages were recorded based on total weight of harvested fruit per date.

Data Analysis

Data were analyzed using both nonparametric methods (Conover 1980) and least squares analysis of variance techniques (Zar 1984). Chemical availability of emamectin benzoate was compared among formulations by chi-square analysis (Conover 1980). Percentage mortality was transformed to the arcsine of the square root to nor- malize error variance. Means were separated by the Waller-Duncan K-ratio t-test (WDKR, Waller & Duncan 1969). Percentage data from ﬁeld experiments were also transformed to the arcsine of the square root and analyzed using standard analysis of variance techniques (Zar 1984). Means were separated by Fisher’s protected LSD (P < 0.05).

440 Florida Entomologist 79(3) September, 1996

RESULTS AND DISCUSSION

Solubility Test Chemical availability differed among the seven formulations tested (X2 = 23.7; df = 6; P < 0.05). Six of the seven formulations resulted in high estimates of availability (79-99%) of emamectin benzoate which did not differ (X2 = 3.1; df = 5; P > 0.05) among formulations. One of the formulations (81) was markedly lower in availability (47%) of emamectin benzoate than all other formulations and accounted for most of the variation among formulations (Table 1).

Glasshouse Tests Trial 1. All of the formulations tested were very effective and comparable (K-ratio = 100; WDKR) at killing both lepidopteran targets when applied at the high rate (8.4 g ai/ha) on all dates, except day 21 when formulation 77 was inferior to formulations 80 and 49 at controlling H. virescens (Tables 2 and 3). High levels of mortality (95- 100%) were achieved up to 21 days after application for all formulations. At the low rate (0.084 g ai/ha), differences in mortality of S. exigua larvae among formulations were noticeable by day 4 (Table 2). On days 4, 7, and 10, formulation 80 was superior to all other formulations at killing S. exigua. On day 14, formulations 79, 80 and 49 were superior to formulations 77 and 78 at controlling S. exigua. On day 21, formulations 80 and 49 were superior to 77 and 78. Mortality of H. virescens did not differ among most formulations on days 0-4 after application when formulations were applied at the low rate (0.084 g ai/ha) (Table 3). On the remaining evaluation dates, no single formulation consistently outperformed all other formulations at killing this pest. On day 7, mortality of H. virescens was greater on plants treated with formulations 79 and 49 than on those treated with formulations 77 and 80. On day 10, mortality was greater on plants treated with formulations 77, 78, and 79 than on those treated with formulation 80. On day 14, mortality was highest on plants treated with formulation 78 followed in decreasing order by those treated with formulations 77, 79, 80, and 49. On day 21, mortality did not differ (K-ratio = 100; WDKR) among most formulations. In general, these data showed that all formulations were comparable in their residual efficacy at controlling both Lepidoptera under glasshouse conditions when applied at the proposed field use rate (8.4 g ai/ha). At the lower rate, formulation 80 was consistently the most effective formulation at controlling S. exigua followed by formulation 79, whereas none of the formulations were consistently superior at controlling H. virescens, although trends in the data suggested that formulation 79 was most effective (although not consistently significant) at controlling this pest. For these reasons, formulations 80 and 79 were selected for advanced testing in trial 2. Trial 2. Differences in the residual efficacy of emamectin benzoate formulations were also observed when the plants were sprayed indoors and moved outdoors. At the high rate (8.4 g ai/ha), mortality of S. exigua and H. virescens did not differ (K-ratio = 100; WDKR) among formulations up to day 10 (Tables 4 and 5). On day 14, higher mortality of S. exigua was found on plants treated with formulation 80 than on those treated with formulation 82. On day 21, higher mortality of S. exigua was found on plants treated with formulations 80, 82, and 49 than on those treated with other formulations. On days 14 and 21, higher mortality of H. virescens was found on plants treated with formulations 80 and 49 than on those treated with other formulations. At the low rate (0.84 g ai/ha), formulation 82 was least effective at controlling S. exigua on day 4; all other formulations resulted in comparable levels of mortality (Ta-

Jansson et al.: Efﬁcacy of Emamectin Benzoate Formulations 441 EMA-

FORMULATIONS

DIFFERENT

WITH -ratio = 100; Waller & Duncan 1969). Waller -ratio = 100;

K 1 -test ( t TREATED

-ratio K PLANTS

PEPPER

Days after Application after Days ON

LARVAE

EXIGUA 1. S. OF TRIAL

, MORTALITY

GLASSHOUSE

0147101421 THE

PERCENTAGE — 9.3(3.1)b 22.0(5.0)b 16.9(6.3)d 7.0(1.9)e 40.3(8.6)d 18.1(6.1)d 53.9(6.9)e Rate, SEM) BENZOATE g ai/ha

± ( EAN MECTIN 2. M Means within the same column followed by the same letter are not signiﬁcantly different by the Waller-Duncan Waller-Duncan Means within the same column followed by letter are not signiﬁcantly different 1 check ABLE MK-0244-077MK-0244-078 0.084MK-0244-079 0.084 100.0(0.0)aMK-0244-080 0.084 100.0(0.0)aMK-0244-049 0.084 100.0(0.0)a 100.0(0.0)aMK-0244-077 0.084 100.0(0.0)a 100.0(0.0)aMK-0244-078 8.4 70.7(9.1)c 100.0(0.0)a 100.0(0.0)aMK-0244-079 88.4(4.0)b 8.4 99.1(0.9)aMK-0244-080 91.3(3.7)b 8.4 57.6(5.1)d 100.0(0.0)a 100.0(0.0)aMK-0244-049 60.2(7.0)cd 8.4 100.0(0.0)a 100.0(0.0)a 16.3(3.2)eNontreated 100.0(0.0)a 69.9(8.9)c 90.6(3.6)b 8.4 100.0(0.0)a 26.3(4.7)de 100.0(0.0)a 82.6(6.0)b 100.0(0.0)a 100.0(0.0)a 73.1(3.9)c 100.0(0.0)a 68.8(6.7)c 54.4(9.7)c 100.0(0.0)a 46.5(10.1)c 100.0(0.0)a 87.1(5.2)b 100.0(0.0)a 98.8(0.8)a 100.0(0.0)a 68.6(5.4)c 100.0(0.0)a 66.0(8.1)d 100.0(0.0)a 81.6(5.4)cd 93.8(3.8)ab 100.0(0.0)a 91.8(2.2)b 98.2(1.8)a 100.0(0.0)a 100.0(0.0)a 99.4(0.6)a 89.9(6.2)bc 98.1(1.9)ab 99.3(0.7)a 98.9(0.8)a 96.2(3.0)ab 97.9(1.5)ab 99.6(0.4)a 100.0(0.0)a 92.9(7.1)ab 98.9(1.1)a 99.3(0.7)ab 99.3(0.7)a 99.6(0.4)a 99.1(0.6)ab 100.0(0.0)a 100.0(0.0)a 100.0(0.0)a 100.0(0.0)a 99.7(0.3)ab Formulation T 442 Florida Entomologist 79(3) September, 1996 OF

FORMULATIONS

DIFFERENT

WITH

-ratio = 100; Waller & Duncan 1969). Waller -ratio = 100; K TREATED

1 -test ( t -ratio PLANTS

K CHICKPEA

Days after Application after Days LARVAE

1. VIRESCENS TRIAL H. ,

GLASSHOUSE

MORTALITY

THE

0147101421 IN

PERCENTAGE BENZOATE

— 0.0(0.0)b 11.1(3.9)b 2.4(0.5)c 19.6(5.6)e 7.4(4.4)e 15.6(3.7)d 9.9(3.3)e Rate, SEM) g ai/ha ± ( EAN EMAMECTIN 3. M Means within the same column followed by the same letter are not significantly different by the Waller-Duncan Waller-Duncan Means within the same column followed by letter are not significantly different 1 check ABLE Formulation MK-0244-077MK-0244-078 0.084MK-0244-079 0.084 100.0(0.0)aMK-0244-080 0.084 100.0(0.0)aMK-0244-049 0.084 100.0(0.0)a 100.0(0.0)aMK-0244-077 0.084 100.0(0.0)a 100.0(0.0)a 100.0(0.0)aMK-0244-078 8.4 100.0(0.0)a 100.0(0.0)a 100.0(0.0)aMK-0244-079 8.4 100.0(0.9)a 95.1(1.9)c 100.0(0.0)aMK-0244-080 8.4 100.0(0.0)a 100.0(0.0)a 96.8(1.5)bcMK-0244-049 97.6(1.9)b 8.4 100.0(0.0)a 100.0(0.0)a 96.6(5.8)abc 100.0(0.0)aNontreated 95.5(2.3)bc 100.0(0.0)a 100.0(0.0)a 96.4(1.9)ab 84.8(2.5)d 99.2(0.8)ab — 100.0(0.0)a 100.0(0.0)a 98.8(1.2)ab 100.0(0.0)a 100.0(0.0)a 49.6(7.7)cd 97.2(1.4)ab 100.0(0.0)a 87.5(4.2)d 92.2(2.6)cd 100.0(0.0)a 100.0(0.0)a 41.8(4.1)d 100.0(0.0)a 100.0(0.0)a 52.9(5.2)cd 100.0(0.0)a 100.0(0.0)a 89.1(4.4)c 93.9(2.6)bc 100.0(0.0)a 100.0(0.8)a 99.2(0.8)ab 100.0(0.0)a 100.0(0.0)a 100.0(0.0)a 44.4(4.9)cd 57.2(4.3)c 100.0(0.0)a 100.0(0.0)a 100.0(0.0)a 100.0(0.0)a 100.0(0.0)a 100.0(0.0)a 98.4(1.1)ab 100.0(0.0)a 100.0(0.0)a 96.5(2.4)ab 100.0(0.0)a 95.2(2.2)b 100.0(0.0)a 100.0(0.0)a T Jansson et al.: Efficacy of Emamectin Benzoate Formulations 443

ble 4). On days 7, 10, and 14, formulation 80 was consistently the most effective (although not consistently significant) formulation at killing S. exigua. Formulations 82 and 49 were the least effective formulations on these days. On day 21, mortality was highest on plants treated with formulation 49 followed by those treated with formulations 80, 81, 82, and 79. Mortality of H. virescens did not differ among most formulations applied at the low rate (0.84 g ai/ha) up to day 4 (Table 5). Between days 7 and 21, no consistent differences in mortality of H. virescens were noted among formulations. Data from the second trial concurred with those from the first trial and showed that all formulations were comparable in their residual efficacy at controlling S. exigua at the recommended field use rate (8.4 g ai/ha). Formulations were comparable in their efficacy at controlling H. virescens at this rate on most dates except days 14 and 21, when formulation 80 was found to be superior to most other formulations tested. At the lower rate, formulation 80 was consistently the most effective formulation at controlling S. exigua followed by formulations 79 and 81 (through day 14). As found in the first trial, none of the formulations were consistently superior at controlling H. virescens at the low rate.

Field Trials

Two dry powder blend formulations (80 and 81) were selected for field testing based on their performance in the glasshouse and on their compositions. Both field trials demonstrated that solid formulations of emamectin benzoate were as effective as the EC formulation at reducing fruit damage from Lepidoptera on tomato (Table 6). In Mexico, tomato pinworm damage did not differ (P > 0.05) among most chemical treatments on both dates. On the first evaluation date, damage was significantly lower on plants treated with formulation 81 applied at 7 day intervals, the EC formulation applied at 7-and 14-day intervals, and formulation 80 applied at 7-day intervals than on those treated with formulation 81 at 14-day intervals and on those treated with the chemical standard, lambda cyhalothrin (Table 6). On the second evaluation date, damage from tomato pinworm did not differ (P > 0.05) among all chemical treatments. Beet armyworm damage did not differ (P > 0.05) among most chemical treatments on both evaluation dates. On the first evaluation date, damage was highest on plants treated with formulation 81 at 14-day intervals; on the second evaluation date, damage was highest on plants treated with lambda cyhalothrin and on those treated with formulation 81 at 7-day intervals. Harvest data showed that formulation 80 applied at 7- and 14-day intervals, formulation 81 applied at 7-day intervals, and the EC formulation applied at 7- and 14-day intervals were the most effective treatments at increasing percentages of marketable fruit (Table 6). Formulation 81 applied at 14- day intervals and the chemical standard, lambda cyhalothrin, produced the lowest percentages of marketable fruit. In North Carolina, all formulations and the chemical standards were effective at reducing fruit damage from H. zea. Percentage fruit damage did not differ among chemical treatments on the first harvest date (Table 6). Percentage of damaged fruit differed (P < 0.05) among chemical treatments on the second harvest date. Damage was lowest on plants treated with formulation 80 at 7-day intervals, which did not differ from those on plants treated with permethrin at 7-day intervals, the EC formulation (49) at 7- and 14-day intervals, and formulation 81 at 7-day intervals. Damage was highest on plants treated with formulation 81 at 14-day intervals. On the third harvest date, superior efficacy was observed on plants treated with formulation 80, the EC, and permethrin at 7-day intervals. Lower efficacy (although not consistently 444 Florida Entomologist 79(3) September, 1996 EMA-

FORMULATIONS

DIFFERENT

WITH -ratio = 100; Waller & Duncan 1969). Waller -ratio = 100;

K -test ( 1 t TREATED

-ratio K PLANTS

PEPPER

Day after Application after Day ON

LARVAE

EXIGUA 2. S.

OF TRIAL

, MORTALITY

GLASSHOUSE

0147101421 THE

PERCENTAGE — 17.64(4.4)c 55.1(8.5)b 40.3(9.1)d 8.3(1.8)d 22.3(4.4)d 17.7(3.5)ef 2.5(2.5)f Rate, SEM) BENZOATE g ai/ha

± ( EAN MECTIN 4. M Means within the same column followed by the same letter are not significantly different by the Waller-Duncan Waller-Duncan Means within the same column followed by letter are not significantly different 1 check ABLE MK-0244-079MK-0244-080 0.84MK-0244-081 0.84MK-0244-082 100.0(0.0)a 0.84MK-0244-049 100.0(0.0)a 0.84 100.0(0.0)aMK-0244-079 0.84 97.6(1.8)b 100.0(0.0)aMK-0244-080 100.0(0.0)a 8.4 97.8(1.7)abMK-0244-081 100.0(0.0)a 99.2(0.8)a 8.4 99.5(0.5)ab 100.0(0.0)aMK-0244-082 74.7(9.7)b 8.4 100.0(0.0)a 100.0(0.0)aMK-0244-049 96.8(2.5)a 95.1(3.4)ab 8.4 100.0(0.0)a 82.6(5.2)cNontreated 100.0(0.0)a 85.6(4.5)b 8.4 100.0(0.0)a 95.1(2.6)b 85.7(5.0)b 100.0(0.0)a 97.9(1.4)a 100.0(0.0)a 62.6(4.1)c 100.0(0.0)a 100.0(0.0)a 25.2(6.1)de 100.0(0.0)a 58.3(11.2)c 100.0(0.0)a 81.4(5.9)b 100.0(0.0)a 47.8(5.2)c 97.5(1.3)a 100.0(0.0)a 2.6(1.3)ef 65.0(6.2)c 100.0(0.0)a 64.3(7.8)c 100.0(0.0)a 100.0(0.0)a 19.1(6.1)cd 38.8(8.0)cd 99.4(0.6)a 97.3(1.4)a 100.0(0.0)a 23.6(4.9)def 100.0(0.0)a 14.3(6.4)cde 15.6(4.8)f 99.4(0.6)a 9.6(4.0)def 100.0(0.0)a 100.0(0.0)a 100.0(0.0)a 94.3(3.5)ab 24.5(10.4)c 98.0(0.8)a 68.1(9.1(b) 100.0(0.0)a 90.2(6.0)a 96.0(2.0)ab 76.8(5.1)b 88.7(2.9)b 95.8(1.6)ab 95.1(1.2)a 92.8(2.2)a Formulation T Jansson et al.: Efficacy of Emamectin Benzoate Formulations 445 OF

FORMULATIONS

DIFFERENT

WITH

-ratio = 100; Waller & Duncan 1969). Waller -ratio = 100; K 1 TREATED

-test ( t -ratio PLANTS

K CHICKPEA

Day after Application after Day ON

LARVAE

2. VIRESCENS TRIAL , H. OF

GLASSHOUSE

0147101421 MORTALITY

THE

PERCENTAGE Rate, g ai/ha BENZOATE

SEM) ± ( EAN EMAMECTIN 5. M Means within the same column followed by the same letter are not signiﬁcantly different by the Waller-Duncan Waller-Duncan Means within the same column followed by letter are not signiﬁcantly different 1 ABLE MK-0244-079R001MK-0244-080A001 0.84MK-0244-081C001 0.84MK-0244-083E001 100.0(0.0)a 0.84MK-0244-049C001 100.0(0.0)a 100.0(0.0)a 0.84MK-0244-079R001 100.0(0.0)a 100.0(0.0)a 0.84 100.0(0.0)aMK-0244-080A001 100.0(0.0)a 8.4 100.0(0.0)a 36.3(6.3)bMK-0244-081C001 100.0(0.0)a 100.0(0.0)a 89.7(3.0)cd 8.4MK-0244-083E001 100.0(0.0)a 96.4(1.9)ab 100.0(0.0)a 96.8(1.7)a 8.4 100.0(0.0)aMK-0244-049C001 86.9(7.0)cd 100.0(0.0)a 8.4 100.0(0.0)a 100.0(0.0)aNontreated check 80.5(4.1)e 53.5(4.6)d 88.1(4.6)d 95.8(1.4)bc 100.0(0.0)a 8.4 100.0(0.0)a 100.0(0.0)a 53.5(6.0)d 97.1(1.5)ab 100.0(0.0)a 100.0(0.0)a 95.0(1.7)bc 82.6(6.0)de 49.7(7.1)c 100.0(0.0)a — 100.0(0.0)a 100.0(0.0)a 97.6(1.2)ab 97.5(1.8)ab 29.3(6.3)f 40.2(8.1)de 100.0(0.0)a 49.1(7.3)c 100.0(0.0)a 100.0(0.0)a 33.6(5.9)ef 100.0(0.0)a 99.0(1.0)ab 18.0(7.5)b 32.5(6.4)d 99.1(0.9)ab 100.0(0.0)a 28.4(5.5)d 99.3(0.7)ab 86.3(3.0)b 98.2(1.8)ab 55.4(6.8)c 12.9(2.9)c 98.4(1.6)ab 98.6(1.0)a 100.0(0.0)a 100.0(0.0)a 86.2(2.7)b 82.1(3.6)b 100.0(0.0)a 100.0(0.0)a 9.2(5.1)c 74.6(4.5)c 99.2(0.8)a 81.6(5.0)b 7.5(2.6)e 92.1(2.7)b 100.0(0.0)a 3.4(1.7)f 2.7(1.4)g 7.7(2.8)e Formulation T 446 Florida Entomologist 79(3) September, 1996 EM-

1 OF

FORMULATIONS

DIFFERENT

WITH

% Damaged Fruit, North Carolina % Damaged Fruit, % Mark 19 Aug. 27 Aug. 12 Sept. TREATED

3 1 SP2 < 0.05) (Zar 1984). PLANTS P

3 ON

SP1 2 LEPIDOPTERA PW2

2 % Damaged Fruit, Mexico % Damaged Fruit, FRUIT PWI

1994. IN

TOMATO

TRIALS Interval, d Interval, Application

DAMAGE

FIELD

Rate, g ai/ha TWO

PERCENTAGE BENZOATE

SEM) ± ( EAN AMECTIN 6. M Means within the same column followed by the same letter are not significantly different by Fisher’s LSD ( Means within the same column followed by letter are not significantly different Fisher’s respectively). (PW1 and PW2, pinworm damage on 8 and 22 Sept. Tomato respectively). (SP1 and SP2, Beet armyworm damage on 8 and 22 Sept. 1 2 3 -Cyhalothrin 1 EC 44.0 7 7.0b 1.0ab 12.2bc 4.7b 83.1bc ABLE MK-0244-080MK-0244-080MK-0244-081 2 WPMK-0244-081 2 WPMK-0244-049 2 WPMK-0244-049 8.4 2 WPλ 8.4 0.16 ECPermethrin 8.4 0.16 ECMethomyl 7 8.4 8.4 14Nontreated check 8.4 7 2 EC 4.5c14 7 6.0bc L 14 0.0b 112.0 0.5b 5.0c 10.0b 5.0c 10.9bc 8.0c 0.5b 0.5b 4.0c — 500.0 7 1.8bc 0.0b 16.2b 0.6c 9.9c 0.5b 87.3abc 7.6c — 91.4a 7 11.6bc 2.5bc 3.9b 8.6a 81.3c 2.4bc 3.1bc 86.2abc 15.0a 1.2a 86.0abc 89.4ab 8.8bc 10.0a 5.0a 2.0a 6.2a 1.4a 3.4a 6.9b 28.0a 10.3c 4.0abc 10.6a 3.2abc 0.0a 2.9ab 8.9b 3.8ab 61.4d 6.7b 0.0a 26.6b 47.7d 40.2c 4.0a 1.9ab 2.5a 0.0a 6.5bc 4.8b Treatment T Jansson et al.: Efficacy of Emamectin Benzoate Formulations 447 significant) was found on plants treated with formulation 81 at 7- and 14-day intervals, formulation 80 at 14-day intervals, the EC at 14-day intervals, and methomyl at 7-day intervals. These studies demonstrated that several solid formulations of the benzoate salt of emamectin were efficacious at controlling two economically important lepidopterous pests, S. exigua and H. virescens, in glasshouse tests. Excellent efficacy was found for up to 14-21 days after application under glasshouse conditions when emamectin benzoate was applied at the proposed field use rate (8.4 g ai/ha). A dry powder blend (80) was shown to be the most efficacious formulation tested. Similar results were found in the field. A dry powder blend formulation (80) was shown to be very efficacious at controlling H. zea, K. lycopersicella, and S. exigua on tomato. Control was comparable to that achieved with the EC formulation, and comparable or superior to lambda cyhalothrin, permethrin, and methomyl. Satisfactory efficacy was also achieved with another dry powder blend (formulation 81), albeit it was slightly less effective than formulation 80 and the EC, especially when applied at 14-day intervals. The lower efficacy of formulation 81 compared with formulation 80 and the EC concurs with data from the residual efficacy tests conducted in the glasshouse. These data also agree with the solubility data for this formulation. This formulation had the lowest percentage of available emamectin benzoate (Table 1), which undoubtedly affected its efficacy at controlling lepidopteran pests. Excellent efficacy of several formulations was found for up to 14 days when use rates were as low as 0.084 and 0.84 g ai/ha under glasshouse and simulated field conditions (i.e., plants sprayed indoors and moved between the glasshouse and outdoors daily), respectively. Such results with this compound are not uncommon in glasshouse environments, especially during the months of March and April in Ohio, when ultraviolet radiation (UV) is low. However, similar results would not be expected in the field because avermectins (e.g., abamectin) are very susceptible to photodegradation (Mac- Connell et al. 1989). They showed that the half-life of abamectin was < 10 h in light. Numerous photodegradates have subsequently been identified for both abamectin (Crouch et al. 1991) and emamectin benzoate (Feely et al. 1992). For these reasons, field use rates of between 8.4 and 16.8 g ai/ha are recommended for the compound (Anonymous 1995). Slower photodegradation undoubtedly occurred in the present study in the glasshouse than what would be expected in the field during the growing season. Glass- houses are known to filter out a large percentage of UV light. Thus, when plants were indoors, emamectin benzoate residues were exposed to minimal levels of UV radiation. It is likely that the reduced photodegradation of emamectin benzoate in the glasshouse optimized the amount that was able to penetrate leaf tissue via translam- inar movement and subsequently prolong the residual efficacy observed. MacConnell et al. (1989) showed that there were marked differences in the half-life of abamectin on Petri dishes and on leaves in light and dark environments and prolonged stability in the dark resulted in greater penetrability into leaves and improved efficacy at controlling mites. Emamectin benzoate is being developed as a broad spectrum lepidoptericide on a wide variety of horticultural crops. Emamectin benzoate has an unprecedented potency against Lepidoptera (Dybas 1988, Dybas & Babu 1988, Mrozik et al. 1989), and has one of the lowest recommended use rates (8.4 g ai/ha) of all insecticides sold commercially or in development (another avermectin, abamectin, currently has the lowest recommended use rate, 5.4 g ai/ha) (Jansson & Dybas 1996). Emamectin benzoate is very compatible with IPM. It is extremely selective at killing a broad spectrum of lepidopterous pests at very low use rates, while conserving natural enemies (D. Cox 448 Florida Entomologist 79(3) September, 1996 et al., unpublished). Although the liquid EC formulation (0.16 EC) is considered a safe product, the development of a solid formulation of emamectin benzoate should further enhance its safety features. Solid formulations can reduce the need for plastic packaging thereby reducing the environmental burden. In addition, the use of solid formulations may eliminate the use of volatile organic solvents as part of a formulation’s composition. Lastly, solid formulations can reduce overall exposure of workers to pesticides, especially when products are loaded into spray equipment.

ACKNOWLEDGMENTS We thank G. Misich, L. Limpel, M. Poling (Ricerca, Inc., Painesville, OH) and M. Alvarez (Merck & Co., Inc., Three Bridges, NJ) for technical assistance. We thank S. J. Gross, H. D. Nalle, and P. Round (Merck & Co., Inc.) for critically reviewing the manuscript. This is Merck Research Laboratories Publication No. 95-MS-2741.

REFERENCES CITED

ANONYMOUS. 1995. Technical data sheet, emamectin benzoate insecticide. Merck Re- search Laboratories, Merck & Co., Inc., Three Bridges, NJ, 11 p. CAMPBELL, W. C., R. W. BURG, M. H. FISHER, AND R. A. DYBAS. 1984. The discovery of ivermectin and other avermectins, pp. 5-20 in P. S. Magee, G. K. Kohn & J. J. Menn [eds.]. Pesticide synthesis through rational approaches. ACS Symp. Ser. No. 255. American Chemical Soc., Washington, DC. CONOVER, W. J. 1980. Practical nonparametric statistics, 2nd ed. J. Wiley, New York. CROUCH, L. S., W. F. FEELY, B. H. ARISON, W. J. VANDEN HEUVEL, L. F. COLWELL, R. A. STEARNS, W. F. KLINE, AND P. G. WISLOCKI. 1991. Photodegradation of avermectin B1a thin films on glass. J. Agric. Food Chem. 39: 1310-1319. DYBAS, R. A. 1988. Discovery of novel second generation avermectin insecticides for crop protection. Intern. Congress. Entomol., Vancouver, B.C., Canada. DYBAS, R. A. 1989. Abamectin use in crop protection, pp. 287-310 in W. C. Campbell [ed.]. Ivermectin and abamectin. Springer-Verlag, New York. DYBAS, R. A., AND J. R. BABU. 1988. 4”-deoxy-4”-methylamino-4”-epiavermectin B1 hydrochloride (MK-243): a novel avermectin insecticide for crop protection, pp. 57-64. in British crop protection conference. Pests and diseases, British Crop Protection Council, Croydon, U.K. DYBAS, R. A., N. J. HILTON, J. R. BABU, F. A. PREISER, AND G. J. DOLCE. 1989. Novel second-generation avermectin insecticides and miticides for crop protection, pp. 203-212 in A. L. Demain, G. A. Somkuti, J. C. Hunter-Cevera & H. W. Ross- moore [eds.]. Novel microbial products for medicine and agriculture. Elsevier, Amsterdam, Netherlands. EDWARDS, M. H., S. A. KOLMES, AND T. J. DENNEHY. 1994. Can pesticide formulations significantly influence pest behavior?: the case of Tetranychus urticae and dico- fol. Entomol. Exp. Appl. 72: 245-253. FEELY, W. F., L. S. CROUCH, B. H. ARISON, W. J. A. VANDEN HEUVEL, L. F. COLWELL, AND P. G. WISLOCKI. 1992. Photodegradation of 4”-(epimethylamino)-4”- deoxyavermectin B1a thin films on glass. J. Agric. Food Chem. 40: 691-696. HARTLEY, G. S., AND I. J. GRAHAM-BRYCE. 1980. Physical principles of pesticide behavior: the dynamics of applied pesticides in the local environment in relation to biological response. Vol. 1 and 2. Academic Press, London. HUDSON, J. L., AND O. R. TARWATER. 1988. Reduction of pesticide toxicity by choices of formulation., p. 124-130 in B. Cross & H. Scher [eds.]. Pesticide formulations. ACS Symp. Ser. No. 371, American Chemical Soc., Washington, DC. JANSSON, R. K., AND R. A. DYBAS. 1996. Avermectins: biochemical mode of action, biological activity, and agricultural importance in I. Ishaaya [ed.], Insecticides with novel modes of action: mechanism and application. Springer-Verlag, New York, (in press). Jansson et al.: Efficacy of Emamectin Benzoate Formulations 449

JANSSON, R. K., AND S. H. LECRONE. 1992. Efﬁcacy of nonconventional insecticides for control of diamondback moth, Plutella xylostella (L.), in 1991. Proc. Florida State Hort. Soc. 104: 279-284. LASOTA, J. A., AND R. A. DYBAS. 1991. Avermectins, a novel class of compounds: implications for use in arthropod pest control. Annu. Rev. Entomol. 36: 91-117. LEIBEE, G. L., R. K. JANSSON, G. NUESSLY, AND J. L. TAYLOR. 1995. Efﬁcacy of emamectin benzoate and Bacillus thuringiensis for control of diamondback moth on cabbage in Florida. Florida Entomol. 78: 82-96. MACCONNELL, J. G., R. J. DEMCHAK, F. A. PREISER, AND R. A. DYBAS. 1989. Relative stability, toxicity, and penetrability of abamectin and its 8,9-oxide. J. Agric. Food Chem. 37: 1498-1501. MROZIK, H., P. ESKOLA, B. O. LINN, A. LUSI, T. L. SHIH, M. TISHLER, F. S. WAKSMUN- SKI, M. J. WYVRATT, N. J. HILTON, T. E. ANDERSON, J. R. BABU, R. A. DYBAS, F. A. PREISER, AND M. H. FISHER. 1989. Discovery of novel avermectins with unprecedented insecticidal activity. Experientia 45: 315-316. TRUMBLE, J. T., W. J. MOAR, J. R. BABU, AND R. A. DYBAS. 1987. Laboratory bioassays of the acute and antifeedant effects of avermectin B1 and a related analogue on Spodoptera exigua (Hübner). J. Agric. Entomol. 4: 21-28. WALLER, R. A., AND D. B. DUNCAN. 1969. A Bayes rule for the symmetric multiple comparisons problem. J. American Statist. Assn. 64: 1484-1503. ZAR, J. H. 1984. Biostatistical analysis. Prentice-Hall, Englewood Cliffs, NJ.

450 Florida Entomologist 79(3) September, 1996

AN AUTOMATED SYSTEM FOR COLLECTION AND COUNTING OF PARASITIZED LEAFMINER (DIPTERA: AGROMYZIDAE) LARVAE

F. L. PETITT1, D. S. SHUMAN2, D. O. WIETLISBACH1 AND J. A. COFFELT2 1The Land, Epcot, Lake Buena Vista, FL 32830

2USDA-ARS, Insect Attractants, Behavior, and Basic Biology Research Lab, Gainesville, FL 32604

The leafminers Liriomyza sativae Blanchard and L. trifolii (Burgess) are serious pests of a wide variety of vegetable and ornamental crops in the United States and many other countries (Minkenberg & van Lenteren 1986, Spencer & Steyskal 1986). The braconid larval-pupal parasitoid, Opius dissitus Muesebeck, parasitizes both L. sativae and L. trifolii (Coly 1984, Lauridsen et al. 1993, Petitt 1993) and occurs commonly in Florida (Schuster & Wharton 1993, Schuster et al. 1991). Opius dissitus has been reared and released for augmentative biological control of L. sativae in greenhouses in The Land at Epcot, Lake Buena Vista, FL since the mid 1980’s (Petitt 1993). In The Land’s insectary, O. dissitus is reared using L. sativae as the host on bush lima bean (Phaseolus lunatus L. cv. Henderson) primary leaves. Leafminer larvae in which O. dissitus has oviposited complete their larval development, cut a hole in the upper leaf surface, crawl out, and drop from the leaves. As larvae drop from the leaves, they are collected in a small vial at the base of a large funnel. The parasitized leafminers remain in vials in the insectary until just before eclosion of the adult parasitoid, at which time the vials are transported to the release site. Research on the life history and biological characteristics of both the host (Petitt & Wietlisbach 1992, 1994) and parasitoid (Petitt & Wietlisbach 1993) has made rearing more cost effective by reducing the amount of time and plant material required per parasitoid produced. Decreased production costs have enabled use of the parasitoid to be expanded to include biological control of L. trifolii in over 100 flower beds (approximately 200,000 susceptible plants per year) in the landscape throughout the Walt Disney World Resort. Mortality problems were encountered in rearing as production expanded and larger numbers of parasitoids were collected per vial. In some instances, eclosing adult wasps could not free themselves from the interior of large masses of puparia which were stuck together. Changing the collection vial several times per day to avoid mortality from overcrowding was inefficient. An automated system was sought to count the larvae and change vials as their capacity was reached. Accurate counts of parasitoids would also facilitate evaluation of augmentative releases. The Electronic Grain Probe Insect Counter (EGPIC) system which was designed to monitor insect infestations in stored product facilities (Shuman et al. 1996) was evaluated as a means of counting L. sativae larvae. The EGPIC system consists of an infrared beam sensor head through which the insects fall, infrared beam generation and detection circuitry, data storage and a user interface. The objectives of this study were 1) to determine whether, and how accurately, the EGPIC system could count L. sativae larvae dropping through the sensor head and 2) to modify the system so that when a parasitoid collection vial is filled to its designated capacity the system responds by placing an empty vial into position at the base of the funnel. Preliminary studies conducted with the EGPIC system showed that it could accurately count leafminer larvae as they dropped through the sensor head. System mod-

Scientiﬁc Notes 451 iﬁcations were made and software was written to integrate the EGPIC sensor head (described in detail by Shuman et al. 1996) into a leafminer collection system (Fig. 1). The system counts parasitized larvae as they drop through a funnel into one of a ring of collection vials mounted on a turntable with a registered advance. A detection/interface circuit connects the sensor head output to a personal computer which directs

Fig. 1. Diagram of the automated counting and collection system for parasitized leafminer larvae dropping from plant foliage.

452 Florida Entomologist 79(3) September, 1996 system operation. When a vial has received the designated number of larvae, the computer rotates the turntable to start ﬁlling the next vial. The system also creates a da- tabase of time-stamped insect counts.

Description of Leafminer Collection System Components The sensor head uses an infrared light emitting diode to produce a beam that is sensed by a phototransistor. The beam traverses a rectangular channel through the sensor head. The fall of an insect through the channel partially masks the beam and results in a signal on the phototransistor output. The channel is 45-mm long; a plastic insert that spans its entire length provides for surface smoothness and minimizes the possibility of insects getting caught in the channel or producing multiple counts. The cross section of the channel (11 × 3 mm) is narrower than the sensor elements’ diam- eters (5 mm) to obscure the weaker edges of the beam, and thus, enhance the unifor- mity of detection sensitivity across the channel. The strength of the beam and the quiescent operation point of the phototransistor are set to optimize performance for this particular larval size. The detection/interface circuitry responds to phototransistor output signals above a threshold level (user adjustable and employing hysteresis for noise immunity) by generating a (digital) count pulse (on a line dedicated to that sensor head) read by the computer. In its current implementation, the computer’s printer (parallel) port is configured by software for system digital signal reads and writes. In this way, the computer can service up to seven sensor head/turntable units. For larger implementations, a commercial, digital I/O board would have to be installed in the computer. The number of units that may be serviced by a single computer with a commercial digital I/O board is dependent upon the rate insects are falling and the speed of the computer, but these factors were not addressed in this study. The system is interrupt driven so the computer is relatively idle (taking care of clocks, other overhead functions, and available for other chores, such as user utilities) until a falling insect causes an interrupt. The interrupt driven design (as opposed to a polling design) also allows the computer to respond immediately to a falling insect so that a quick succession of counts can be acquired without error. The interface circuitry generates an interrupt pulse (on a common line for all sensor heads) to notify the computer when any sensor head output is detected. The computer then checks all the lines dedicated to individual sensor heads for a count pulse so that the computer can then determine and record through which sensor head an insect dropped. In addition, the computer periodically initiates a test of system operability by momentarily reducing the output of the infrared beam generator to simulate the fall of insects si- multaneously through all sensor heads, and then checks for appropriate interface circuitry outputs. The computer’s functions are supervisory as well as data acquisition, storage, and presentation. A configuration screen allows the user to change various parameters affecting data acquisition. For example, the ‘Retrigger Threshold’ is the amount of time that must pass between two consecutive insect counts from the same sensor head in order for the second count to be registered. The purpose of this threshold is to prevent one insect from being counted twice, and thus, it also determines the maximum insect count rate. The default count rate is 10 insects per second. The ‘Turntable Rotation Control Signal’ can be specified to accommodate different vial sizes and the ‘Insects Per Vial’ parameter can be set to any value desired. During data acquisition, a user interface/status screen displays operational data in real-time and allows for data file annotations. The resulting data file contains time- stamped insect counts, and all other system events, such as starting acquisition, self-

Scientiﬁc Notes 453 testing, retriggers, insect counts during turntable rotation, and data backup to hard disk. Additional user options include data backup time interval, auto archiving to any drive, and an audible indication of insect counts. The information in a data ﬁle can be presented in many ways, such as summarized, ordered by time or sensor head, and stripped of system events, and it can be exported to a printer or a spreadsheet program.

System Veriﬁcation and Operation

A leafminer collection system with two sensor head/turntable units was initially tested by setting the ‘Insects per Vial’ parameter at 20 insects and running the system for eight days, during which time 53 and 50 vials were collected on units 1 and 2, respectively. The mean number of larvae collected per vial (±S.E.M.) was 19.5 (±0.2) for unit 1 and 19.8 (±0.2) for unit 2. The number collected on each turntable unit ranged from 17 to 23 insects. Given that these results were considered acceptable for the desired purpose, this two-unit system was installed at The Land at Epcot on February 17, 1994 and the system is still operating at present. Three further sets of counts were conducted at five, 13, and 19 months after instal- lation at The Land. In each case, counts were made of actual numbers of pupae per cup for ten vials that were filled when the ‘Insects per Vial’ parameter was set at 200 insects. The mean (±S.E.M.) of the ten counts were 201.2 (±2.6), 197.6 (±2.8), and 200.6 (±2.6) insects. Counts ranged from 184 to 214 insects per vial for the 30 vials. Overall, the accuracy was well within what would be acceptable for this purpose. The system has been very reliable. It has been used for the past 20 months to count and collect about 13,000 parasitoids per week into vials containing 200 each. Only three operational problems have occurred during this entire period. In each case, the rectangular channel became obstructed (once by a group of larvae, once by a piece of bean leaf, and once by soil from the potted lima beans). Of course, there is an upper limit to the number of larvae that can pass through the channel per unit of time without it being obstructed; however, no attempt was made to quantify that upper limit. The system has facilitated wider scale and more efficient use of the parasitoid because of easier and quicker distribution and has greatly improved our ability to evaluate parasitoid releases.

SUMMARY

Efﬁciency of augmentative biological control of Liriomyza leafminers has been improved by development of an automated system for collection and counting parasitized larvae as they drop from plant foliage to pupate. The system uses an infrared beam to count larvae as they drop through a funnel into one of several collection vials on a turntable. When a vial has received the designated number of larvae, the turntable rotates so that the next vial will begin to ﬁll. Operation and tests of the system for over two years have shown that it is very reliable and accurate.

ACKNOWLEDGMENTS

We thank Lori Brasington, Marian Coffey, and Yuqing Fan of The Land at Epcot, Walt Disney World, and Hok Chia, Brian Hook and Betty Weaver of the USDA Insect Attractants and Basic Biology Research Laboratory, Gainesville, Florida for their assistance on this project.

454 Florida Entomologist 79(3) September, 1996

REFERENCES

COLY, E. V. 1984. La mouche mineuse des cultures maralchères Liriomyza trifolii (Burgess) au Sénégal. Camberen. Dakar. Projet FAO/TCP/SEN/2202. (Centre pour le développement de l’horticulture). 32 p. LAURIDSEN, L., D. BORDAT, AND P. ROBERT. 1993. Susceptibility of Opius dissitus, Muesebeck, (Hym. Braconidae) and Hemiptarsenus varicornis, (Girault), (Hym. Eulophidae) at two strains of Paecilomyces fumosoroseus (Wize), Brown and Smith, (Hyphomycetes), in “Liriomyza”: Conference on leaf-mining flies in cultivated plants, Montpellier, France, March 24-25. MINKENBERG, O. P. J. M., AND J. C. VAN LENTEREN. 1986. The leafminers Liriomyza bryoniae and L. trifolii (Diptera: Agromyzidae), their parasites and host plants: a review. Agric. Univ. Wageningen Paper 86-2. PETITT, F. L. 1993. Biological control in the integrated pest management program at The Land, Epcot Center, pp. 129-133 in Proc. Work. Group Integrated Control Glasshouses, Pacific Grove, Calif. Bull. Int. Org. Biol. Control/West Palearct. Reg. Sect. Vol. 16(2). PETITT, F. L., AND D. O. WIETLISBACH. 1992. Intraspecific competition among same- aged larvae of Liriomyza sativae (Diptera: Agromyzidae) in lima bean primary leaves. Environ. Entomol. 21: 136-140. PETITT, F. L., AND D. O. WIETLISBACH. 1993. Effects of host instar and size on parasitization efficiency and life history parameters of Opius dissitus. Entomol. Exp. Appl. 66: 227-236. PETITT, F. L., AND D. O. WIETLISBACH. 1994. Laboratory rearing and life history of Li- riomyza sativae (Diptera: Agromyzidae) on lima bean. Environ. Entomol. 23: 1416-1421. SCHUSTER, D. J., AND R. A. WHARTON. 1993. Hymenopterous parasitoids of leaf-mining Liriomyza spp. (Diptera: Agromyzidae) on tomato in Florida. Environ. En- tomol. 22: 1188-1191. SCHUSTER, D. J., J. P. GILREATH, R. A. WHARTON, AND P. R. SEYMOUR. 1991. Agromyz- idae (Diptera) leafminers and their parasitoids in weeds associated with tomato in Florida. Environ. Entomol. 20: 720-723. SHUMAN, D. S., J. A. COFFELT, AND D. WEAVER. 1996. A computer-based electronic fall-through probe insect counter for monitoring infestation in stored products. Transactions of the ASAE. (In Press). SPENCER, K. A., AND G. C. STEYSKAL. 1986. Manual of the Agromyzidae (Diptera) of the United States. U.S. Dept. Agric. Handb. No. 638.

Scientiﬁc Notes 455

DIURNAL PATTERNS OF OVIPOSITIONAL ACTIVITY IN TWO PSEUDACTEON FLY PARASITOIDS (DIPTERA: PHORIDAE) OF SOLENOPSIS FIRE ANTS (HYMENOPTERA: FORMICIDAE)

M. A. PESQUERO1, S. CAMPIOLO2, H. G. FOWLER2 AND S. D. PORTER3 1Instituto de Biociencias, Universidade Estadual Paulista, 18610 Botucatu, Sao Paulo, Brazil

2Instituto de Biociencias, Universidade Estadual Paulista, 13500 Rio Claro, Sao Paulo, Brazil

3Center for Medical, Agricultural, and Veterinary Entomology, USDA-ARS, P.O. Box 14565, Gainesville, FL 32604

At least 18 species of Pseudacteon phorid parasitoids attack fire ants in the Sole- nopsis saevissima complex (Williams & Banks 1987, Pesquero et al. 1993, Disney 1994, Porter et al. 1995a, 1995b). The high species richness of these phorid parasitoids, and the behaviors exhibited by fire ants in their presence (Porter et al. 1995a) suggest a long evolutionary history. The absence of natural enemies, such as phorids, may explain why exotic North American populations of fire ants (Solenopsis invicta Buren) have much higher densities than those found in their native Brazil (Porter et al. 1992). In spite of the high species richness of phorids recorded on fire ants in South Amer- ica, little is known about their behavior and coexistence. Some species of phorids attack different sized workers than others (Campiolo et al. 1994), but are there also other ways they partition their host resource? Here, we document diurnal activity patterns of two common species of Pseudacteon attacking the fire ant Solenopsis saevissima (Fr. Smith), a close relative of S. invicta (Trager 1991), in sub-tropical Brazil. Studies were conducted on the new campus of the Universidade Estadual Paulista in Rio Claro, SP, Brazil (23°S). Colonies of S. saevissima were maintained in petri dish observation nests in Fluon®-coated plastic trays (35 × 40 × 5 cm). Every month for a year (with the exception of November and December) two colony trays were taken to the field to attract phorids. These trays were agitated to elicit activity of the ants and placed 30 m apart in the shade on a lawn. During a 3 h period once a week, aspirator collections of all phorids present at trays were performed, alternating 10 min periods between trays. Collections were made from 600-1800 hours, such that at the end of the month each 3-h segment of the day was covered. Observations were not made at night because the flies were not active after dark. At the beginning and end of each sample period, a digital thermometer was used to register air temperature in the shade, and a sling psychrometer was used to determine relative humidity. Phorids were identified under a dissecting microscope using a key to neotropical species (Borgmeier 1969). Seven species of Pseudacteon occurred at colonies exposed in trays throughout the year, and collected individuals were deposited in the collection of H.G.F. Of the 492 females collected, P. tricuspis Borgmeier was the most abundant species (73%) followed by P. litoralis Borgmeier (17%), P. obtusus Borgmeier (4%), P. curvatus Borgmeier (3%), P. pradei Borgmeier (1.6%), P. solenopsidis Schmitz (1.2%), and P. wasmanni Schmitz (0.2%). Both P. tricuspis and P. litoralis were collected consistently throughout the year, unlike most of the other species (Pesquero et al. 1995). The proportion of all female Pseudacteon flies collected in hourly periods from sun- rise (Fig. 1) demonstrates that P. litoralis is active during early morning and late

456 Florida Entomologist 79(3) September, 1996 evening, while P. tricuspis is more active during mid-day. Consequently, the abun- dances of these were significantly negatively correlated (r = -0.991, Pearson r is reported here and subsequently). The logarithm of the number of P. tricuspis females captured was significantly related to air temperature (r = 0.646, P < 0.001), soil temperature (r = 0.668, P < 0.001), and humidity (r = -0.423, P = 0.028), but none of these were significantly correlated with the logarithm of females for P. litoralis (r = -0.055, r = -0.188, and r = -0.101, respectively). Differences in activity patterns may reflect physiological limitations; however, the weak correlations found between measured environmental variables and the abundance of P. litoralis suggest that other factors are probably responsible for establishing observed diel patterns of abundance and activity. It is very likely that light intensity is used as a cue to regulate activity in this species. This does not discount the influence of light intensity, however, on the activity patterns of P. tricuspis. Unfortu- nately, light intensity was not measured during our studies.

Fig. 1. Daily activity patterns of Pseudacteon litoralis and P. tricuspis ﬂies collected attacking ﬁre ants during 10 months of observations in Rio Claro, São Paulo, Brazil. Note different scales on y-axes. Hourly means are shown ±SEM (n = 7 for hours 1-9 and n = 6 for hours 10-12.

Scientiﬁc Notes 457

These observations on daily patterning of activity by two sympatric species of Pseudacteon parasitoids of ﬁre ants suggest that more than one species may be needed for introduction in biological control programs. Furthermore, variation in daily patterning of ovipositional activity and worker-size selection may explain, in part, how a number of Pseudacteon species coexist as parasitoids of the same ant host. Portions of these observations were ﬁnanced by the USDA and the Brazilian Na- tional Science Council (CNPq). We thank Maria Thereza Fink, Nozar Pinto, and Mar- celo Schlindwein for their encouragement and assistance. Jim Hoy and Lloyd Morrison read the paper and provided a number of useful suggestions.

SUMMARY

Two phorid parasitoids of Solenopsis ﬁre ants in Brazil were active at different times of the day. Pseudacteon litoralis was most active in the early morning and evening while P. tricuspis was most active during the afternoon.

REFERENCES CITED

DISNEY, R. H. L. 1994. Scuttle flies: the Phoridae. Chapman & Hall, London. BORGMEIER, T. 1969. New or little-known phorid flies, mainly of the neotropical region (Diptera, Phoridae). Studia Entomol. 12: 33-132. CAMPIOLO, S., M. A. PESQUERO, AND H. G. FOWLER. 1994. Size-selective oviposition by phorid (Diptera: Phoridae) parasitoids on workers of the fire ant, Solenopsis saevissima (Hymenoptera: Formicidae). Etologia 4: 85-86. PESQUERO, M. A., S. CAMPIOLO, AND H. G. FOWLER. 1993. Phorids (Diptera: Phoridae) associated with mating swarms of Solenopsis saevissima (Hymenoptera: For- micidae). Florida Entomol. 76: 179-181. PESQUERO, M. A., H. G. FOWLER, S. CAMPIOLO, AND S. D. PORTER. 1995. Seasonal activity of species of Pseudacteon (Diptera: Phoridae), parasitoids of fire ants (So- lenopsis) (Hymenoptera: Formicidae) in Brazil. Cientifica (in press). PORTER, S. D., H. G. FOWLER, AND W. P. MACKAY. 1992. Fire ant mound densities in the United States and Brazil (Hymenoptera: Formicidae). J. Econ. Entomol. 85: 1154-1161. PORTER, S. D., R. K. VANDER MEER, M. A. PESQUERO, S. CAMPIOLO, AND H. G. FOWLER. 1995a. Solenopsis (Hymenoptera: Formicidae) fire ant reactions to attacks of Pseudacteon flies (Diptera: Phoridae) in southeastern Brazil. Ann. En- tomol. Soc. America 88: 570-575. PORTER, S. D., H. G. FOWLER, S. CAMPIOLO, AND M. A. PESQUERO. 1995b. Host speci- ficity of several Pseudacteon (Diptera: Phoridae) parasites of fire ants (Hy- menoptera: Formicidae) in South America. Florida Entomol. 78: 70-75. TRAGER, J. C. 1991. A revision of the fire ants, Solenopsis geminata group (Hy- menoptera: Formicidae: Myrmicinae). J. New York Entomol. Soc. 99: 141-198. WILLIAMS, D. F., AND W. A. BANKS. 1987. Pseudacteon obtusus (Diptera: Phoridae) attacking Solenopsis invicta (Hymenoptera: Formicidae) in Brazil. Psyche 94: 9-13.

458 Florida Entomologist 79(3) September, 1996

POTENTIAL OF BEAUVERIA BASSIANA AND PAECILOMYCES FUMOSOROSEUS (DEUTEROMYCOTINA: HYPHOMYCETES) AS BIOLOGICAL CONTROL AGENTS OF THRIPS PALMI (THYSANOPTERA: THRIPIDAE)

1 1 2 A. CASTINEIRAS , J. E. PEÑA1, R. DUNCAN AND L. OSBORNE 1Tropical Research and Education Center University of Florida, IFAS 18905 SW 280th St. Homestead Florida 33031

2Central Florida Research and Education Center University of Florida, IFAS 2807 Binion Road, Apopka 32703-9598

The melon thrips, Thrips palmi Karny, is one of the most important vegetable pests damaging pepper, eggplant, bean and cucurbits in South Florida. Eggs are laid in the leaf tissue. Life cycle consists of two aerial feeding stages and two subterranean non-feeding stages. Adults emerge from the soil and move to the leaves of the host plant (Walker 1992). Literature on biological control of thrips is mostly about predators (Riudavets 1995) and parasitoids (Loomans et al. 1995). There have been only a few reports on the efficacy of entomopathogenic fungi. Neozygites parvispora (MacLeod & Carl) Rem. & Keel. (Saito et al. 1989) and Verticillium lecanii (Zimm.) Viégas (Saito 1992) have been documented infecting T. palmi in greenhouses in Japan. Also, an unidentified species of the genus Hirsutella Patouillard was found in Trinidad, British West Indies, infecting approximately 80% of T. palmi populations in the field (Hall 1992). To determine the potential of Beauveria bassiana (Bals.) Vuill. and Paecilomyces fumosoroseus (Wize) as biological control agents of T. palmi, strains BbH and BbHa of B. bassiana and strain 97 of P. fumosoroseus (Table 1) were tested. Beauveria bassiana and P. fumosoroseus inocula were cultured on Dodine medium (Oatmeal agar plus 0.8 g dodine and 0.2 g tetracycline) and grown on rice in an incubator at 26 ± 1°C, 75-85% RH and a photoperiod of 12:12 (L:D). Green bean plants (Phaseolus vulgaris L. cv. Opus) were grown singly in pots in a greenhouse at 26 ± 2°C and 80 ± 5% RH. When plants had 3 to 4 leaves, they were in- oculated with 50 T. palmi larvae taken from a laboratory colony.

TABLE 1. STRAIN ORIGIN OF BEAUVERIA BASSIANA AND PAECILOMYCES FUMOSOROSEUS TESTED ON THRIPS PALMI.

Strains Isolated from Locality

B. bassiana BbH Cosmopoltes sordidus Germ. Homestead, Florida (Coleoptera: Curculionidae) B. bassiana BbHa Bephrateloides cubensis (Ashm.) Homestead, Florida (Hymenoptera: Eurytomidae) P. fumosoroseus 97 Mealybugs Apopka, Florida (Homoptera: Pseudococcidae)

Scientiﬁc Notes 459

Four groups of 10 infested bean plants were tested. Three groups of plants were treated with the entomopathogenic fungi, each one with a different strain, by spraying one ml of conidial suspensions (2.0 × 108 conidia per ml) on each plant. High conidial concentrations were used expecting a high mortality on T. palmi. Conidial suspensions were obtained by washing 100 g of rice with mycelium in 400 ml of sterile 0.05% Tween 80 water solution. One group of plants (control) was sprayed with sterile 0.05% Tween 80 water solution. Plants were maintained in the same greenhouse and conditions for 24 h, then one leaf of each plant was placed in one 135 mm petri dish lined with moist filter paper. Plates were sealed with parafilm and incubated for 72 h at 26 ± 1°C and a photoperiod of 12:12 (L:D). Infection by the fungus was confirmed by microscopic examination of mycelium visible externally on the insect cuticle. Percent mortality was calculated. On the same days infected larvae on incubated leaves were counted, a sample of one leaf was taken from each plant in the greenhouse and checked for infected thrips larvae. Data were analyzed using Statistical Analysis System general linear models (SAS 1985) for balanced ANOVA. Means were separated by a Waller-Duncan k-ratio t-test. No infected insects were found on the non-incubated leaves taken from the greenhouse. On the incubated leaves, B. bassiana BbHa caused the highest mortality but the percent of infected larvae was rather low (Table 2). It has been documented that a very high relative humidity (95%) is necessary for conidial germination of entomopathogenic fungi (Ramoska 1984, Walstad et al. 1970) and the average relative humidity in the greenhouse was below 85%. P. fumosoroseus 97 was not a good candidate for biological control of T. palmi because only a few insects (0.20%) were infected by the fungus. Because B. bassiana BbHa was the strain that induced the highest mortality on the thrips in the previous experiment, it was further tested in the soil. Bean plants were grown on PRO-MIX BX® (Les Tourbières Premier Ltée, Rivière- du-Loup, Québec, Canada) potting soil (75-85% Canadian Sphagnum Peat Moss, pH=6.5) in 49×34×5 cm plastic flats in a greenhouse (26 ± 2°C and 80 ± 5% RH) until 3 to 4 leaves developed. Plants were infested by placing bean leaves on top of them with T. palmi taken from a bean field in Homestead, Florida. Potting soil was infested with thrips pupae ten days later with plants that were cut and left to dry on the flats. The dried plants were removed from the flats 4 days later. Eight flats taken at random were sprayed with 100 ml of a spore suspension (3.1 × 108 spores per ml) obtained as described before, and eight flats also taken at random (control), were sprayed with 100 ml of sterile 0.05% Tween 80 water solution. Mean

TABLE 2. PERCENT MORTALITY OF THRIPS PALMI LARVAE TREATED WITH BEAUVERIA BASSIANA AND PAECILOMYCES FUMOSOROSEUS.

Entomopathogens % Mortality ± S.E1

B. bassiana BbHa 24.23 ± 0.84 a B. bassiana BbH 12.90 ± 0.27 b P. fumosoroseus 97 0.20 ± 0.02 c Control 0.00 ± 0.00 c

1Means followed by the same letter are not signiﬁcantly different according to a Waller-Duncan k-ratio t-test on arcsine transformed data (p>0.001, k-ratio=100). Untransformed means are presented.

460 Florida Entomologist 79(3) September, 1996 temperature and relative saturation value of the soil (monitored daily) were 22.6 ± 2°C and 47.5 ± 3% RS, respectively. Soil fungus concentration was determined 24 h after treatment by taking 5 g soil samples from each flat. Samples were suspended in 30 ml of sterile 0.05% Tween 80 water solution. Serial suspensions (10-1 to 10-8) were plated on Dodine medium and incubated as in the previous experiment. The number of colony forming units (CFU) was determined after 6 days. The effect of the pathogen on thrips survival was evaluated by placing one ground emergence trap (Peña & Duncan 1992) per flat and counting the number of emerging adults at 3, 4, 6, 9, and 12 days after treatment. Traps consisted of an opaque vinyl chloride pipe (20.5 cm diam, 15 cm high) covered with a transparent plastic plate coated with Tanglefoot® (The Tanglefoot Co.Grand Rapids, MI). New plates with Tan- glefoot® were placed on top of the pipes after each counting. Means were separated using Statistical Analysis System t-test procedures (SAS 1985). Concentration of the fungus in the potting soil 24 h after spraying was 1.2 × 107 CFU per ml. The number of adult thrips caught in the traps from sprayed flats was always significantly lower than in control flats (Table 3). Emerging adult thrips populations were 50.12%, 53.03% and 40.09% lower in the treated flats on the third, fourth and sixth days after spraying compared with the controls. Because all replicates were conducted concurrently under the same conditions, and flats were taken randomly, differences in adult emergence can be attributed to the action of B. bassiana BbHa in the soil. This study shows that there may be potential for epizootics of B. bassiana in populations of T. palmi under certain conditions. Beauveria bassiana is a soil fungus and soil provides environmental conditions favorable to conidia (Gaugler et al. 1989). Be- cause high aerial populations of the melon thrips are related to low rainfall periods (Etienne et al. 1990, Cooper 1991) and fungal pathogens need high relative humidity (Ramoska 1984, Walstad et al. 1970), the soil inhabiting pupal stage seems to be the most susceptible to fungal infection. However, much more information is needed to determine the impact of entomopathogens on pupal mortality, and on the frequency of pathogen inoculations to effectively reduce T. palmi populations in the field. Thanks to R. M. Baranowski (Tropical Research and Education Center, University of Florida, Homestead, FL) and Roberto Pereira (Dept. of Entomology and Nematol- ogy, University of Florida) for reviews of the earlier version of this manuscript. This is Florida Agricultural Experiment Station Journal No. R-04892.

TABLE 3. CUMULATIVE NUMBER OF ADULT THRIPS PALMI EMERGED FROM FLATS TREATED WITH BEAUVERIA BASSIANA BBHA AND UNTREATED (CONTROL).

Emerged Adults ± S.E.1

Days after Treated with B. Untreated t-value Treatment bassiana BbHa (Control) (df =14) Sig. level

3 97.87 ± 15.81 196.25 ± 15.81 2.28 P < 0.03 4 125.00 ± 20.15 266.50 ± 45.05 2.86 P < 0.01 6 254.62 ± 41.35 431.37 ± 53.16 2.62 P < 0.02 9 413.75 ± 48.14 587.75 ± 59.88 2.26 P < 0.04 12 429.62 ± 47.64 612.62 ± 63.27 2.31 P < 0.03

Scientiﬁc Notes 461

SUMMARY

The potential of Beauveria bassiana (strains BbH and BbHa) and Paecilomyces fumosoroseus (strain 97) as biological control agents of the melon thrips, Thrips palmi, was studied in greenhouse experiments. Twenty-four percent mortality was found on thrips larvae treated with B. bassiana BbHa but infection only developed when leaves with larvae were incubated after being sprayed with the pathogen. Mortality by P. fumosoroseus was very low (0.20%). A reduction of 50% in adult emergence was observed when potting soil with pupae was sprayed with B. bassiana BbHa.

REFERENCES CITED

COOPER, B. 1991. Status of Thrips palmi (Karny) in Trinidad. FAO Plant Protection Bulletin. 39: 45-46. ETIENNE, J., J. GUYOT, AND X. VAN WATERMEULEN. 1990. Effect of insecticides, predation, and precipitation on populations of Thrips palmi on aubergine (eggplant) in Guadeloupe. Florida Entomol. 73: 339-343. GAUGLER, R., S. D. COSTA, AND J. LASHOMB. 1989. Stability and efficacy of Beauveria bassiana soil inoculations. Envirom. Entomol. 18:412-417. HALL, R. A. 1992. New pathogen on Thrips palmi in Trinidad. Florida Entomol. 75: 380-383. LOOMANS, A. J. M., AND J. C. VAN LENTEREN. 1995. Biological control of thrips pests: a review on thrips parasitoids. Wageningen Agric. Univ. Papers. 95: 89-200. PEÑA, J. E., AND R. DUNCAN. 1992. Sampling methods for Prodiplosis longifila (Diptera: Cecidomyiidae) in limes. Envirom. Entomol. 21: 996-1001. RAMOSKA, W. A. 1984. The influence of relative humidity on Beauveria bassiana infectivity and replication in the chinch bug, Blissus leucopterus. J. Invert. Pathol. 43: 389-394. RIUDAVETS, J. 1995. Predators of Frankiniella occidentals (Perg.) and Thrips tabaci Lind.: a review. Wageningen Agric. Univ. Papers. 95: 43-87. SAITO, T. 1992. Control of Thrips palmi and Bemisia tabaci by a mycoinsecticidial preparation of Verticillium lecanii. Proceedings of the Kanto Tosan Plant Pro- tection Society. 39: 209-210. SAITO, T., T. KUBOTA, AND S. SHIMAZU. 1989. A first record of the entomopathogenic fungus, Neozygites parvispora (MacLeod and Carl) Rem. and Kell., on Thrips palmi Karny (Thysanoptera: Thripidae) in Japan. Appl. Entomol. and Zool. 24: 233-235. SAS INSTITUTE, INC. 1985. SAS User’s Guide: Statistics. SAS Inst. Cary, N.C. 957 pp. WALKER, A. R. 1992. Biology and population ecology, in D. J. Girling [ed.] Thrips palmi,a literature survey with an annotated bibliography. International Insti- tute of Biological Control. Ascot, Berks, UK. 37 pp. WALSTAD, J. D., R. F. ANDERSON, AND W. J. STAMBAUGH. 1970. Effects of environmental conditions on two species of muscardine fungi (Beauveria bassiana and Metarhizium anisopliae). J. Invert. Pathol. 16: 221-226.

462 Florida Entomologist 79(3) September, 1996

DIFLUBENZURON RESIDUE: REDUCTION OF DIAPREPES ABBREVIATUS (COLEOPTERA: CURCULIONIDAE) NEONATES

W. J. SCHROEDER U. S. Dept. of Agriculture, Agricultural Research Service Horticultural Research Laboratory 2120 Camden Road, Orlando, FL 32803

The sugarcane rootstock borer weevil, Diaprepes abbreviatus (L.), is a pest of sugarcane and citrus in the West Indies and Florida (Hall 1995). Previous tests at the U.S. Horticultural Research Laboratory in Orlando indicated that the insect growth regulator diflubenzuron (1-(4-chlorophenyl)-3-(2,6-difluorobenzoyl)urea) significantly reduced the reproductive potential of D. abbreviatus when applied to citrus foliage (Schroeder et al. 1976, Lovestrand & Beavers 1980, Schroeder et al. 1980). The effect was similar to that reported by Moore & Taft (1975) for the boll weevil, Anthonomus grandis Boheman. Eggs from treated weevils had embryonic development but larvae failed to hatch. Micromite®, a commercial product containing diflubenzuron, was registered in Florida for use on bearing citrus (May 1995) for control of the citrus rust mite, Phyllocoptruta oleibora (Ashmead). In previous studies, diflubenzuron was applied to citrus foliage and D. abbreviatus adults were allowed to feed and oviposit on the treated foliage (Schroeder et al. 1976). This study eliminates feeding as a factor and limits the effect of diflubenzuron to the residue present on the leaf surface used as the oviposition site. Adult D. abbreviatus were obtained from a laboratory colony maintained on artificial diet (Beavers 1983). Newly emerged adults (30 females and 10 males) were transferred to a 76 × 40 × 35 cm wood and screen cage with green beans and water. Sour orange, Citrus aurantium L., seedlings in 4 liter pots were maintained outside in full sun during March and April 1995 in Orlando, FL. The trees were sprayed to runoff with Micromite® 25W at 149 and 298 grams a.i./1000 liters with 1% 435-66 Citrus Spray Oil® (v/v) and water for the control trees. The rates represent one and two times the rate recommended for rust mite control. There were 10 trees for each of the 3 treatments. Five pairs of mature leaves were removed at random from each treatment, beginning 17 days after treatment and continuing weekly thereafter. The pairs of leaves were held together with a paper clip to form an oviposition site and the petioles were placed in a 30 ml cup filled with water. The cups were arranged at random in a 24 × 27 cm tray and placed into a cage with adult weevils for oviposition. There were 5 cups per each of the 3 treatments for each week. Leaves were removed after 4 days and each pair placed in a 100 ml vial for egg hatch (about ten days). Neonate larvae were counted for each treatment; the test was terminated after seven weeks. Arc- sin squareroot transformed data were subjected to ANOVA and means were separated by Student-Newman-Keuls multiple range test. There were 3027, 1867 and 13,212 neonate larvae recovered from leaves treated with the 1×, 2× rate and control, respectively. This is an average 77% and 86% reduction for all time periods with no significant difference between the two rates (P<0.05) (Fig. 1). Diflubenzuron affects reproduction of D. abbreviatus, and it should be used as part of an integrated pest management program. The citrus blue-green weevils, Pach- naeus opalus and P. litus have a similar life cycle and diflubenzuron may also affect reproduction for these species.

Scientiﬁc Notes 463

Fig. 1. Percent reduction in D. abbreviatus neonates from eggs laid on diﬂubenzu- ron treated foliage.

The research was funded in part by the Florida Citrus Production Research Advisory Council, Florida Department of Agriculture and Consumer Services—grant No. 942-43. Mention of trademark, warranty, proprietary product, or vendor does not constitute a guarantee by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products or vendors that may also be suitable.

SUMMARY The insect growth regulator diﬂubenzuron (Micromite®) signiﬁcantly affected the reproductive potential of D. abbreviatus for more than a month after application to citrus foliage.

REFERENCES CITED

BEAVERS, J. B. 1983. Biology of Diaprepes abbreviatus (Coleoptera: Curculionidae) reared on artificial diet. Florida Entomol. 65: 263-269. HALL, D. G. 1995. A revision to the bibliography of the sugarcane rootstalk borer weevil, Diaprepes abbreviatus (Coleoptera: Curculionidae). Florida Entomol. 78: 364-377. LOVESTRAND, S. A., AND J. B. BEAVERS. 1980. Effect of diflubenzuron on four species of weevils attacking citrus in Florida. Florida Entomol. 63: 112-115. MOORE, R. F., JR., AND H. M. TAFT. 1975. Boll weevils: Chemosterilization of both sexes with busulfan plus Thompson-Hayward TH-6040. J. Econ. Entomol. 68: 96-98. SCHROEDER, W. J., J. B. BEAVERS, R. A. SUTTON, AND A. G. SELHIME. 1976. Ovicidal effect of Thompson-Hayward TH-6040 in Diaprepes abbreviatus on citrus in Florida. J. Econ. Entomol. 69: 780-782. SCHROEDER, W. J., R. A. SUTTON, AND J. B. BEAVERS. 1980. Diaprepes abbreviatus: Fate of diflubenzuron on nontarget pests and beneficial species after application to citrus for weevil control. J. Econ. Entomol. 73: 637-638.

464 Florida Entomologist 79(3) September, 1996

A PHOTOGRAPHIC TECHNIQUE FOR CONSTRUCTING LIFE TABLES FOR BEMISIA ARGENTIFOLII (HOMOPTERA: ALEYRODIDAE) ON POINSETTIA

MARK S. HODDLE1, ROY VAN DRIESCHE1, JOHN SANDERSON2 AND MIKE ROSE3 1Department of Entomology, University of Massachusetts, Amherst, MA 01003

2Department of Entomology, Cornell University, Ithaca, NY 14853

3Department of Entomology, Texas A&M University, College Station, TX 77843

Whiteflies (Homoptera: Aleyrodidae) are serious pests in a variety of agricultural crops (Onillon 1990). Several species of whitefly have been the target of classical biological control efforts (Gould et al. 1992, Summy et al. 1984), while others have been subject to inundative biological control (Hoddle & Van Driesche 1996). A common crit- icism of biological control is the lack of quantitative evaluation studies after natural enemies have been released (Luck et al. 1988). Life table studies provide a powerful technique for such evaluations because they provide a detailed description of age specific mortality of individuals in the population (Carey 1993). When information on the pest’s fecundity is available, the effect of the natural enemy can be expressed in terms of its effect on the pest’s population growth rate (Van Driesche & Bellows 1996). Due to the sessile nature of immature whiteflies, life tables can be constructed from photographs of cohorts of nymphs on leaves (Gould et al. 1992, Summy et al. 1984). Summy et al. (1984) noted that the photographic technique has several advantages over sampling whitefly cohorts with a 10× hand lense: 1) reduced variability in sampling, 2) increased accuracy of lifestage determination, 3) improved accuracy as whitefly cohorts become larger, 4) decreased data collection time, and 5) permanent sampling record should verification of results be needed at a later date. Hoddle & Van Driesche (1996) used a numbering technique to follow the fates of individual whiteflies. This method had the following disadvantages when compared to the photography method: 1) numbering of whiteflies on leaves and data collection are very slow, 2) leaves can be damaged when whiteflies are numbered, 3) nymphs need to be adequately spaced so numbers can be written beside them, 4) numbers can fade and disappear over the course of the evaluation, 5) fate of individual nymphs cannot be verified at a later date, and 6) data cannot be re-analyzed to address different hypotheses. The Floricultural Program at the University of Massachusetts, Amherst, has been developing an integrated pest management program for Bemisia argentifolii Bellows & Perring (the silverleaf whitefly) on greenhouse grown poinsettia. One aspect of the project is evaluation of aphelinid parasitoids for B. argentifolii control. We elected to construct life tables for B. argentifolii on poinsettia in the presence and absence of aphelinid parasitoids using a photographic method. We felt it necessary to describe our photographic technique, modified from Gould et al. (1992) and Summy et al. (1984), in detail because instructions on how to establish whitefly cohorts, use the photographic method, and construct life tables from the resulting slides were not available in other publications, lacked sufficient detail to be of immediate use, or were impractical. The photographic technique described here should be appropriate for any invertebrate whose immature stages are sessile and found in large numbers, e.g. scales (Homoptera: Coccoidea), and data from photography should be amenable to time specific stage frequency analysis (Manly 1990).

Scientiﬁc Notes 465

To establish whitefly cohorts, we chose poinsettia plants whose petioles extended 1-2 cm beyond the rim of the pot so that the leaf to be photographed could be placed on a flat surface when the plant was laid on its side. One suitable leaf each on 10-12 plants per greenhouse was tagged. A clip cage modified from Mowry (1993) was placed on each tagged leaf and the cage perimeter marked with an indelible marker. To establish whitefly patches of different densities, one to four mating pairs of whiteflies were placed in each cage for 2-3 days (at 25°C) for oviposition, after which cage and adults were removed. The number of eggs within the marked perimeter of the cage was recorded using a dissecting microscope. After 7-10 days in the greenhouse, tagged leaves were examined for nymphal eclosion. The number of first instars that settled from eggs were counted, and photography in the greenhouse commenced. The part of the tagged leaf on which most nymphs settled was photographed. An area 35mm × 23mm around the settled nymphs was delineated using a photographic slide frame. The four inside corners of the slide frame were marked with a red indelible marker. The four red dots lined up with the camera viewing area when the recommended camera set up was used. A label was placed within the marked area on the leaf with the same number and color as the tag on the petiole. The label was made by placing tape on a microscope slide and cutting out small squares (5mm × 5mm) with a razor blade. Squares were numbered with an indelible marker and placed on the leaf (Fig. 1.) The camera used was a Nikon F3® outfitted with a 55mm macrolens, a SB21a macro speedlight®, one PK11a extension tube®, and Fuji Velvia® 50asa slide film. F- stop and aperture settings were at 16 and 22 respectively. To check the camera set up in the laboratory, the area enclosed by a slide frame was marked on a piece of graph

Fig. 1. A camera-ready poinsettia leaf.

466 Florida Entomologist 79(3) September, 1996 paper. The camera was positioned over the marked area and focused by moving the camera either toward or away from the marked area. (If the camera viewing area does not exactly match the marked area when focused, a template can be constructed from the number of squares enclosed by the viewing area on the graph paper to mark the area to be photographed instead of the slide frame.) Photography commenced immediately after nymphal eclosion and settlement in the greenhouse. The plant was placed on its side and the leaf positioned on a ﬂat surface (such as the bottom of a shallow box) so the underside of the leaf faced up. The leaf was held in place with small, ﬂat weights. The camera viewing area was aligned with the four red dots and focused by moving the camera toward or away from the leaf. Two photographs were taken of each cohort in case one of the photographs was unus- able; photography was repeated two times per week. Photography of cohorts ceased

Fig. 2. Leaf maps showing the fates of individual whitefly nymphs exposed to parasitoids on each photographic date. 1 = settled first instar, 2 = second instar, 3 = third instar, 4 = fourth instar, P = pupa, P* = parasitized pupa, P⇑ = emerged parasitoid, C = whitefly pupal case, ? = immature whitefly disappeared from leaf, and D = undetermined death of an immature whitefly.

Scientiﬁc Notes 467

TABLE 1. A SUMMARY LIFE TABLE CONSTRUCTED FROM BI-WEEKLY PHOTOGRAPHS OF WHITEFLY COHORT NUMBER THREE, AS MAPPED IN FIG. 2.

No. Alive No. Dying Stage in Stage in Stage Cause of Mortality

Eggs/crawlers 15 3 undetermined death: 3 Settled crawlers 12 3 undetermined death: 2 disappeared: 1 Second instar 9 2 undetermined death: 2 Third instar 7 Fourth instar 7 2 undetermined death: 1 disappeared: 1 Pupae 5 3 parasitized: 3 (wasps emerged) Adult Whiteﬂies 2 —

when all nymphs died, emerged as adult whiteflies, produced adult parasitoids or disappeared due to unknown causes. At each photographic session the date of photography, plant number (from the pot), film roll number, photograph number, and cohort number (from petiole tag or label on leaf) were recorded. This system provided a cross referencing scheme should labels fall off either the leaf or petiole. When the film was sent for developing, the number of each roll of film was written on the processor’s en- velop to determine date of photography. The film processor was instructed to number each slide as it was developed so that cohort number could be determined should the label be illegible. The photographic date and cohort number were recorded on each slide, and slides were catalogued according to cohort number. Slides were analyzed by cohort in chronological order using a backlit dissecting microscope (10×). The fate of each individual whitefly nymph was recorded on a leaf map drawn for each photographic date (Fig. 2). The number of nymphs entering each instar, the number dying in each instar, and cause of mortality for each individual was summarized (Table 1.) Occasionally, some nymphs from the original egg mass developed outside the photographed area. The theoretical number of eggs required to have produced the number of settled first instars actually photographed was calculated by dividing the number of first instars photographed by the proportion of total nymphs that emerged and settled on the leaf. Individual cohort life tables were combined to form summary life tables for treatments or time periods as needed for specific experiments. Support for this work was provided by the University of Massachusetts IPM Pro- gram and USDA/NRICGP grant # 9402481. Vincent D’Amico III provided the artwork.

SUMMARY A technique for establishing whiteﬂy cohorts, photographing settled whiteﬂy nymphs, and life table construction from photographed nymphs on greenhouse grown poinsettia is presented.

REFERENCES CITED

CAREY, J. R. 1993. Applied demography for biologists with special emphasis on insects. Oxford University Press, Oxford. 206 pp.

468 Florida Entomologist 79(3) September, 1996

GOULD, J. R., T. S. BELLOWS, JR., AND T. D. PAINE. 1992. Evaluation of biological control of Siphoninus phillyreae (Haliday) by the parasitoid Encarsia partenopea (Walker), using life-table analysis. Biological Control 2: 247-265. HODDLE, M. S., AND R. VAN DRIESCHE. 1996. Evaluation of Encarsia formosa (Hy- menoptera: Aphelinidae) to control Bemisia argentifolii (Homoptera: Aley- rodidae) on poinsettia (Euphorbia pulcherrima): A life table analysis. Florida Entomologist 79: 1-12. LUCK, R. F., B. M. SHEPARD, AND P. E. KENMORE. 1988. Experimental methods for evaluating arthropod natural enemies. Ann. Rev. Entomol. 33: 367-391. MANLY, B. F. J. 1990. Stage-structured populations: sampling, analysis and simula- tion. Chapman and Hall, London. 187 pp. MOWRY, T. M. 1993. A method for confining small insects to plant surfaces. J. Agric. Entomol. 10: 181-184. ONILLON, J. C. 1990. The use of natural enemies for the biological control of whiteflies, pp. 287-313 in D. Gerling [ed.] Whiteflies: their bionomics, pest status and management. Intercept Ltd., Andover. 348 pp. SUMMY, K. R., M. R. DAVIS, W. G. HART, AND F. E. GILSTRAP. 1984. Use of close-up photography in non-destructive monitoring of citrus blackfly cohorts. J. Rio Grande Valley Hortic. Soc. 37: 55-60. VAN DRIESCHE, R. G., AND T. S. BELLOWS, JR. 1996. Biological control. Chapman and Hall, New York. 539 pp.

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468 Florida Entomologist 79(3) September, 1996

SCAPTERISCUS ABBREVIATUS (ORTHOPTERA: GRYLLOTALPIDAE), A MINOR PEST ON ST. CROIX, US VIRGIN ISLANDS

J. H. FRANK1 AND J. L. W. KEULARTS2 1Entomology & Nematology Department, University of Florida, Gainesville, FL 32611-0630, USA

2Cooperative Extension Service, University of the Virgin Islands, St. Croix, VI 00850

Incorrect identification of mole crickets in many West Indian publications during the last 100 years has caused confusion of two kinds. First, the zoogeography of mole crickets in the Caribbean is unclear although Nickle & Castner (1984) clarified identity of the species in Puerto Rico and other islands. Second, it is unclear which species, if any, are important pests on many of the islands. Scapteriscus didactylus (Latreille) is, or was, a major pest of many crops in Puerto Rico (Barrett 1902) and is a pest in parts of Hispaniola (Frank et al. 1987). It has been reported from the U.S. Virgin Islands of St. John and St. Thomas without indication of pest status, but not from St. Croix (Ivie & Nickle 1986). Adults can fly. Scapteriscus vicinus Scudder, an immigrant from South America, is a major pest of pasture- and turfgrasses and vegetable seedlings in the southeastern USA. There are no confirmed records of its existence in the West Indies, although for decades this name was used erroneously by agricultural entomologists for S. didactylus in Puerto Rico. Four specimens collected in Christiansted, St. Croix, in 1939 were reported to

Scientific Notes 469 belong to this species (Beatty 1944) but their identification is dubious, and their current location is unknown (Nickle & Castner 1984, Ivie & Nickle 1986). Adults can fly. Scapteriscus abbreviatus Scudder, an immigrant from South America, is a major pest of turfgrasses in Florida in the limited areas where it occurs. It was reported to occur in Puerto Rico without specific mention of its pest status (Wolcott 1936, Castner & Fowler). It was reported from St. Croix by Miskimen & Bond (1970), without indication of year of collection or pest status, but has not been reported from St. John or St. Thomas. Verification of its existence on St. Croix rests only on 3 specimens collected in Christiansted in 1940 (Nickle & Castner 1984, Ivie & Nickle 1986). Adults cannot fly. For 3 days in June 1991, we tried to augment the information for St. Croix provided by Ivie & Nickle (1986) in three respects. Which, if any, mole cricket species is a pest on St. Croix? Does a flying mole cricket (S. didactylus or S. vicinus) occur on St. Croix? Does S. abbreviatus, which apparently has not been collected for over 50 years, still occur there? We visited commercial agricultural areas (mainly tomato fields) and horticultural areas (nurseries for ornamental plants). We examined lawns and flower beds in urban areas and all three of the island’s golf courses: Buccaneer Golf Course (BGC), Caram- bola Golf Club (CGC), and The Reef Golf Course (RGC). We looked for mole crickets along banks of streams and marine estuaries. We operated an UV light trap at dusk. We examined only sites that appeared suitable for habitation by mole crickets. When we discovered mole cricket galleries, we used highly diluted detergent in an attempt to flush mole crickets to the soil surface. We also examined the insect collection of the Cooperative Extension Service, University of the Virgin Islands, and of the USDA- ARS, both at Kingshill, St. Croix. Soil conditions in June 1991 were extremely dry because of prolonged drought. Vegetables, ornamental plants, and the turf of two of the golf courses were kept alive by irrigation. At one of the golf courses (RGC), the turf of the fairways was dead due to irrigation restrictions. There was a trickle of water in a stream in the northwest of the island; streams elsewhere were dry. Saltwater intrusion into wells and the low water level in a reservoir were the immediate causes of the irrigation restrictions. However, long-term decline (over decades) in the island’s water supply has caused loss of most of its agriculture; where once grew fields of sugarcane is now semi-arid brush. Long-term and short-term drought can be expected to reduce mole cricket populations or even eliminate them locally. We found mole crickets only in the moister, northwestern part of St. Croix and only at two localities. All were S. abbreviatus, and 29 specimens were collected. Five were on a golf course (CGC) in sand and turf around the perimeter of a sand trap (damage to the turf was trivial), and seventeen were in drying mud in a low-lying wet area. Seven were found on a farm growing vegetables and ornamental plants; they were in a raised bed of sweet pepper seedlings where the heavy field soil had been mixed with sand, and the bed had been wetted by irrigation. Pepper seedlings were about 15 cm high, and about a third of them had been killed, evidently by mole crickets. No mole crickets were collected at UV light. None was found in the insect collection of the Cooperative Extension Service or of the USDA at Kingshill, St. Croix. Apart from Beatty’s (1944) statement, there is no evidence of a flying mole cricket on St. Croix. We think it likely that all the Scapteriscus specimens collected in Christiansted in 1939-1940 belonged to S. abbreviatus, but that Beatty (1944) erroneously reported some of them as S. vicinus and did not mention S. abbreviatus because he (correctly) believed all specimens to be conspecific. We thank J. L. Castner and T. J. Walker (Univ. Florida) for criticizing a draft of this note. This is Florida Agricultural Experiment Station journal series no. R-05047.

470 Florida Entomologist 79(3) September, 1996

SUMMARY

A search for mole crickets on St. Croix in June 1991 revealed only Scapteriscus abbreviatus, which was damaging vegetable seedlings and turf. This adventive (= non- indigenous) species must be considered only a minor pest because it occupied a very small area of the island and caused minor damage. Low soil moisture in most agricultural and horticultural areas of St. Croix probably restricts population spread.

REFERENCES CITED

BARRETT, O. W. 1902. The changa, or mole cricket (Scapteriscus didactylus Latr.) in Porto Rico. Puerto Rico Agric. Exp. Stn. Bull. 2: 1-19. BEATTY, H. S. 1944. The insects of St. Croix, V.I. J. Agric. Univ. Puerto Rico 28: 114- 172. CASTNER, J. L., AND H. G. FOWLER. 1984. Distribution of mole crickets (Orthoptera: Gryllotalpidae: Scapteriscus) and the mole cricket parasitoid Larra bicolor (Hymenoptera: Sphecidae) in Puerto Rico. Florida Entomol. 67: 481-484. FRANK, J. H., R. E. WOODRUFF, AND C. A. NUÑEZ. 1987. Scapteriscus didactylus (Or- thoptera: Gryllotalpidae) in the Dominican Republic. Florida Entomol. 70: 478- 483. IVIE, M. A., AND D. A. NICKLE. 1986. Virgin Islands records of the changa, Scap- teriscus didactylus (Orthoptera: Gryllotalpidae). Florida Entomol. 69: 760-761. MISKIMEN, G. W., AND R. M. BOND. 1970. The insect fauna of St. Croix, United States Virgin Islands. Scientiﬁc Survey of Puerto Rico and the Virgin Islands (New York Acad. Sci.) 13: 1-114. NICKLE, D. A., AND J. L. CASTNER. 1984. Introduced species of mole crickets in the United States, Puerto Rico and the Virgin Islands. Ann. Entomol. Soc. America 77: 450-465. WOLCOTT, G. N. 1936. “Insectae Borinquenses” a revised annotated check-list of the insects of Puerto Rico. J. Agric. Univ. Puerto Rico 20: 1-600.

Book Reviews 471

BOOK REVIEWS

DOWNIE, N. M., AND R. H. ARNETT, JR. 1996. The Beetles of Northeastern North America. The Sandhill Crane Press, Gainesville, FL. Vol. 1, p. i-xv, 16-880 (Introduc- tion; Suborders Archostemata, Adephaga, and Polyphaga, thru Superfamily Cantharoidea), Vol. 2, p. i-x, 891-1721 (Polyphaga: Series Bostrichiformia through Curculionoidea). Hardback. 17.8 × 25.4 cm (7 × 10 in.). ISBN 1-877743-11-9 (set). $175.

Two publications have been essential to every coleopterist, amateur or professional, interested in the beetle fauna of the continental USA. They are The Coleopter- ists’ Bulletin, founded by Ross Arnett in 1947 (the apostrophe was omitted later from the title), and The Beetles of the United States (a Manual for Identification) (BOTUS) written by Ross Arnett and published (first edition) in 1960-1963 by Catholic Univer- sity of America Press (now out of print). For those interested in the fauna of northeastern North America, Arnett has completed the hat trick by co-authorship of The Beetles of Northeastern North America (BONENA). Arnett has accomplished a major feat by publication of BONENA. I cannot name anyone who has done more for coleopterists in the USA. Now, is anyone brave enough to attempt a companion book on the Coleoptera of the southeastern USA? BONENA defines northeastern North America as Greenland, St. Pierre and Mique- lon, eastern Canada (Ontario eastward), and the northeastern states of the USA (CT, DC, DE, IL, IN, MA, MD, ME, MI, NH, NJ, NY, OH, PA, VT, and WI). Whereas BOTUS provided keys to adult beetles to the level of genus, BONENA provides keys to the level of species and is the only book to do so for all families of Coleoptera for that area. The only precedents in North America are W. S. Blatchley’s (1910) An Illustrated Descrip- tive Catalogue of the Coleoptera or Beetles known to occur in Indiana (The Nature Pub- lishing Co., Indianapolis) and M. H. Hatch’s (1953-1971) 5 volumes, The Beetles of the Pacific Northwest (University of Washington Press, Seattle). When Hatch (1953-1971) undertook a similar task, he based it upon his interpretation of the existing literature and specimens available to him, some of which seemed to belong to undescribed species. I am most familiar with his volume 2 (Staphylini- formia) in which he assigned 2 sections to be written by collaborators with specialist knowledge. These sections, and the rest of the book, were written with new generic di- agnoses, new keys, new illustrations (a very useful part of the book), and descriptions of new species. Even so, he found the huge subfamily Aleocharinae to be too compli- cated, needing too much study to resolve, so he omitted it. A quarter of a century later, some parts of Hatch’s works remain useful although no revisions were published. His volume 2 is now mainly of historical interest to specialists. This is because of subsequent revisionary studies of genera and tribes, revealing not only many additional species, but also that Hatch had inadvertently misinterpreted parts of the older literature. Hatch could only have avoided misinterpretation by examining type specimens and, therefore, would not have finished the work in his lifetime. BONENA begins with a manuscript by the late Norville Downie, which was completed by Downie with help from Arnett in 1970-1994. The authors had several advantages over Hatch. They adapted text and illustrations from BOTUS. They incorporated new information (from taxonomic revisions by many authors) which was published after the appearance of Hatch’s work, so they had less pioneering work to do than did Hatch. In the later years of their work, they had access to word-processing equipment, so they could more readily than Hatch incorporate additions and changes to produce an accurate, thorough, and up-to-date work.

472 Florida Entomologist 79(3) September, 1996

BONENA will age as new revisionary studies are published by specialists in several families. Aging can be corrected for by publication of revised subsequent editions of BONENA to maintain its currency. Its advantage is, or should be, that it combines the knowledge of thousands of earlier specialist publications and saves the purchaser from need to acquire this earlier literature. It is not entirely adequate, in my opinion, because it is inadequately illustrated. In some families of Coleoptera it will be possible to identify specimens using the unillustrated keys in BONENA. Yet, every modern revisionary study of Staphylini- formia includes not only illustrations of genitalic structure but, usually, other diag- nostic structures also, because these are necessary to reliable identification. BONENA has habitus illustrations of few genera and, unlike Hatch (1953-1971) and comparable European works, lacks illustrations of species-specific characters. There- fore, users of BONENA will have to acquire copies of the earlier publications in order to identify specimens in some families. It is possible that BONENA was not copiously illustrated in order to save the cost of space. A 50% expansion of space might have been necessary to provide an adequate number of illustrations. In a printed edition this would cause a 50% increase in publication cost and might deter buyers. If, however, the book were to be produced on CD- ROM, the expansion in space could have been incorporated at no additional publication cost and, in fact, a CD-ROM edition could have been produced for much less than the cost of the printed edition. That Arnett did not produce the book on CD-ROM (at lower cost) instead of on paper (at higher cost, even though price per page is modest compared with books from major publishers) suggests that he was concerned not only with preferences of potential buyers (for paper vs CD-ROM) but also with piracy; it would cost pirates less to copy a magnetic version than a printed version. It is possible that BONENA was not copiously illustrated because of the cost of producing new illustrations. It is a sad reflection on economics that such an important work could not afford to employ an illustrator. However, BONENA is based on earlier taxonomic works, most of whose authors would (in my opinion) have given permission to use their illustrations to the BONENA co-authors simply for an acknowledgment (a few of them evidently were contacted and did so). This would have solved much of the problem of illustration. Instead of contacting more authors for such permission, the BONENA authors seem to have relied largely on illustrations from BOTUS and on old illustrations with expired copyright. The illustrations from BOTUS that were included are useful and generally are well-reproduced in BONENA, but other illustrations have been taken from other sources and are of poor quality. Some are poorly reproduced (partially faded) or misaligned. Some have erroneous captions or are mis- placed. A bad example is 3 illustrations (p. 407, 427, 459) taken from Blatchley (1910 p. 483, 484, 462), who in turn had taken them from Erichson (1840, Genera et Species Staphylinorum . . . plate 2). Erichson and Blatchley ascribed them to Lispinus an- gustatus Erichson, Glyptoma crassicorne Erichson, and Megalopinus cephalotes (Erichson), which do not occur in North America, but BONENA ascribes them to the North American Scopaeus exiguus Erichson (wrong subfamily), Thoracophorus costa- lis (Erichson) (wrong genus), and Megalopinus caelatus (Gravenhorst) (wrong species). The illustrations on p. 376 and p. 385 are transposed. The text should have been checked more thoroughly for typographical errors. Er- rors are not just in figure captions (e.g., p. 369, 407, 425, 427, 880) and literature cited (e.g., all 5 publications by Puthz, p. 562) and names of organisms and authors, but in ordinary English words and even in the eulogy to Norville Downie. There are more such errors than in all of Hatch’s 5 volumes although they are now easier to detect, by word-processor.

Book Reviews 473

BONENA does not contain descriptions of previously-undescribed species. It adapts keys from the existing literature. Keys adapted from recent taxonomic revisions should work, though in some places with difﬁculty because of inadequate illustration. Keys adapted from 19th century literature, for example from publications by T. L. Casey, may not work well, if at all, but they do represent the current status of knowledge of some taxa. Despite these criticisms, I would buy a copy of the book although I have little call to identify specimens from the Northeast. I would do this because of the wealth of information it provides, even though some of the information is incorrect in this edition. Additions and corrections can be made in a second edition: users can help by writing to Arnett to point out errors. He has done a valuable service to coleopterists by producing it, and they should reciprocate by buying it and helping him to improve it. The book ought to become and remain the standard work on Coleoptera of the region in- deﬁnitely. Ross: in whom will you vest copyright of your work? Will you make it possible for impecunious coleopterists to continue your tradition, for example a 6th edition of Downie, Arnett et al. in 2050, or will you, by copyrighting your work, make them start from scratch? J. H. Frank Entomology & Nematology Department University of Florida Gainesville, FL 32611-0630

Book Reviews 473

HOY, M. A. 1994. Insect Molecular Genetics. Academic Press; San Diego. xxii + 546p. ISBN 0-12-357490-0. Paperback. $39.95.

This book introduces entomologists who have had limited training in genetics, bio- chemistry, and molecular biology to the field of insect molecular genetics. It was written for the expressed purpose of making this complex discipline both accessible and palatable to the DNA novice and, to this end, the author succeeds smashingly. The 15 chapters of this book are organized in 3 parts. Each chapter begins with an overview of the material to be covered and ends with relevant references. Part I, Genes and Genome Organization in Eukaryotes (five chapters), presents the “nuts and bolts” of molecular genetics and establishes a general framework for understanding the more difficult concepts developed in later chapters. The first 3 chapters teach the “ABC’s” of DNA and cell biology. Chapters 4 and 5 focus specifically on insect ge- nomes and provide basic information on chromosomal and mitochondrial DNA, genes, genome organization, and development in insects. As a matter of necessity, many of the examples used throughout this book to illustrate various aspects of insect molecular genetics are drawn from the Drosophila literature. The author has made a special effort, however, to emphasize recent molecular studies on other insect species. Part II, Molecular Genetic Techniques (also 5 chapters), describes the methods used by molecular biologists to manipulate and analyze nucleic acids. Chapters 6-9 cover the fundamentals of DNA cloning, sequencing, and the polymerase chain reac- tion (PCR). Essential concepts are portrayed by simple flow charts, tables, and dia- grams, and many of the procedural details are presented in “cook book” format. This wealth of technical information is presented in such an organized and straightfor- ward manner that the DNA neophyte should be able to digest the material without feeling overwhelmed or intimidated. The final chapter of Part II (Chapter 10) provides general information on the use of a transposable element (P-element) for transferring modified genes to Drosophila and, thereby sets the stage for later discussions on insect genetic engineering.

474 Florida Entomologist 79(3) September, 1996

The “meat” of the book from the entomological perspective is contained in Part III (Applications to Entomological Problems). This part builds on the conceptual foundation that was established in previous sections and offers something for the experienced and budding insect molecular geneticist, alike. The material presented is a blend of what has been done in the past, present research needs, and what could be accomplished in the future. The molecular details of sex determination and behavior in insects are explored in Chapters 11 and 12. Chapters 13 and 14 describe various molecular genetic techniques, explain how these molecular tools have been used to an- swer questions relevant to insect systematics and ecology, and discuss which type of molecular analysis is best suited for particular applications. As a finale, Chapter 15 takes the reader on an entomological tour of “Jurassic Park”. It is an imaginative ex- position on the future role of genetic engineering for modifying both pest and beneficial arthropods for use in pest management programs. A comprehensive glossary and a brief historical summary of significant events and accomplishments in genetics and molecular biology are included at the end of the book. As the author points out in the preface, entomologists have been reluctant to em- brace the discipline of molecular genetics, primarily because of fears about technical difficulty and a complete inability to relate to the Drosophila literature. This attitude is certainly understandable, considering the fact that many entomologists have had little or no training in genetics and/or molecular biology. This book cracks opens the molecular door and gives those entomologists who are bold enough to peek inside a glimpse of how these powerful techniques can be used to study practical problems in insect systematics, ecology, physiology and pest management. The motivational carrot has been offered. It is now up to the entomological community to pull the cart forward. Sharon E. Mitchell USDA/ARS Genetic Resources Conservation Unit University of Georgia Agricultural Experiment Station Griffin, GA 30223-1797

474 Florida Entomologist 79(3) September, 1996

DRAKE, V. A., AND A. G. GATEHOUSE (eds.). 1995. Insect Migration: Tracking Re- sources Through Space and Time. Cambridge University Press, Cambridge. xvii + 478 p. ISBN 0-521-44000-9. Hardback. $74.95.

Migration is a fundamental component of many insect life cycles, and multidisci- plinary studies have begun to clarify some major deficiencies in our understanding of it. Two recent books succeed in summarizing these studies and fitting them into patterns that are roughly complete or at least have the missing pieces identified. One is by Hugh Dingle and deals with all of animal migration (Migration: the Biology of Life on the Move, Oxford Univ. Press, New York, 1996, 474 p.). The other, the subject of this review, has 41 authors and deals only with insects. Insect Migration stems from a symposium at the XIX International Congress of Entomology in Beijing (1992). The coherence of its 21 chapters and the currentness of its references demonstrate the editors’ success in persuading the symposium participants to modify and update their contributions. The book has a slight geographical bias toward eastern Asia and Australia meant to counterbalance the biases toward Af- rica and North America of previous reviews. (Little is reported about insect migration in South America, but little is known.) The species discussed usually migrate above their flight boundary layer, where wind direction is the principal determinant of di-

Book Reviews 475 rection of movement. Weak fliers have little choice but to use the wind for transport; strong fliers, such as Monarch butterflies and desert locusts, can progress in any direction near the ground or they can take advantage of favorable winds at higher altitudes and travel fast at low cost. Many migratory species are most damaging in geographic areas they cannot oc- cupy continuously because temperature or moisture is only seasonally or erratically favorable. One theme of the book is the evolutionary dilemma posed by insects that move into temporarily favorable habitats from which neither they nor their descen- dants are known to return (the so-called “pied piper” effect, proposed by Rabb & Stin- ner in 1979). Such one-way migration can be viewed as an inadvertent consequence of the insects using uncertain winds as a transport system, or it can be viewed as a consequence of our knowing too little about the insects’ movements. Indeed, in a few cases further study has revealed return movements in what were thought to be pied piper species. In other cases, a nomadic life style may be a means of continually exploiting habitats of erratic suitability. Insect Migration has three main groups of chapters. The first and largest group describes insect migration in relation to weather and climate, with one or more chapters on Africa and Europe, North America, eastern Asia, and Australia. In West Africa seasonal reversal of windborne migration has been demonstrated for bugs, moths, and flies as well as several species of grasshoppers. Much migration in Africa is more complex and involves seasonally changing wind patterns in area of shifting habitat suitability. The complexity of windborne migration is well illustrated by studies of desert locust (Schistocerca gregaria) and African armyworm (Spodoptera exempta). Their movements cannot be simply summarized, and there is no easily grasped return of migrant genes from their source areas. Some migrations are fatal, with insects carried into deserts, poleward in autumn, and out to sea. Yet staying put does not insure survival either. Eastern North America is much more favorable for regular to-and-fro seasonal migration than is Africa and Europe, there being no Sahara or Mediterranean. Spring poleward migrations are on northward, fast-moving, low-level jets at altitudes of 200 to 1000 m and take 2 to 3 nights. Autumn equatorward migrations are nearer the surface (100-300m) on slower southward airflows and are limited by how rapidly temperatures fall behind the front. Passage of successive fronts may be required for migrants to reach overwintering areas. Circumstantial evidence supports a north-in-spring, south-in-fall migration for many pest species. Such migrations have been confirmed experimentally for potato leafhopper (Empoasca fabae) and black cutworm (Agrotis ipsilon). Two planthoppers in eastern Asia migrate northward and northeastward in spring and summer attacking rice in areas where they cannot overwinter. Some migration to the southwestward in autumn has been detected, but whether these are significant contributors to the gene pool of individuals that move northeastward the following year is uncertain. Three chapters deal with migrations of the oriental armyworm (My- thimna separata) in northeastern China, Korea, and Japan. In China there seems to be enough return migration to make the northward migrations adaptive. Whether the outbreaks in Korea and northern Japan are an adaptation is not addressed. Three chapters deal with important Australian migrants: common armyworm (Mythimna convecta), native budworm (Helicoverpa punctigera) and Australian plague locust (Chortoicetes terminifera). In each case adaptive return migration is possible but unproved. Especially in the case of the native budworm, which is adapted to the ephemeral environments of the arid inland, migration may be the only way to maintain a permanent, though nomadic, population.

476 Florida Entomologist 79(3) September, 1996

A second group of chapters concerns adaptations for migration. A. G. Gatehouse and X. -X. Zhang review the literature on potential for migration, including when migration occurs relative to reproduction, reproductive status of migrants sampled during flight, duration of pre-reproductive period, and free or tethered flight performance. They conclude that not much is known but that females of some species vary extensively in what it takes to initiate facultative migration and in migratory potential itself. K. Wilson outlines types of temporal and spatial variation in habitat and their expected effect on the evolution and maintenance of migratory potential. He reports that many studies suggest adaptive variation in migratory potential, but that few provide a convincing association with habitat heterogeneity and these mostly with temporal heterogeneity. J. Colvin describes laboratory studies of the genetics and ontogeny of two correlates of migratory potential in the cotton bollworm (Helicov- erpa armigera): duration of tethered flight and of the pre-reproductive period. J. N. McNeil et al. discuss the physiological integration of migration in Lepidoptera—specifically, the roles of juvenile hormone titer and the accumulation and mobilization of lipids. R. Dudley, in a chapter dealing with the aerodynamics and energetics of migratory flight, notes that data on free flying migrants are few and describes how a motor- boat can be used to collect such data on butterflies and day-flying moths as they migrate across large lakes. A third group of chapters treats forecasts of migrant pests. Two chapters in this group concern general problems and techniques of forecasting, specifically operational aspects and the use of computer-based geographic information systems. Other chapters deal with specific forecasting systems—for brown planthopper in China, rice planthoppers in Japan, locusts and grasshoppers in West Africa and Madagascar, and desert locust throughout its invasive area. The book concludes with a “holistic conceptual model” of insect migration by V. A. Drake, A. G. Gatehouse, and R. A. Farrow. They identify these four components of a migration system and list for each the processes that are involved: migration arena (the space, geographical and vertical, within which a population’s migration takes place), population trajectory (space/time population demography that results from migration), migration syndrome (the physiological, morphological, and behavioral traits that implement migration and determine the fitness of the migrants), and genetic complex (the genes that underlie the migration syndrome and their interactions and modes of inheritance). In this manner they succeed in describing, classifying, and il- lustrating the complexities of insect migration. Their conceptual model becomes an organizing framework for migration studies that accommodates what is known and reveals what is not. In words borrowed from the title, this book tracks studies of insect migration through space (worldwide) and time (the past 25 years). It summarizes and integrates the results of these studies and cites the important primary literature. Insect Migra- tion belongs on the bookshelf of those interested in insect ecology and life histories as well as those who must propose control strategies for migratory insect pests. Thomas J. Walker Department of Entomology and Nematology University of Florida Gainesville, Florida 32611-0620