Bionomics of Orasema Simplex (Hymenoptera: Eucharitidae), a Parasitoid of Solenopsis ﬁre Ants (Hymenoptera: Formicidae) in Argentina

Biological Control 48 (2009) 204–209

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Biological Control

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Bionomics of Orasema simplex (Hymenoptera: Eucharitidae), a parasitoid of Solenopsis ﬁre ants (Hymenoptera: Formicidae) in Argentina

L. Varone *, J. Briano

USDA-ARS South American Biological Control Laboratory, Bolivar 1559 (1686) Hurlingham, Buenos Aires Province, Argentina article info abstract

Article history: Biological characteristics of the parasitoid Orasema simplex Heraty (Hymenoptera: Eucharitidae), a poten- Received 17 June 2008 tial candidate for the biological control of fire ants in the United States were investigated. Female survi- Accepted 9 October 2008 vorship, fertility and oviposition preferences were studied in the laboratory. Naturally parasitized Available online 17 October 2008 colonies were examined to determine offspring sex ratio, development success and time, and to artificially parasitize healthy ant colonies. In addition, field studies were carried out to establish natural ovi- Keywords: position substrates and adult activity patterns. Orasema simplex female survivorship was 3.6 ± 1.5 days. Orasema simplex Newly emerged females contained 613.5 ± 114.0 mature eggs. The adult development success in natural Solenopsis richteri parasitized colonies was 22.2% with a female-biased sex ratio (4:1). The time required from planidia to Solenopsis invicta Fire ants adult was 29.5 ± 5.4 days. In the field, adults were mostly found around the ant nests at midday. A broad Biological control range of plant species was observed as oviposition substrates. The transfer of planidia to healthy ant colonies was achieved but the development success was very low. Orasema simplex appears to have a limited potential as a fire ant biocontrol agent because of cosmetic damage to a wide variety of plants used for oviposition. However, further studies are necessary to evaluate the real damage exerted by oviposition punctures. Published by Elsevier Inc.

1. Introduction Initially, the planidia burrows under the host cuticle and swells slightly indicating limited feeding by the parasitoid. Upon pupa- The species of Eucharitidae (Hymenoptera) are parasitoid wasps tion, first instar larva becomes external (Heraty, 1994a, 2000). of ants (Das, 1963; Johnson et al., 1986; Williams and Whitcomb, The parasitoid completes development when the host enters the 1974). Almost all species of Orasema are parasitoids of ants in pupal stage (Johnson, 1988; Heraty et al., 1993). Orasema imma- the genera Solenopsis, Pheidole, Tetramorium and Wasmannia tures are treated by host workers as ant brood, since they acquire (Heraty, 1994b). The first studies on the biology and habits of the host colony odors that allows acceptance inside the colony (Vander Eucharitidae were carried out by Wheeler (1907), who found sev- Meer et al., 1989). When development is complete, adults emerge eral species of Orasema associated with ants of the genera Pheidole within the ant nest (Heraty, 1985). and Solenopsis in Texas and Colorado. Eucharitids have an unusual In Argentina and Brazil, unidentified Orasema spp. have been re- life cycle. Females lay their eggs in plant tissue near ant nests ported ovipositing in several plants (Parker, 1942; Tocchetto, (Wheeler, 1907; Johnson et al., 1986). The site of oviposition and 1942). They were first reported parasitizing fire ants of the Solenop- the range of plants used by the different eucharitid species are sis saevissima complex in 1964 in Uruguay (Silveira-Guido et al., uncertain. Therefore, the presumed specificity for oviposition 1964) and later in Brazil (Williams and Whitcomb, 1974). The im- plants may be a consequence of the scarce data available (Johnson, ported fire ants, Solenopsis invicta Buren and S. richteri Forel are two 1988). In general, the part of the plant the eucharitids utilized for species of this complex that were accidentally introduced in the oviposition is highly variable, including overwintering buds, open- United States from South America and became serious medical ing flower buds, stems of blossom clusters, seed pods and leaves and economic pests (Adams, 1986; Logfren 1986a,b; Drees et al., (Clausen, 1940). The emerged planidia attach themselves to a for- 1992; Lard et al., 2001; Pereira et al., 2002). Since then, several nat- aging worker ant or intermediate host and are carried to the nest ural enemies have been under study: two species of microsporidi- phoretically. Once there, they are transferred to an ant larva. an pathogens, several species of phorid flies, a congeneric parasitic ant and more recently the mermithid nematode Allomermis sole- nopsii (Briano et al., 1995a,b, 1997, 2002a; Calcaterra et al., 1999; * Corresponding author. Fax: +54 11 4452 1882x104. Oi et al., 2005; Orr et al., 1995; Pesquero et al., 1995; Porter, E-mail address: [email protected] (L. Varone). 1998, 2000; Williams et al., 1999; Porter and Varone unpublished).

1049-9644/$ - see front matter Published by Elsevier Inc. doi:10.1016/j.biocontrol.2008.10.003 L. Varone, J. Briano / Biological Control 48 (2009) 204–209 205

The flies Pseudacteon tricuspis Borgmeier, Pseudacteon curvatus 2.2. Field observations Borgmeier, Pseudacteon litoralis Borgmeier, and Pseudacteon obtusus Borgmeier have been released (Porter et al., 2004; Vazquez et al., 2.2.1. Parasitism rate 2006; Graham et al., 2003; Gilbert et al., 2008) and the microspo- Individuals of Orasema simplex were obtained from parasit- ridian Vairimorpha invictae Jouvenaz and Ellis is still in quarantine. ized colonies collected from February 2005 to May 2007 at the Orasema was also listed as a potential candidate for the biological 24 positive sites found in Argentina (Varone and Heraty, unpub- control of Solenopsis and investigations on this organisms started at lished). The colonies were excavated, put in 10 L dusted buckets the South American Biological Control Laboratory (SABCL) in 2005 and transported to the laboratory for later flotation and separa- as part of the biological control program against fire ants. tion from the soil (Banks et al., 1981). The ant brood was sepa- In our recent surveys in Argentina, O. simplex Heraty was the rated from the colony using sorting sheets (Banks et al., 1981) eucharitid species most commonly found parasitizing fire ants, and then observed under the dissecting scope for the presence with a wide distribution but with low abundance and persistence of parasitoids. in the field (Varone and Heraty, unpublished). The objective of this Once detected, the Orasema larvae and pupae were returned to study was to determine the bionomics of O. simplex in the labora- their original host colonies that were previously reduced to 100– tory and to conduct field trials to detect natural oviposition sub- 200 workers and a small amount of Orasema-free brood. These strates and adult activity patterns. Such information is essential small fragments of colonies were kept in rearing chambers at to assess the real potential of O. simplex as a biological control 30 ± 2 °C in plastic vented containers with food and humidity agent of imported fire ants in the United States. sources, and checked daily for the emergence of the adult parasitoids. The development time from planidia to adult was estimated 2. Materials and methods and the sex ratio of emerged adults recorded. The CBS was selected to conduct ecological studies because of 2.1. Laboratory studies the high occurrence and persistence of O. simplex (Varone and Heraty, unpublished). The site chosen was the entrance of the sta- 2.1.1. Survivorship and fertility tion, a 250 m long–20 m wide dirt road surrounded by a xerophytic Orasema simplex female survivorship was estimated with 30 forest. newly emerged females (obtained from field collected colonies) ex- posed to different plant species as oviposition substrates. The spe- 2.2.2. Adult activity cies tested were: (1) Eupatorium aff. laevigatum L. (Asteraceae), (2) Adult presence was monitored observing 36 fire ant mounds in Vinca rosae L. sp. (Apocynaceae), (3) Impatiens sp. (Balsaminaceae), the morning (8–10 h; 19.2 ± 1.7 °C), at noon (11–13 h; 24.8 ± and (4) Viola sp. (Violaceae). No plants were available in the control 0.2 °C) and in the afternoon (15–17 h; 27.6 ± 1.6 °C) for three treatment; 4–9 replicates were considered for each treatment. consecutive days. Each unmated female was placed in a plastic vented bottle at- Each mound and the surrounding area (1 m diameter) were ob- tached to a stem of the potted test plant or, in the controls, placed served during 2–3 min; individuals found were captured with an in a plastic container with humid tissue paper. All females fed hon- aspirator, marked with latex paint and released. The volume of ey–water solution and were checked daily. Potted plants were each mound was estimated as a hemisphere with the formula 3 placed in a rearing chamber at 25 ± 2 °C and 14–10 h L–D. Fertility V = (2/3)pR (V, volume; R, radius) to evaluate a size preference was estimated by dissecting other 10 females immediately after of O. simplex. Sixteen of the 36 mounds were excavated and trans- emergence and by counting the mature oöcytes in the ovarioles. ported to the laboratory in dusted buckets for later brood examination. 2.1.2. Transfer of planidia Several tests with different approaches were conducted to arti- 2.2.3. Oviposition substrates ficially transfer newly emerged planidia from the Corrientes Bio- Natural oviposition substrates were recorded in three different logical Station (CBS, S 27° 33,1730;W58° 40,7710) to receptor habitats with high occurrence of O. simplex: (1) the entrance of the laboratory S. invicta colonies that were free of the parasitoid. The CBS, (2) a pasture in Colonia Hughes, Entre Ríos (S 32° 22,7850;W approaches were: (1) plants with oviposition marks and/or plani- 58° 16,6780) and (3) abandoned land in Concepción del Uruguay, dia were potted in the field, transported to the laboratory and ex- Entre Ríos (S 32° 27,7610;W58° 14,1960). In each place, 10–15 fire posed to ant colonies in buckets, (2) planidia collected in leaves in ant mounds were randomly selected and plants occurring within the field were transferred by hand (n = 50–80 per replication) to the surrounding area (1 m diameter) of each mound were exam- receptor host brood with the ratio of 1 planidia/larva, and (3) plant ined for Orasema oviposition marks. All broadleaf plants were indi- leaves with planidia were introduced into small fragments of ant vidually checked and grasses were examined for 10 min. Plants colonies. In the second and third approaches, the receptor brood with flowers were directly pressed and dried for identification; and workers were maintained in plastic vented boxes with humid- other plants were transplanted to pots and transported to the lab- ity and food sources. After 2–3 weeks, the brood was separated and oratory to allow flowering for later identification. The presence of observed for the presence of Orasema. Each approach was repli- planidia was observed frequently and some of them were success- cated 8–10 times with different receptor colonies. fully reared on healthy ant colonies.

2.1.3. Oviposition substrates 2.3. Statistical analysis Oviposition substrates were tested with non-choice trials. Twenty seven females were conﬁned individually until death in a Female survivorship was analyzed with one-way ANOVA. Ant 2 L plastic vented bottle with arbitrarily selected plants of eco- volumes and parasitism of Orasema was compared with a two- 2 nomic-ornamental value (5–7 replicates). Plant species used were: sample t test. A v test was used to determine if the proportion corn, soybeans, Vinca rosae, lemon and red pepper. After female of parasitized colonies was independent of the presence of Orasema death, plants were examined for the presence of oviposition marks in the vicinity of the mounds and if the presence of parasitized col- and later emergence of planidia. Tests were conducted in a green- onies was independent of the season. Statistica 6.0 and Statistix 7 house, mostly in the summer. software were used to run the tests. Means are reported ±SD. 206 L. Varone, J. Briano / Biological Control 48 (2009) 204–209

Table 1 Table 2 Plants with oviposition marks of Orasema simplex in non-choice laboratory tests and Mean survivorship of Orasema simplex with four oviposition substrates. in field surveys. Plant substrate Replicates Female survivorship (x ± SD) Clase and order Family Species Eupatorium aff. larvigatum 7 3.8 ± 1.3 Laboratory tests Vinca rosae 9 3.4 ± 1.0 Liliopsida (Monocot) Impatiens sp. 4 2.9 ± 1.4 Poales Poaceae Zea mays L. Viola sp. 4 2.9 ± 1.2 Magnoliopsida (Dicot) No substrate 6 4.5 ± 1.4 Fabales Fabaceae Glycine max L. Gentianales Apocynaceae Vinca rosae L. Means do not differ significantly (P = 0.28). Sapindales Rutaceae Citrus limon (L.) Burn Solanales Solanaceae Capsicum annuum L. Field tests entire egg load in the fully developed ovaries at the time of emer- Liliopsida (Monocot) gence and that the adults do not feed. In many species, the eggs are Asparagales Smilacaceae Smilax campestris Griseb Poales Poaceae Paspalum unispicatum all laid in one day, so there is little need for food (Clausen, 1941). (Scribn. & Merr.) Nash, In addition, across parasitoid wasp taxa, life-span is negatively P. denticulatum Trin., correlated with the proportion of oöcytes mature upon emergence, P. notatum Fluegge and so pro-ovigenic wasps (with most egg load mature at emergence) P. dilatatum Poir Magnoliopsida (Dicot) tend to be shorter-lived than synovigenic ones (that continue Asterales Asteraceae Grindelia pulchella Dann. Stevia maturing eggs throughout life) (Jervis et al., 2001). aff. entreriensis Hieron Although Jervis et al. (2001) estimated that most Hymenoptera Eupatorium aff. laevigatum L. (98%) are synovigenic species, the short survivorship and high Fabales Fabaceae Sesbania virgata (Cav.) Pers. number of eggs upon emergence reported here strongly suggests Gentianales Asclepiadaceae Asclepias curassavica L. Lamiales Verbenaceae Verbena montevidensis Spreng. that O. simplex is a pro-ovigenic species. This pro-ovigenic strategy Malvales Malvaceae Sida rhombifolia L. and the large number of eggs produced would be a way to counter- Scrophulariales Scrophulariaceae Stemodia aff. lanceolata Benth act the low probability of the phoretic transport provided by foraging worker ants (see also Price, 1975). Moreover, most pro-ovigenic species are associated with hosts with aggregated distribution (Jervis et al., 2001) and fire ants are 3. Results and discussion clearly aggregated in incipient colonies although mature colonies are often regularly dispersed (Adams and Tschinkel, 1995). 3.1. Laboratory studies 3.1.2. Transfer of planidia 3.1.1. Survivorship and fertility The successful transfer of planidia was obtained at very low Overall O. simplex female survivorship was estimated at 3.6 ± 1.5 rates only by placing them (together with plant tissue) in frag- days (range: 1–7) and was similar for all plants tested (Table 2, mented receptor colonies with abundant brood. The number of F = 1.32; df = 4, 25; P = 0.28). However, when no oviposition sub- new adults emerged was 1–5 from the original 50–70 planidia strate was available, the survivorship was 4.5 ± 1.4 days, slightly transferred. Despite the low success, this is the first report of arti- higher. In spite of providing the females honey-water solution, no ficial transmission of this parasitoid to non parasitized fire ant col- feeding was observed. onies. The rearing of O. simplex under laboratory conditions should The mean number of mature oöcytes found in the ovarioles at be improved to facilitate further investigation on its life cycle and emergence was 613.5 ± 114.0 (range: 470–742) (Fig. 1). When artificial propagation. the female emerged, the exuviae remained longer on the head Although the association of planidia with intermediate hosts, and antennae. Only females that got rid of it seemed to have the Thysanoptera or Hemiptera, has been reported (Das, 1963; Clau- oöcytes developed and countable. Otherwise, the eggs appeared sen, 1941; Beshear, 1974; Johnson et al., 1986; Heraty et al., as a white amorphous tissue. Similar fertility was reported for Ora- 1993), we saw no indication of such a mechanism in the Ora- sema coloradensis Gahan, with an average number of eggs of 478 sema–Solenopsis association. (Johnson et al., 1986). However, these females were collected in The developmental time from the transfer of planidia to the field at unknown ages. adult emergence was 29.5 ± 5.4 days (n = 12). The duration of According to Clausen (1940), the total egg capacity of eucharitid the pupal stage was 8.2 ± 3.8 days (n = 68). This is the first accu- females ranges from 1000 to >10,000 eggs. Previous observations rate estimation of part of the life cycle of O. simplex as parasitoid (Johnson, 1988) indicated that females of most species have the of fire ants.

Fig. 1. Orasema simplex female with removed egg complement (left) and mature oöcytes (right) at emergence. L. Varone, J. Briano / Biological Control 48 (2009) 204–209 207

and in a hole made by the ovipositor. Immediately after oviposition, the egg punctures were hardly visible; a few days after, the tissue surrounding the egg cavity dried up and turned brown (Fig. 2). Ovipositions made in fruits can cause cosmetic damage (Tocchetto, 1942; Roberts, 1958). The bacterium Pseudomonas savastanoi (E. Smith), which causes the olive tree tuberculosis, was isolated from tumors associated with ovipositions punctures of O. aenea (Nicolini, 1950). A similar damage of O. assectator Kerrich was reported in tea leaves in Assam, India, where affected leaves seemed to lose the good tea character (Das, 1963). However, the real deleterious effect of the punctures in leaves and entire plants has not been determined yet and should be deeply investigated, since it is a key factor for the potential of this organism as biocontrol agent.

3.2. Field observations

Fig. 2. Oviposition punctures of Orasema simplex in Vinca rosae in the laboratory. 3.2.1. Parasitism rate At positive sites, 144 (34.1%) Solenopsis colonies were parasitized with O. simplex, with a total of 99 larvae, 1130 pupae and 183 adults. The mean number of individuals per parasitized colony was 4.4 ± 7.4 larvae (range 1–27), 13.6 ± 21.3 pupae (range 1–102) and 4.0 ± 5.9 adults (range 1–24). The field parasitism rates were not significantly different across the seasons (Fig. 3, 37.8% in summer, 27.2% in autumn, 37.7% in winter, and 26.7% in spring, v2 = 4.96; df =3;P = 0.17). Although O. simplex was almost always found parasitizing worker brood, once it was found attached to a sexual female larva (Fig. 4). Frequently, sexual brood in the colonies was less abundant than worker brood, therefore the real inci- dence of O. simplex on sexual brood remained unclear and should be further investigated. Another intensive collection of Orasema on fire ants, conducted in summer 1984 by Wojcik et al. (1987), revealed that 41% of the colonies were parasitized with up to 598 individuals per colony. The great variability in the intracolonial rate of parasitism might be the consequence of the unusual life cycle of the parasitoid, in which the success of the planidia to reach the ant larvae depends Fig. 3. Seasonal distribution of Solenopsis colonies parasitized with Orasema on the encounter rate with foraging ants. simplex. The rates of parasitism did not differ significantly (v2 = 4.9; df =3;P = 0.17). A total of 314 adults of O. simplex were recovered from parasitized immature ants after returning to their original fragmented 3.1.3. Oviposition substrates colonies, representing a development success of 22.2%. The sex ra- In the laboratory O. simplex laid eggs on a broad range of sub- tio of the emerged adults was female biased, with a 4:1 proportion. strates. Incisions marks were observed in all the plants tested As previously observed by Vander Meer et al. (1989), in our exper- (Table 1) and in all cases, eggs successfully developed to first instar iments, immature wasps were tended by host colony workers as larvae. As in other eucharitids, the eggs were deposited individu- they were own brood, not showing any aggressive behavior against ally in rows, mostly along the borders of the under side of leaves the intruders, even to newly-emerged adults. However, several

Fig. 4. Larva of Orasema simplex attached to a worker brood (left) and to sexual brood (right). 208 L. Varone, J. Briano / Biological Control 48 (2009) 204–209 adults O. simplex were found partially preyed, supporting the hypothesis of the loss of host-speciﬁc compounds on adults Ora- sema soon after emergence (Vander Meer et al., 1989). A similar re- sponse was obtained by Briano et al. (2002b) when trying to artiﬁcially propagate the parasitic ant Solenopsis daguerrei (Sant- schi) into clean receptor colonies of S. richteri.

3.2.2. Adult activity At the CBS entrance, the adult activity of O. simplex was concen- trated at noon. A total of 28 adults (24 females and 4 males) were observed, 23 (82.1%) at noon, 1 (3.6%) in the morning (female) and the remaining 4 (14.3%) (females) in the afternoon. Only one marked female was recaptured the same day and no mating was observed. The presence of adult wasps in the vicinity of ant colonies was not a good indicator of parasitism. Wasps were observed flying around in 10 out of the 16 mounds excavated at the end of the field observations; 6 (60%) of those 10 colonies were parasitized. On the other hand, 2 (33%) of the remaining 6 excavated mounds with no O. simplex around were parasitized. Although these proportions looked different, the difference was not significant (Fig. 5, v2 = 1.07; df =1;P = 0.3), probably because of the small sample size. In addition, the volumes of parasitized and non parasitized ant mounds Fig. 6. Ant mound volumes versus parasitism of Orasema simplex. Volumes were not significantly different (P=0.7). were not different (Fig. 6, t = 0.3; df = 34; P = 0.7) suggesting that a size preference did not exist. Furthermore, a great variability in the ant mound volume was observed. O. coloradensis and O. viridis Ashmead were also found into buds and stems (Johnson et al., 1986). In addition, during the field sur- 3.2.3. Oviposition substrates veys conducted for this work, oviposition punctures and eggs of As in the laboratory tests, O. simplex showed a broad range of O. aenea were observed on leaves of blueberry, Vaccinium corymbo- substrates for oviposition. Pooling the three field sites, 13 out of sum L., in a plantation in Concordia, Entre Ríos province. Other the 15 plant species found around the mounds were used as sub- plants were reported with oviposition marks of, probably, O. aenea strates (Table 1). This wide range might be indicating that the (Tocchetto, 1942). plants are used only as a physical support for the eggs. Moreover, In summary, O. simplex showed a short survivorship and high the two plants without oviposition marks had their leaves either fertility, with a great proportion of mature oöcytes at, or shortly with dense hairs (Solanum sp.) or with a hard surface (palm) that after, female emergence, suggesting a pro-ovigenic strategy. might impede the oviposition process. Female wasps showed a broad range of oviposition substrates The part of the plant used by other eucharitids for oviposition either in the field or in the laboratory. Rearing techniques must varies with the species. Leaves have been reported as oviposition be improved to successfully transfer the planidia to new colonies. substrates for other Orasema spp.: unidentified leaves for O. color- Orasema simplex appears to have a limited potential as a fire ant adensis Gahan and O. smithi Howard (Clausen, 1940) and leaves of biocontrol agent because of cosmetic damage to a wide variety of Ilex paraguayensis St. Hill and Olea europea L. for O. aenea Gahan plants used for oviposition. However, the real damage exerted by (Gahan, 1940; Nicolini, 1950, respectively). Moreover, eggs of oviposition punctures in plants of economic and ecological importance should be evaluated. Studies on field host range are in pro- gress. Field sites with high presence of this parasitoid and with high intracolonial rate of parasitism should be detected to study the detrimental effect on fire ant colonies and populations. This information will be essential to better understand the real potential of this agent for the biological control of the imported fire ant in the United States.

Acknowledgments

We deeply thank John Heraty (University of California, River- side, CA) for specimens’ identification and for reviewing the manuscript. We also acknowledge Luis Calcaterra (SABCL) for his valuable help during the first part of the research, Sonia Cabrera (SABCL) for her field and laboratory assistance and Daniel Iele (SABCL) and Alicia Delgado (Universidad Nacional de Córdoba, Argentina) for helping with the field data collection. We thank Sanford Porter (CMAVE, Gainesville, FL) for his useful suggestions when reading the manuscript.

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