Dispersal Of the Delayed Action Insecticide ...

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Taking into account that the importance of leaf-cutting ants as pests, know- ... 3, 2007 of this insecticide in the colony, and as a reference in future studies, such as ... ganochlorine) as the AI (Echols 1966; Amante 1968; Cherrett & Sims 1969).
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Dispersal Of the Delayed Action Insecticide Sulfluramid In Colonies of the Leaf-Cutting Ant Atta sexdens rubropilosa (Hymenoptera: Formicidae) by Luiz C. Forti1, Dênis R. Pretto1, Nilson S. Nagamoto1,*, Carlos R. Padovani2, Roberto S. Camargo1, Ana Paula P. Andrade1

ABSTRACT Toxic baits are the most used control method for leaf-cutting ants due to their high effectiveness and because they are considered the safest for humans. Taking into account that the importance of leaf-cutting ants as pests, knowing the process by which dispersal and worker contamination is achieved becomes essential to understand several aspects about the functioning of a bait-borne AI (active ingredient) used in toxic baits. It has been established that an effective toxic bait should have a delayed- action AI, but its dispersion among the different sizes of workers is unknown. Workers of different sizes are involved in quite different tasks such foraging, cultivation of symbiotic macrofungus or control of deleterious microfungi. Therefore, we prepared a toxic bait containing the delayed-action AI sulfluramid and a dye (Rhodamine B) as an AI tracer in order to study dispersal and contamination in colonies, evaluated at different periods and in rlation to different workers' sizes. Both field and laboratory colonies were evaluated. The great level of contamination, about 50% at 24 hours, in all sizes of workers demonstrates that worker contact with toxic bait is intense within this period. The distribution in field and laboratory colonies was similar. This contamination pattern is probably enough to cause the colony to die because of contamination of smaller workers, leading to the loss of control of the aggressive microfungi, which can quickly overgrow the symbiotic fungus culture. The dispersal dynamics of AI in leafcutting ant workers is important for investigations on the mode of action

Laboratório de Insetos Sociais-Praga, Defesa Fitossanitária, FCA/UNESP, Fazenda Exp. Lageado, Rua José Barbosa de Barros 1780, Zip Code 18610-307, PO Box 237, Botucatu, SP, Brazil. *Corresponding author. email: [email protected] 2Dept. Bioestatística, IB/UNESP, Zip Code 18618-000, PO Box 510, Botucatu, SP, Brazil.

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of this insecticide in the colony, and as a reference in future studies, such as those attempting to reduce the concentration of AIs in baits to reduce their environmental impact, or for facilitation of new AI screening. Keywords: Atta sexdens rubropilosa, toxic bait, leaf-cutting ant, insecticide, Rhodamine B.

INTRODUCTION The leaf-cutting ants (Hym.: Formicidae: Attini) are serious defoliating pests in neotropical agriculture (Cherrett 1986; Boaretto & Forti 1997), demanding effective methods for control on a huge scale. Hot fogging and toxic baits are among the most effective treatments and the latter is the most frequently-used control method because it is considered to be the safest for humans and easy to use (Boaretto & Forti 1997; Forti et al. 1998; MachadoNeto et al. 1999). In Brazil, approximately 12,000 tons of toxic baits are used per year (Boaretto & Forti 1997). Toxic bait for leaf-cutting ants consists of dehydrated citrus pulp mixed with vegetable oil plus the active ingredient (AI), formulated into pellets (Amante 1968; Cherrett & Sims 1969; Forti et al. 1998). It has been demonstrated that AIs should have delayed action in adult workers in order for the bait to be effective (Nagamoto et al. 2004, 2007; N.S. Nagamoto & L.C. Forti, in prep.). It is also preferable that the bait work by ingestion with small or no contact action, be odorless and non-repellent, easily dispersed through the colony and relatively quickly degraded in the environment, with low toxicity to vertebrates, thus being environmentally acceptable (Peregrine & Cherrett 1974; Forti et al. 1998). The citrus pulp is an very atractive substrate for leaf-cutting ants (Mudd et al. 1978; Verza et al. 2006) and repellent or fast-acting AI probably reduces the usually good citrus-based bait carrying and its distribution inside colonies (Pretto & Forti 2000; Forti et al. 2003; Moreira et al. 2003; Nagamoto et al. 2004). The first effective toxic baits against leaf-cutting ants used dechlorane (organochlorine) as the AI (Echols 1966; Amante 1968; Cherrett & Sims 1969). Although serious concerns about commercial use of this AI appeared about ten years later due to its organochlorine nature (Waters et al. 1977), in Brazil, due to the fact that these ants were very serious pests and the absence of a good substitute, baits with this AI were sold until 1993 (Forti et al. 1998).



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The potential of sulfluramid (fluoroaliphatic sulfonamide) was discovered when it was used as a surfactant to suspend toxicant formulations against fire ants (Hym.: Forrmicidae: Solenopsidini) and it has shown delayed toxicity in these ant workers (Vander Meer et al. 1985). For leaf-cutting ants, this AI showed very good results in several tests in colonies. subsequently, it was used to replace dechlorane and is still sold today (Cameron 1990; Zanuncio et al. 1992; Forti et al. 1998, 2003). The most recent effective AI discovered was fipronil (phenylpyrazole) (Forti et al. 1996; White 1998; Grosman et al. 2002); however, baits using this AI are produced at a smaller commercial scale and are less effective against some leaf-cutting ants species like Atta capiguara (Forti et al. 2003). It was demonstrated in initial laboratory research with each of these three AIs that they exhibit delayed action against fire ants (Lofgren et al. 1962; Vander Meer et al. 1985; Collins & Callcott 1998). However, because of difficulties in the development of reliable methods for evaluation of this parameter in leaf-cutting ants, delayed action of sulfluramid, fipronil and dechlorane against these ants was demonstrated only recently (Nagamoto et al. 2004, 2007). For leaf-cutting ants, fipronil has delayed action in a smaller range of concentrations than sulfluramid, although for fire ants these two AIs are similar in levels of delayed activity over the range of concentrations (Vander Meer et al. 1985; Collins & Callcott 1998; Nagamoto et al. 2004; N.S.N. & L.C.F., unpubl. data). The process by which workers become contaminated with toxic baits is closely associated with their behavioral and feeding habits. Workers cut living plant parts, which are taken into the colony where they are cleaned and subsequently chewed into pulp. Such pulp is incorporated into the fungal sponge (substrate + cultivated fungus). Later, smaller workers inoculate hyphae of the cultivated macrofungus (Basidiomycota), Leucoagaricus gongylophorus, into the newly added material (Weber 1972; Andrade et al. 2002; Camargo et al. 2003; Nagamoto et al. 2004). Although adult workers only ingest liquids (Littledyke & Cherrett 1976; Paul & Roces 2003), this intensive substrate processing can provide opportunities for fat-and water-soluble substances to be ingested (Peregrine et al. 1972); this is probably also true for toxic bait AIs (Andrade et al. 2002; Nagamoto et al. 2004).

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This cultivated fungus is an obliged symbiont of these ants because it is the main food for ant workers and larvae (Weber 1972). The fungal sponge is also colonized with several microfungi, which has been observed to overgrow the fungal sponge when workers are experimentally removed (Currie et al. 1999; Rodrigues et al. 2005a), or after application of toxic baits (Forti et al. 1998; Rodrigues et al. 2005b), or in response to unknown factors (Currie et al. 1999). Among these microfungi, Escovopsis spp. is frequently reported and potentially can cause a dramatic reduction in ant colony growth (Currie et al. 1999; Currie 2001; Nagamoto et al. 2003; Rodrigues et al. 2005a,b). Therefore, microfungi can be regarded as probable synergists of toxic baits (Rodrigues et al. 2005a; Camargo et al. 2006). Knowing the process by which the insecticide is dispersed and workers become contaminated allows us to understand several aspects about the action mode of delayed-action AIs. In particular, sulfluramid is the most interesting AI to study because it is the most widely used and effective AI for leaf-cutting ants (Forti et al. 1998, 2003; Nagamoto et al. 2004, 2007), and it has delayed activity over a wide range of concentrations (Nagamoto et al. 2004; N.S.N. & L.C.F., unpubl. data). Studies specifically targeted for leaf-cutting ants control are very important for future screening of AIs and development or improvement of toxic baits. The objective of this research was to evaluate the dispersal of a delayed-action AI of toxic bait in leaf-cutting ant colonies. For this, we used sulfluramid as the AI, the dye Rhodamine B as an AI tracer, and Atta sexdens rubropilosa colonies.

Bait preparation

MATERIAL AND METHODS

Rhodamine B dye was selected as the AI tracer since it: 1) is non-toxic and non-repellent for insects in general; 2) is water soluble; 3) has fluorescent properties when exposed to ultraviolet light and is a very powerful dye, allowing it to be evaluated even at very low concentrations (Spurr 1995; Blanco et al. 2006; Moczek & Cochrane 2006). It was verified that it accumulates in the digestive tract of leaf-cutting ant workers (L.C.F., pers. obs.).



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Rhodamine B was tested at concentrations of 0.1, 0.2, 0.3, 0.4, and 0.6% (w/w) in non-toxic baits to evaluate its possible influence on bait retrieval by workers. Although none of the concentrations interfered with retrieval, we chose 0.4% because it facilitates dye solubility in water and because it was the ideal concentration for visual evaluation (see tracer detection method). The toxic bait was prepared as follows: industrial citrus pulp was ground and passed through a sieve; sulfluramid (GX071HB; Griffin Corporation, Valdosta, GA, USA) at 0.3% w/w, soybean oil at 5% w/w and Rhodamine B 0.4% w/w were added. Water was added to the mixture and the mass was extruded into 2.5 mm diameter by 2.0 to 6.0 mm-long pellets and dried in a lab oven at 40ºC.

Dispersal in laboratory colonies

Preliminarily, worker population sample sizes had to be defined for the various size categories, since the ant population of each size category was unknown for our laboratory colonies to be used in this research (about one liter of fungus sponge). To accomplish this, 3 colonies were killed with sulphuric ether and the ants were grouped according to their head width categories (in mm), as set out by Wilson (1980): very small (0.8 to 1.1), small (1.1 to 1.8), medium (1.8 to 3.3), and large (3.3 to 4.6). The population composition per size category was: 2,823 ± 111.0 very small; 763 ± 57.2 small, 638 ± 35.0 medium, and 209 ± 21.1 large workers. In order of tracer evaluation feasibility, based on these data, we defined that about 100 workers were adequate for tracer evaluation for the three smaller size categories and about 50 were adequate for large workers. After sample sizes were established, 12 laboratory colonies received the toxic bait. At 24, 48, 96, and 144 hours after bait application, 3 colonies for each time interval were killed with sulphuric ether and samples of workers were obtained, which were stored at approximately -10ºC. Later, these individuals were examined for dye presence (see tracer detection method).

Dispersal in field colonies

Colonies located at the São Paulo farm (Botucatu, SP, Brazil), in Eucalyptus spp. area, were used. In order to evaluate the effect of time in the worker contamination process, we chose to use two colonies, with loose soil areas of 31 m2 (C1) and 176 m2 (C2). To be sure that the application of bait would be

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done in the tunnels of the selected colonies, entrance holes selected for bait application were preliminarily mapped with pieces of soda straws impregnated with citrus pulp (Fowler et al. 1993). The bait dose applied followed the commercial recommendation (10g/m2 loose soil), and ant samples were obtained from the fungus chambers after 24 and 48 hours (C1 and C2, respectively). The nests were excavated using hand tools at the central portion of the loose soil mound, and samples were obtained down to a 3 m depth in relation to the soil surface. All material contained inside fungus chambers was collected, placed in plastic bags, identified, and then stored at -10°C. The numbers of fungus chambers sampled were 113 and 140 (C1 and C2, respectively). Three to four (C1) or four to five (C2) workers for each category size (very small, small, medium, large) were sampled in each chamber. A total of about 13 or 18 workers from each fungus chamber sampled were separated from the collected material and evaluated for the presence or absence of dye. Consequently, 1,493 and 2,521 individuals (C1 and C2, respectively) were evaluated. The higher amount of workers sampled in C2 was due to the greater size of its fungus chambers compared with C1 and laboratory colonies.

Tracer detection method

Because the Rhodamine B becomes easily impregnated in protein tissues, we chose to use swine intestine because it is practical and easily found. Industrial swine intestine was cut into 30-cm-long pieces and washed with neutral detergent. Then, acrylic strips 35 × 3 cm (length × width) were coated with intestine to facilitate handling and were dried at room temperature (25 ±3 ºC). The workers sampled from the colonies were placed in plastic bags, and then washed with distilled water to remove any dye that might have been attached to the external surface of their bodies. These workers were dried on filter paper and placed onto the acrylic strips coated with swine intestine. We choose to evaluate the dye contained in workers' gasters because most ingested fluids are stocked within this part and dyes appear quickly in this area (Erthal et al. 2004; L.C.F. & N.S.N., pers. obs.). Gasters were squeezed with tweezers over strips. Compressing this body region of the ants causes their digestive tracts to rupture and, if there is any dye inside, it will impreg-



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nate the intestine. After that, the intestine was removed from the strip and exposed to ultraviolet light in a dark environment and the number of dye markings was recorded.

Statistical analysis

The study on the association between size categories, treatments, and labeled and non-labeled workers was conducted using Goodman’s test (G test) for contrasts between and within multinomial populations (Goodman 1964, 1965). Lower case letters were used to indicate results for comparisons between groups within a fixed response category, while upper case letters were used in comparisons between response categories within a group. All considerations in the present work were discussed at the 5% significance level.

RESULTS Laboratory colonies

All bait pellets were carried into the fungus garden in the first 24 hours. The tracer was verified in all category sizes (Table 1). The very small workers differed statistically from the other sizes at the first evaluation periods (24 and 48 h) although not in the later ones (72 and 144 h). A reduction in the percentage of workers with tracers in the colony with time was observed. This can be clearly observed in the 144-hour treatment, which was statistically different from the treatments of 24 and 48 hours for all worker sizes. This reduction was probably due to product elimination, as also verified in fire ants (Wendel & Vinson 1978; Sorensen et al. 1980). The percentage of dyed very small-sized workers decreased more slowly than other worker sizes. The waste of a large part of the colonies was labeled with the dye one hour after applying the bait. Since during this period most colonies have started to incorporate the bait into the fungus, the presence of dye in the waste chamber can potentially be explained by regurgitation of infrabucal pellets in this chamber; this infrabucal material can be produced during removal of undesirable impurities, like microfungi spores, attached in bait granules (Quinlan & Cherrett 1978) or because of intense activity of allo- and self-grooming after the workers became externally dyed from contact with the bait.

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Table 1. Proportions of labeled and non-labeled workers in laboratory colonies of Atta sexdens rubropilosa.

Treatment Category 24 hours Very Small Small Medium Large

Labeled 0.534 a B 0.689 b C 0.744 b C 0.734 b C

48 hours

Very Small Small Medium Large

0.425 a B 0.677 b C 0.626 b BC 0.619 b BC

0.575 b A 0.323 a A 0.374 a AB 0.381 a AB

334 313 313 155

72 hours

Very Small Small Medium Large

0.518 a B 0.452 a B 0.541 a B 0.516 a AB

0.482 a A 0.548 a B 0.459 a B 0.484 a BC

332 310 316 159

144 hours

Very Small Small Medium Large

0.270 a A 0.277 a A 0.324 a A 0.385 a A

0.730 a B 0.723 a C 0.676 a C 0.615 a C

318 336 321 122

Workers

Non-labeled 0.466 b A 0.311 a A 0.256 a A 0.266 a A

Total 322 312 312 154

Lower case letters: comparison between categories (sizes) with fixed treatments (hours) and contamination. Upper case letters: comparison between treatment (hours) with fixed categories (sizes) and contamination.

There was a reduction in leaf cutting 72 and 96 hours after applying the bait, and the onset of intoxication symptoms could be observed. At the end of the experiment (144 hours), we verified great mortality of workers, while survivors were not very active.

Field colonies

The numbers of dyed, medium- and large-sized workers were higher than smaller sizes in the two times, 24 and 48 hours (Table 2), which was the same as in the laboratory colonies.

DISCUSSION If we compare the difference in contamination tendency over time between laboratory and field colonies, it can be hypothetically stated that in field colonies, the tendency is for the distribution and the processing of the toxic bait to be more delayed because their size is greater.

Forti, L.C. et al. — Dispersal of Sulfuramid in Leaf-Cutting Ant Colonies



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Table 2. Proportions of labeled and non-labeled workers in field colonies of Atta sexdens rubropilosa. Treatment Category

Labeled

Workers

Non-labeled

Total

24 hours

Very Small Small Medium Large

0.331 a A 0.330 a A 0.464 b A 0.511 c A

0.669 c A 0.670 c A 0.536 b A 0.489 a B

375 373 375 370

48 hours

Very Small Small Medium Large

0.381 a A 0.427 a A 0.539 b A 0.623 b B

0.619 b A 0.573 b A 0.461 a A 0.377 a A

636 632 633 620

Lower case letters: comparison between categories (sizes) with fixed treatments (hours) and contamination. Upper case letters: comparison between treatment (hours) with fixed categories (sizes) and contamination.

On the other hand, it can also be stated that the final distribution of toxic bait and its AI is very similar in both small laboratory colonies and adult field colonies, with the only difference being the delay in bait load and distribution. This corroborates with the fact that toxic bait evaluation in laboratory colonies uses colonies about half the size of those used in the present work (Forti et al. 1993), in which the time required for distribution of toxic bait is usually lower than an hour, whereas in the field this would take several hours to occur (L.C.F. & N.S.N., pers. obs.). The bait pellets are carried by larger workers to the fungus chamber, where smaller ants work with the pellets (Andrade 2002; Andrade et al. 2002). The fact that the smaller workers are less contaminated initially can be explained by the fact that larger ants are involved in primary activities with substrates, like foraging, and that smaller workers are those more involved in implanting the fungus hyphae on substrates (Andrade et al. 2002). Because the smaller workers are involved in microfungi control, their contamination can be very important in loss of microfungi control, commonly reported in baited colonies (Forti et al. 1998; Nagamoto et al., 2003; Rodrigues et al. 2005b). To attempt to control detrimental microorganisms, the workers spend a large amount of time licking: a) the symbiotic fungus hyphae, b) themselves

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and each other, and c) the substrate (Stahel & Geijskes 1939; Currie & Stuart 2001; Currie 2001; Andrade et al. 2002). For the substrate, licking activity is also important to remove waxes which can impair the fungal cultivation (Garcia et al. 2005). Processing the substrate in small pieces and hydrating it for incorporation into the fungus garden is also time-consuming (Andrade 2002; Andrade et al. 2002). This pattern of intensive substrate licking and treatment applies to all attractive substrates, including toxic baits, so it can be stated these activities are crucial for the toxic baits' AI to contaminate the workers (Andrade 2002; Andrade et al. 2002; Nagamoto et al. 2004). These intensive activities also serve to explain the high percentage of dyed workers. Trophallaxis could be an alternative explanation for dispersal in other ants; however, most authors state that trophallaxis is less prevalent in leaf-cutting ants when compared with ant genera that typically forage on liquids (Andrade et al. 2002; Paul & Roces 2003; Nagamoto et al. 2004). Although it is known that workers distribute bait pellets uniformly through almost all fungus chambers (Pretto & Forti 2000), colony size under field conditions is a trait that must not be overlooked, as some of the colonies may contain thousands of fungus chambers (Autuori 1947; Moreira et al. 2004). Faster acting AIs may decrease the bait distribution and also improve its rejection (Camargo et al. 2003; Forti et al. 2003, Lopes et al. 2003), lowering the efficiency of these baits in larger field colonies. The observed contamination of small, medium, and large workers demonstrates that worker contact with toxic bait particles or already dyed fungus or labeled workers is intense within the first 24 hours, possibly enough to cause the colony to because of synergism with the loss of control of the aggressive microfungi in the fungal sponge, which quickly overcomes the symbiotic fungus (Rodrigues et al. 2005a; Camargo et al. 2006). It would be interesting to know if there are some worker size classes that are more important for colony control than others, because it is known that in faster-acting AIs the bait rejection rate is higher, meaning the smaller workers are potentially less contaminated, allowing them to maintain control of the microfungi.



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Concluding remarks Our methodology, because of the similar results in both small laboratory colonies and huge field colonies, can be considered good enough for purposes of toxic bait AI dispersal studies. The dispersal and contamination dynamics in leaf-cutting ant colonies are important for investigations on the mode of action of delayed-action AIs (like sulfluramid) in the colonies, and as a reference in future studies such as those attempting to reduce the concentration of this insecticide in commercial baits aiming to reduce impacts on the environment. For example, it is possible that reducing the AI concentration in a bait would not necessarily mean that the insecticide concentration dispersed in workers decreases to a point that its effectiveness is compromised. Future research into the possible synergism with toxic bait and microfungi is also important for better understanding the action of toxic baits for leaf-cutting ants. Comparisons of the present work with future studies on the dispersal of other AIs may contribute toward an understanding about the mechanisms of action of toxic baits in the colonies, facilitating the development of new commercial baits. Sulfluramid is currently suitable to be used as a leaf-cutting ant toxic bait AI, since it has delayed action over a wide range of concentrations (Nagamoto et al. 2004), is widely distributed in small to adult colonies and among the four workers size classes (this work), and is environmentally acceptable (Forti et al. 1998).

ACKNOWLEDGMENTS We are grateful to Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for a fellowship granted to the senior author (grant 301167/2003-6); to Nelson Carneiro, José Carlos Santos (in memoriam), and Everaldo Correa for collaboration in the field work; to the Atta Kill company for financial assistance (in part), and to Brian Taylor of CSU, Chico, for assistance with the translation.

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