Biology of eurytoma sivinskii, an unusual eurytomid (hymenoptera) parasitoid of fruit fly (diptera: tephritidae) pupae.

Translation provided by the authors.

Eurytoma sivinskii Gates and Grissell, an unusual eurytomid pupal parasitoid of Diptera, was recently discovered in the vicinity of Tejeria, Veracruz, Mexico, where it was recovered from pupae of the West Indian fruit fly, Anastrepha obliqua (Macquart) (Diptera: Tephritidae) (Gates & Grissell 2004). Most species of the large and widespread parasitoid genus Eurytoma attack gall-forming Cynipidae (Hymenoptera) and Diptera (Tephritidae and Cecidomyiidae) (DiGiulio 1997), and numerous other arthropod taxa and plants. In this case, instead of attacking hosts hidden within a plant structure, E. sivinskii forages for hosts (i.e., pupae) in and on the soil (Mena-Correa 2005).

Because of E. sivinskii's peculiar foraging behavior for tephritid hosts of economic importance, its potential as a biological control agent is under investigation (J. Mena-Correa, J. Sivinski, R. Ramirez-Romero, M. Gates & M. Aluja, unpublished). Some basic parameters of the parasitoid oviposition behavior and development in pupae of the Mexican fruit fly Anastrepha ludens (Loew) are determined herein. Type of parasitism (ecto- or endoparasitism), number of emerged adults per host, duration of immature stages, host stage attacked, parasitism related to female age, and propensity to superparasitize were studied. The locations of eggs laid by the parasitoid on the host pupae, location of adult parasitoid emergence through the host puparium, and how these locations are related to survival, developmental rate, competition among ovipositing females, and sex of the resulting adult were analyzed.

MATERIALS AND METHODS

Insect Cultures and Experimental Conditions

This study was carried out at the Instituto de Ecologia A.C., Xalapa, Veracruz, Mexico. Environmental conditions were kept at 27 [+ or -] 2[degrees]C, 75 5% RH, with a 12:12 h photoperiod. The E. sivinskii colony was maintained on 2-d-old pupae from an Anastrepha ludens colony kept for > 200 generations (Aluja et al. 2008). Pupae were placed on the surface of a layer of unsterilized clay soil and exposed to parasitoids for 6-8 d. This soil was gathered from the area where E. sivinski was originally discovered in Tejeria, Veracruz. Adults of E. sivinskii were placed in Plexiglas cages (30 x 30 x 30 cm) after emergence and fed ad libitum with honey and water. To avoid prior oviposition experience, parasitoids to be used in experiments were not exposed to host pupae.

Parasitoids were transferred from emergence cages to experimental cages (23 x 23 x 23 cm) by capturing them in glass vials. The experimental cages were prepared 24 h before the beginning of tests to minimize insect stress. Host pupae were always manipulated with flexible forceps to avoid damage. After pupae were parasitized, they were placed in plastic cups (200 mL) containing vermiculite moistened with water mixed with sodium benzoate at 2 g / L which prevented fungal contamination. Cups were covered with a lid.

Duration of Immature Stages

Development stages of E. sivinskii have been described elsewhere (Gates et al. 2008). Recorded images with Image Pro-Plus[R] software were examined to determine duration of each stage. All specimens were monitored until d 23 when adult females began to emerge as described by (Mena-Correa 2005).

Specimens used for the determination of the duration of immature stages stemmed from the 47-49th generations of our colony. To rear the parasitoids, we exposed 300 mL of A. ludens pupae (ca. 7000 pupae) to 3 E. sivinskii cohorts kept in 30 x 30 x 30 Plexiglas cages (200 females and 100 males per cage). Twenty-four h after exposure to parasitoids, pupae were removed and placed in 500 mL plastic vials with humidified vermiculite and covered with a lid. That same day (i.e., 24 h after exposure to parasitism) and from then on every 24 h over a 23-d period pupae were dissected individually in search of immature stages of the parasitoid. Twenty six eggs, 169 larvae (all stages represented), 40 prepupae, and 127 pupae were recovered. All specimens were placed in recently prepared Carnoy fixing solution (60 mL absolute alcohol, 30 mL chloroform and 10 mL acetic acid) for 24 h. Subsequently, they were washed and preserved in 70% EtOH in a hermetic glass flask until needed for evaluations. All dissections were made in a physiological solution to avoid tissue contraction (Martinez 2002).

Determination of the Host Stage Attacked

First-, second- and third-instars, as well as 3, 8, and 14-d-old A. ludens pupae at different developmental stages were exposed to E. sivinskii adults inside 200-mL plastic cups. Twenty-five A. ludens larvae or pupae were presented to E. sivinskii cohorts of 10 females and 5 males (8-d-old) for 5 h. Each treatment was replicated 5 times.

Rates of parasitism among treatments were compared via a one-way analysis of variance (Zar 1998) with developmental stage as the independent factor. Consecutively, we compared the rates of parasitism in host pupae alone with a one-way analysis of variance (Zar 1998) with pupal age as the independent factor. Data were arc-sin [square root of x]-transformed prior to analysis (Zar 1998). Parasitism Related to Female Age

To obtain E. sivinskii adults of every age, 150 mL of A. ludens pupae (3-to 5-d-old) were introduced daily into colony cages for 24 h. After this period, each batch of pupae was placed in a separate cage and newly emerged E. sivinskii adults were recovered every day and age-classified. Then, we tested 1-to 27-d-old females to determine if there was an age effect on ability to parasitize pupae. Individuals (10 females and 5 males) within each age class were exposed to twenty 3-d-old A. ludens pupae over a 15-d-period. Each age-treatment was replicated 5 times, and the number of parasitoids recovered from each treatment was recorded.

Rates of parasitism among treatments were compared via a one-way analysis of variance (Zar 1998) with female age as the independent factor. When significant differences were found, a Tukey's test was performed (Zar 1998). Data were arc-sin [square root of x]-transformed before analysis (Zar 1998).

Determination of Superparasitism

Different host pupal densities (i.e., 1, 5, and 10 pupae) were exposed for 5 h to adult parasitoid cohorts of 10 females and 5 males (6-to 9-d-old). After exposure, hosts were dissected with saline solution and a stereo-microscope and the number of eggs per host was recorded. Dissections were performed either the same day or 24 h after parasitoid exposure. Each treatment had 5 replicates.

We analyzed with a two-way ANOVA test the mean number of eggs per parasitized pupae, the rate of parasitized pupae (attacked pupae / exposed hosts), and the rate of pupae with more than 1 parasitoid egg (pupae with more than 1 egg / attacked pupae), with host density and exposure time as independent factors. The rates of parasitized pupae and of pupae with more than 1 egg were arc-sin transformed prior to analysis (Zar 1998). In addition, the randomness of egg distributions in 5- and 10-pupae lots was detemined by Chi-square tests, following the generation of expected egg/pupae frequencies through Poisson distributions (Zar, 1998). The nature of the distributions, random, dispersed or clumped, were then identified by dividing the variances by the means and comparing these ratios to a random distribution where the ratio = 1.0 (Zar, 1998).

Locations of Eggs and Adult Emergence from the Puparium After determining that multiple eggs were deposited in a single host, we ran an additional experiment to detect if the initial position of eggs in the host body influenced the parasitoid emergence, rate of parasitism, and sex ratios. Four-d-old A. ludens pupae were exposed to 2 E. sivinskii cohorts (total of 50 and 150 six- to 9-d-old females and males, respectively). Exposed host pupae were recovered after 24 h and examined under saline solution 24 h later to identify the number of parasitoid eggs and their position in the host body. Based on the number of parasitoid eggs and their position, host pupae were classified and then placed individually in plastic containers (diameter: 4 cm; height: 2 cm) with humidified vermiculite and covered with a lid that allowed airflow. Pupae were observed daily for 30 d and the number of parasitoids that emerged per host, their sex and the site of emergence on the host body (anterior, medium, or posterior) recorded.

The number of days it took adults to emerge was compared by a two-way ANOVA, with the number of eggs per host and the initial location of the eggs in the pupae as independent factors. Prior to this, data were mean-rank transformed. We compared the rates of emergence among emergence positions (i.e., location of emergence hole), considering the initial position of eggs and the number of eggs per host by chi-square tests. When significant differences were found, multiple two-by-two comparisons were performed with a significant threshold level, which was corrected according to the Dunn-Sidak method (Sokal & Rholf 1995). We compared parasitoid sex-ratios against a 1:1 ratio with a chi-square goodness-of-fit test. In addition, for each set of experiments, parasitoid sex-ratios were compared among pupae with different numbers of eggs and egg positions in the pupae, based on a chi-square test (Zar 1998). Finally, a possible correlation between the location of adult emergence (i.e., emergence hole), egg location, and number of eggs was tested by an r Spearman test (Zar, 1998).

RESULTS

Duration of Immature Instars and Stages

The life cycle (egg to adult) was completed in 23.1([+ or -] 2.1) d (mean [+ or -] S.E.) at 27 [+ or -] 2[degrees]C. Minimum and maximum durations for the egg stage were 0 (where 0 < 24 h) and 4 d. The larval stage lasted between 9 and 18 d. Minimum and maximum durations for the prepupal stage were 0 and 13 d, respectively, and those for the pupal stage were 8 and 14 d.

Minimum and maximum periods for larval instars were 1 and 4 d (first instar), 2 and 7 d (second and third instars), 0 and 8 d (fourth instar) and 4 and 13 d (fifth instar), respectively. The minimum and maximum periods of the pupal instars were 0 and 9 d (first instar), 8 and 14 d (second instar), 0 and 6 d (third instar), 4 and 7 d (fourth instar) and 0 and 3 d (fifth instar). Male emergence began on the 20t' d following parasitoid exposure. Females started to emerge 23 d after exposure.

Host Stage Attacked

Parasitism of E. sivinskii in pupae was higher than in larvae (F = 839.31; df = 1; P < 0.001). In no case were first and second instar larvae parasitized. However, 1 third-instar larva out of 625 exposed yielded a single parasitoid. When rates of parasitism at different pupal ages were compared, significant differences were observed (F = 24.43; df = 2; P < 0.001). Eight-d-old pupae exhibited the highest rate of parasitism (98.1 [+ or -] 4) (Fig. 1).

Parasitism Related to Female Age

There were no differences in rates of parasitism by female E. sivinskii of different ages (F = 1.284; df = 26; P = 0.181) (Fig. 2).

Type of Parasitism

Eurytoma sivinskii was clearly ectoparasitic since 100% of the eggs were laid within the internal cavity of the puparium and on the surface of the pupa.

Determination of Superparasitism

Eurytoma sivinskii will lay more than 1 egg per host. In the first experiment, of the total parasitized pupae, 25.42% contained 1 egg, 32.20% had 2, 16.94% had 3, 13.56% had 4, 6.77% had 5, 3.39% had 6, and 1.70% had 8 eggs. Host density did not affect the number of eggs per pupae (F = 0.277; df = 2; P = 0.763), the rates of parasitism (F = 3.062; df = 2; P = 0.084), or the rates of pupae with more of 1 egg (F = 0.205; df = 2; P = 0.817) (Table 1). The numbers of eggs / pupae were not randomly distributed in either the 5 or 10 pupae lots ([chi square](5)= 15.9, df = 2, P < 0.005; [chi square](10)= 12.6, df = 4, P < 0.01). These non-random distributions were due to clumping as evidenced by variance/ mean rations of 1.9 and 1.6, respectively. Eggs were oviposited on the posterior and middle regions of the host. No oviposition was ever detected in the anterior region.

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

Locations of Eggs and Adult Emergence through the Puparium

In these experiments, the maximum number of eggs per host was 5. Thus, the following combinations of egg number vs. location in host were statistically compared: (a) 1 egg-posterior, (b) 2 eggs-posterior, (c) 3 eggs-posterior, (d) 4 eggs-posterior, (e) 5 eggs-posterior, (f) 1 egg-middle, (g) 2 eggs-middle, (h) 3 eggs-middle, (i) 4 eggs-middle and 0) 5 eggs-middle. The number of eggs (F = 6.468; df = 4; P < 0.001) and their location (F = 3.958; df = 1; P = 0.048) had a significant influence of on the time of adult emergence. In pupae containing 5 eggs, adult emergence was significantly delayed when compared to pupae containing 1, 2, or 3 eggs. In addition, eggs oviposited in the middle of the pupae resulted in more rapid development than eggs in the posterior (Table 2). Peak female and male eclosion were observed at 23.9 ([+ or -]1.8) and 21.4 ([+ or -]1.6) d, respectively. Adults were significantly more likely to emerge from the middle of the host puparium, independent of the initial position of the eggs (i.e., medium or posterior) or the number of eggs (i.e., 1, 2, or 5 eggs).

When sex-ratios were compared against a 1:1 distribution, we observed a female-bias when host pupae contained 2, 4, and 5 eggs ([chi square] = 4.00, 6.760, and 18.614; df = 1; P < 0.05) but not when host pupae contained 1 and 3 eggs ([chi square] = 1.666 and 0.104; df = 1; P > 0.05). Regarding the initial position of oviposited eggs, sex-ratio was female-biased in both middle ([chi square] = 4.312, df = 1; P < 0.05) and posterior ([chi square] = 14.720, df = 1; P < 0.001) positions. No relationship was observed between the emergence location and the number of eggs (R = 0.047; P = 0.558) or the location of eggs (R = 0.005; P = 0.948).

DISCUSSION

Eurytoma sivinskii is a solitary ectoparasitoid of fruit fly pupae, particularly those midway in development. Its females could superparasitize, under laboratory conditions, by ovipositing up to 8 eggs per host. Adults emerged in a shorter period of time when hosts originally contained a single parasitoid egg, although there was no lower likelihood of adult emergence when multiple eggs were present. However, longer development could inflict other costs such as increased exposure to pathogens, predators, or hyperparasitoids (Benrey & Denno 1997).

Eggs were commonly laid in the middle and posterior region of the host, but not in the anterior third. Adult parasitoids derived from eggs laid in the posterior region developed more rapidly, but adult sex ratio and percent of emergence were the same in both posterior and medially laid eggs. Oviposition sites might be influenced both by local differences in the resistance offered by the puparium and by advantageous placement of the egg on the pupa itself. Spalangia endius Walker (Pteromalidae) females are as likely to drill in the anterior or posterior portions of young Musca domestica L. pupae, but as the puparium toughens with age, parasitoids are more likely to drill in the posterior, where the vulnerable anal spiracles are located, and these drilling are more likely to be successful (King 2001). For S. endius larvae, there appears to be no developmental advantage to an initially posterior or anterior placement, which may be due to the capacity of the hatchlings to wander to optimal feeding sites (King 2001). However, initial location does seem to matter in another pupal parasitoid of Diptera, Nasonia uitripenis Walker (Pteromalidae) (Rivers & Yoder 1996), where oviposition in the posterior portion of Sarcophaga bullata Parker (Sarcophagidae) puparia results in increased larval oxygen consumption, weight, and lipid content. Parasitized S. bullata have elevated lipid levels and the effect on the parasitoid was most pronounced when eggs were laid in the posterior portion (Rivers & Yoder 1996). At the same time, envemonation in the anterior parts of host leads to more rapid death and greater developmental disruption (Rivers & Denlinger 1994), which might also have nutritional consequences for the parasitoid. Unlike E. sivinskii, oviposition position in N. uitripenis has no effect on developmental rate.

The exit sites of adult E. sivinskii are concentrated in the middle of the A. ludens puparium. This contrasts strongly with the exit sites of S. endius which are mostly in the anterior end of the host (King 2001), where the puparium is likely to have weak cleavage lines that would have allowed the fly host itself to emerge. The reasons that E. sivinskii leaves from the middle are not entirely clear, but other parasitoids concentrate their penetration activities on the middle of the tephritid puparium. In the fruit fly specialist Coptera haywardi (Ogloblin) (Diapriidae), nearly all ovipositions are in the middle regions of Anastrepha suspensa (Loew) puparia, while the generalist chalcid, Dihrinus himalayanus Westwood, frequently oviposits in both the middle and the posterior (Sivinski et al. 1998). Perhaps the relatively strong preference for the middle by the tephritid specialist reflects a structural weakness in the Anastrepha puparial design that is also exploited by emerging adults.

ACKNOWLEDGMENTS

Larissa Guillen, Andrea Birke, and Guadalupe Cordova (Instituto de Ecologia, A.C., Xalapa, Veracruz, Mexico (INECOL) helped organize experiments and provided critical advice along the way. We gratefully acknowledge the technical help of Armando Torres Anaya, Cecilia Martinez Arcos, Jovita Martinez Tlapa, and Sandi Mendez Trejo (INECOL) who helped rear the parasitoids and their hosts. We gratefully acknowledge the useful suggestions for improvement by James L. Nation, Alberto Anzures-Dadda and 2 anonymous referees that helped us produce a better quality paper. Alberto Anzures-Dadda also provided invaluable help while preparing the revised version of this paper. Financial support for this study was principally furnished by the Mexican Campana Nacional contra las Moscas de la Fruta (Secretaria de Agricultura, Ganaderia, Desarrollo Rural y Pesca--Instituto Interamericano de Cooperacion para la Agricultura (SAGARPA-IICA). Additional funds were provided by the United States Department of Agriculture--Agricultural Research Service (USDA-ARS) and INECOL. We acknowledge partial financial support from the Comision Nacional para el Conocimiento y Uso de la Biodiversidad (CONABIO; Proyect No. FB325/H296/96) and the Consejo Nacional de Ciencia y Tecnologia (CONACyT)/Fondo del Sistema de Investigacion del Golfo de Mexico (SIGOLFO, Project No. SIG96001/ 96-01-003-V). Information reported here forms part of the undergraduate thesis of JMC (Universidad Nacional Autonoma de Mexico [UNAM]), directed by MA.

REFERENCES CITED

ALUDA, M., J. SIVINSKI, S. OVRUSKI, L. GUILLEN, J. LANCINO, M. LOPEZ, A. TORRES-ANAYA, G. GALLEGOS-CHAN, AND L. RUIZ. 2008. Colonization and domestication of seven species of native new world hymenopterous larval-prepupal and pupal fruit fly (Diptera: Tephritidae) parasitoids. Biol. Sci. Tech. (In Press).

BENREY, B., AND R. F. DENNO. 1997. The slow-growth-high-mortality hypothesis: a test using the cabbage butterfly. Ecology 78: 987-999.

DIGUILIO, J. 1997. Eurytomidae, pp. 477-495 In G. Gibson, J. Huber, and J. Wooley [eds], Annotated Key to the Genera of Nearctic Chalcidoidea (Hymenoptera). NRC Research Press, Ottawa, CA.

GATES, M. W, AND E. E. GRISSELL. 2004. A new species of Eurytoma (Hymenoptera: Eurytomidae) attacking the mango fruit fly, Anastrepha obliqua (Macquart) (Diptera: Tephritidae), pp. 147-159 In K. Rajmohana, K. Sudheer, P. Girish-Kumar, and S. Santhosh [eds.], Perspectives on Biosystematics and Biodiversity. Harvest Ledia Services, Calicut, India.

GATES, M., J. MENA-CORREA, J. SIVINSKI, R. RAMIREZ-ROMERO, G. CORDOVA-GARCIA, AND M. ALUDA. 2008. Description of the immature stages of Eurytoma sivinskii Gates and Grissell (Hymenoptera: Eurytomidae), an ectoparasitoid of Anastrepha (Diptera: Tephritidae) pupae. Entomol. News (In Press).

KING, B. 2001. Parasitization site on the host of the parasitoid wasp Spalangia endius (Hymenoptera: Pteromalidae). Environ. Entomol. 30: 346-349.

MARTINEZ, I. 2002. Metodologias. Tecnicas basicas de anatomia microscopica y de morfometria para estudiar los insectos. Bol. Soc. Entomol. Mexicana 30: 187-195.

MENA-CORREA, J. 2005. Biologia Basica de Eurytoma sivinskii (Hymenoptera: Chalcidoidea: Eurytomidae). Tesis, Universidad Nacional Autonoma de Mexico. Mexico, D.F., Mexico.

RIVERS, D., AND D. DENLINGER 1994. Developmental fate of the flesh fly, Sarcophaga bullata envemonated by the pupal ectoparasitoid, Nasonia vitripennis. J. Insect Physiol. 40: 121-127.

RIVERS, D., AND J. YODER 1996. Site-specific effects of parasitism on water balance and lipid content of the parasitic wasp Nasonia vitripennis (Hymenoptera: Pteromalidae). Europ. J. Entomol. 93: 75-82.

SIVINSKI, J., K. VULINEC, E. MENEZES, AND M. ALUDA. 1998. The bionomics of Coptera haywardi (Ogloblin) (Hymenoptera: Diapriidae) and other pupal parasitoids of tephritid fruit flies (Diptera). Biol. Control. 11:193-202.

SOKAL, R. R., AND F. J. ROHLF. 1995. Biometry: The Principles and Practice of Statistics in Biological Research. W. H. Freeman and Co. New York.

ZAR, J. H. 1998. Biostatistical Analysis. Pearson Education. New Jersey. J. MENA-CORREA (1), J. SIVINSKI (2), M. GATES (3), R. RAMIREZ-ROMERO (1) AND M. ALUJA (1,4)

(1) Instituto de Ecologia, A.C., Apartado Postal 63, 91000 Xalapa, Veracruz, Mexico (2) Center for Medical, Agricultural and Veterinary Entomology, USDA-ARS, P.O. Box 14565, Gainesville, FL 32604, USA (3) Systematic Entomology Laboratory, PSI, ARS, USDA, do Smithsonian Institution, National Museum of Natural History, PO Box 37012, Washington, DC 20013- 7012, USA (4) Corresponding author: [e-mail]

TABLE 1. MEAN NUMBER ([+ or -] S.E.) OF OVIPOSITED EGGS PER PUPAE,
RATES OF PARASITISM, AND OF PUPAE CONTAINING MORE THAN 1 EGG AFTER
EXPOSURE OF DIFFERENT DENSITIES OF ANASTREPHA LUDENS PUPAE TO EURYTOMA
SIVINSKII FEMALES.

Host         Mean number        Mean parasitism      Mean number of
density        of eggs             rate(b)(%)        pupae with more
             per pupae(a)                            of one egg(c)(%)

1          2.20[+ or -]1.3      100[+ or -]0.0       60.0[+ or -]54.8
5          1.96[+ or -]1.5      60.0[+ or -]6.9      56.0[+ or -]51.8
10         2.54[+ or -]0.9      78.0[+ or -]16.4     77.8[+ or -]15.7

(a) Non significant differences (F=0.277; 2 of; P=0.763).

(b) Non significant differences (F =3.062; 2 of; P =0.084).

(c) Non significant differences (F =0.205; 2 of; P =0.817).

TABLE 2. LIFE HISTORY PARAMETERS OF EURYTOMA SIVINSKII DEVELOPED IN
ANASTREPHA LUDENS PUPAE CORRELATED WITH THE INITIAL POSITION OF
OVIPOSITED EGGS AND THE NUMBER OF OVIPOSITED EGGS.

                                    Proportion (%) of adult emergence
Initial         Mean ([+ or -]       in relation to position in pupae
position        S.E.) time to
of eggs         adult emergence       Anterior   Medium    Posterior

Medium         23.5[+ or -]2.6 a       25.4 a    49.3 b     25.4 a
Posterior      22.7[+ or -]1.6 b       14.0 a    65.6 b     20.4 a

No of
eggs

1              23.4[+ or -]1.7 a        8.6 b    74.3 a     17.1 b
2              22.6[+ or -]1.7 a       16.7 b    75.0 a      8.3 b
3              22.8[+ or -]2.0 a       28.9 a    28.9 a     42.1 a
4              23.6[+ or -]2.4 ab      24.0 a    52.0 a     24.0 a
5              24.4[+ or -]2.2 b       15.4 b    65.4 a     19.2 b

Initial       Total adult       Sex-ratio (%)
position      emergence (%)      males)
of eggs

Medium         41.9 a           33.3 a
Posterior      58.1 b           30.1 a

No of
eggs

1              21.9 a           42.9 a
2              22.5 a           33.3 ab
3              23.8 a           41.2 a
4              15.6 a           24.0 ab
5              16.3 a            7.7 bc

Different letters for each parameter within treatment indicate
significant differences at (P < 0.05).