First large-scale study reveals important losses of managed honey bee and stingless bee colonies in Latin America.

Over the last quarter century, increasing honey bee colony losses motivated standardized large-scale surveys of managed honey bees (Apis mellifera), particularly in Europe and the United States. Here we present the first large-scale standardized survey of colony losses of managed honey bees and stingless bees across Latin America. Overall, 1736 beekeepers and 165 meliponiculturists participated in the 2-year survey (2016-2017 and 2017-2018). On average, 30.4% of honey bee colonies and 39.6% of stingless bee colonies were lost per year across the region. Summer losses were higher than winter losses in stingless bees (30.9% and 22.2%, respectively) but not in honey bees (18.8% and 20.6%, respectively). Colony loss increased with operation size during the summer in both honey bees and stingless bees and decreased with operation size during the winter in stingless bees. Furthermore, losses differed significantly between countries and across years for both beekeepers and meliponiculturists. Overall, winter losses of honey bee colonies in Latin America (20.6%) position this region between Europe (12.5%) and the United States (40.4%). These results highlight the magnitude of bee colony losses occurring in the region and suggest difficulties in maintaining overall colony health and economic survival for beekeepers and meliponiculturists.

When a Tritrophic Interaction Goes Wrong to the Third Level: Xanthoxylin From Trees Causes the Honeybee Larval Mortality in Colonies Affected by the River Disease.

The "River Disease" (RD), a disorder impacting honeybee colonies located close to waterways with abundant riparian vegetation (including Sebastiania schottiana, Euphorbiaceae), kills newly hatched larvae. Forager bees from RD-affected colonies collect honeydew excretions from Epormenis cestri (Hemiptera: Flatidae), a planthopper feeding on trees of S. schottiana. First-instar honeybee larvae fed with this honeydew died. Thus, we postulated that the nectars of RD-affected colonies had a natural toxin coming from either E. cestri or S. schottiana. An untargeted metabolomics characterization of fresh nectars extracts from colonies with and without RD allowed to pinpoint xanthoxylin as one of the chemicals present in higher amounts in nectar from RD-affected colonies than in nectars from healthy colonies. Besides, xanthoxylin was also found in the aerial parts of S. schottiana and the honeydew excreted by E. cestri feeding on this tree. A larva feeding assay where xanthoxylin-enriched diets were offered to 1st instar larvae showed that larvae died in the same proportion as larvae did when offered enriched diets with nectars from RD-colonies. These findings demonstrate that a xenobiotic can mimic the RD syndrome in honeybee larvae and provide evidence of an interspecific flow of xanthoxylin among three trophic levels. Further, our results give information that can be considered when implementing measures to control this honeybee disease.

Effects of Synthetic Acaricides and Nosema ceranae (Microsporidia: Nosematidae) on Molecules Associated with Chemical Communication and Recognition in Honey Bees.

Acaricides and the gut parasite Nosema ceranae are commonly present in most productive hives. Those stressors could be affecting key semiochemicals, which act as homeostasis regulators in Apis mellifera colonies, such as cuticular hydrocarbons (CHC) involved in social recognition and ethyl oleate (EO) which plays a role as primer pheromone in honey bees. Here we test the effect of amitraz, coumaphos, tau-fluvalinate and flumethrin, commonly applied to treat varroosis, on honey bee survival time, rate of food consumption, CHC profiles and EO production on N. ceranae-infected and non-infected honey bees. Different sublethal concentrations of amitraz, coumaphos, tau-fluvalinate and flumethrin were administered chronically in a syrup-based diet. After treatment, purified hole-body extracts were analyzed by gas chromatography coupled to mass spectrometry. While N. ceranae infection was also shown to decrease EO production affecting survival rates, acaricides showed no significant effect on this pheromone. As for the CHC, we found no changes in relation to the health status or consumption of acaricides. This absence of alteration in EO or CHC as response to acaricides ingestion or in combination with N. ceranae, suggests that worker honey bees exposed to those highly ubiquitous drugs are hardly differentiated by nest-mates. Having determined a synergic effect on mortality in worker bees exposed to coumaphos and Nosema infection but also, alterations in EO production as a response to N. ceranae infection it is an interesting clue to deeper understand the effects of parasite-host-pesticide interaction on colony functioning.

Sub-lethal effects of the consumption of Eupatorium buniifolium essential oil in honeybees.

When developing new products to be used in honeybee colonies, further than acute toxicity, it is imperative to perform an assessment of risks, including various sublethal effects. The long-term sublethal effects of xenobiotics on honeybees, more specifically of acaricides used in honeybee hives, have been scarcely studied, particularly so in the case of essential oils and their components. In this work, chronic effects of the ingestion of Eupatorium buniifolium (Asteraceae) essential oil were studied on nurse honeybees using laboratory assays. Survival, food consumption, and the effect on the composition of cuticular hydrocarbons (CHC) were assessed. CHC were chosen due to their key role as pheromones involved in honeybee social recognition. While food consumption and survival were not affected by the consumption of the essential oil, CHC amounts and profiles showed dose-dependent changes. All groups of CHC (linear and branched alkanes, alkenes and alkadienes) were altered when honeybees were fed with the highest essential oil dose tested (6000 ppm). The compounds that significantly varied include n-docosane, n-tricosane, n-tetracosane, n-triacontane, n-tritriacontane, 9-tricosene, 7-pentacosene, 9-pentacosene, 9-heptacosene, tritriacontene, pentacosadiene, hentriacontadiene, tritriacontadiene and all methyl alkanes. All of them but pentacosadiene were up-regulated. On the other hand, CHC profiles were similar in healthy and Nosema-infected honeybees when diets included the essential oil at 300 and 3000 ppm. Our results show that the ingestion of an essential oil can impact CHC and that the effect is dose-dependent. Changes in CHC could affect the signaling process mediated by these pheromonal compounds. To our knowledge this is the first report of changes in honeybee cuticular hydrocarbons as a result of essential oil ingestion.

Phenolic Fingerprinting, Antioxidant, and Deterrent Potentials of Persicaria maculosa Extracts.

Persicaria maculosa (Polygonaceae) (known as lady's thumb) is an annual morphologically variable weed that is widely distributed in Chile. The purpose of this study was to investigate the antifeedant potential of methanolic (MeOH), ethanolic (EtOH), and dichloromethane (DCM) extracts from the aerial parts of this plant collected in the Valparaíso and Curicó provinces (Chile) and relate this activity to the antioxidant capacity and the presence of phenolic compounds in the extracts. A phenolic profile based on HPLC-ESI-MS/MS allowed the identification of 26 phenolic compounds, most of them glycosyl derivatives of isorhamnetin, quercetin, and kaempferol. In addition, the total phenolic content (TP), total flavonoids (TF), and antioxidant activity measured by 2,2-diphenyl-1-picrylhydrazyl (DPPH), superoxide anion scavenging (O2-), ferric-reducing antioxidant power (FRAP), and cupric-reducing antioxidant capacity (CUPRAC) of the extracts are reported. The antifeedant potentials of the plant extracts were tested against Epilachna paenulata, Pseudaletia adultera, Macrosiphum euphorbiae, and Diaphorina citri insects for the first time. The activity against the aphid M. euphorbiae was significant for the DCM extracts of plants from Valparaíso and Curicó (settling % = 23% ± 4% and 23% ± 5%, respectively). The antifeedant activities against the beetle E. paenulata and the lepidoptera P. adultera were significant for Valparaíso extracts, especially when tested against E. Paenulata (IFP = 1.0 ± 0.0). Finally, the MeOH and EtOH extracts from Valparaíso plants reduced the diet consumption of the psilid D. citri (p < 0.05). The results showed that P. maculosa is a good source of flavonoids with some antioxidant capacities and has potential interest as botanical eco-friendly alternative with deterrent activity.

Chemical Composition, Antimicrobial Activity, and Mode of Action of Essential Oils against Paenibacillus larvae, Etiological Agent of American Foulbrood on Apis mellifera.

This study aimed to characterize the chemical composition of Aloysia polystachia, Acantholippia seriphioides, Schinus molle, Solidago chilensis, Lippia turbinata, Minthostachys mollis, Buddleja globosa, and Baccharis latifolia essential oils (EOs), and to evaluate their antibacterial activities and their capacity to provoke membrane disruption in Paenibacillus larvae, the bacteria that causes the American Foulbrood (AFB) disease on honey bee larvae. The relationship between the composition of the EOs and these activities on P. larvae was also analyzed. Monoterpenes were the most abundant compounds in all EOs. All EOs showed antimicrobial activity against P. larvae and disrupted the cell wall and cytoplasmic membrane of P. larvae provoking the leakage of cytoplasmic constituents (with the exception of B. latifolia EO). While, the EOs' antimicrobial activity was correlated most strongly to the content of pulegone, carvone, (Z)-ß-ocimene, δ-cadinene, camphene, terpinen-4-ol, elemol, ß-pinene, ß-elemene, γ-cadinene, α-terpineol, and bornyl acetate; the volatiles that better explained the membrane disruption were carvone, limonene, cis-carvone oxide, pentadecane, trans-carvyl acetate, trans-carvone oxide, trans-limonene oxide, artemisia ketone, trans-carveol, thymol, and γ-terpinene (positively correlated) and biciclogermacrene, δ-2-carene, verbenol, α-pinene, and α-thujene (negatively correlated). The studied EOs are proposed as natural alternative means of control for the AFB disease.

Essential oil from Eupatorium buniifolium leaves as potential varroacide.

Beekeeping has experienced a great expansion worldwide. Nowadays, several conventional pesticides, some organic acids, and essential oil components are the main means of chemical control used against Varroa destructor, an ectoparasite that may contribute to the colony collapse disorders. Varroa resistance against conventional pesticides has already been reported; therefore it is imperative to look for alternative control agents to be included in integrated pest management programs. A good alternative seems to be the use of plant essential oils (EOs) which, as natural products, are less toxic and leave fewer residues. Within this context, a bioprospecting program of the local flora searching for botanical pesticides to be used as varroacides was launched. A primary screening (driven by laboratory assays testing for anti-Varroa activity, and safety to bees) led us to select the EOs from Eupatorium buniifolium (Asteraceae) for follow up studies. We have chemical characterized EOs from twigs and leaves collected at different times. The three E. buniifolium EOs tested were active against Varroa in laboratory assays; however, there are differences that might be attributable to chemical differences also found. The foliage EO was selected for a preliminary field trial (on an experimental apiary with 40 hives) that demonstrated acaricidal activity when applied to the hives. Although activity was less than that for oxalic acid (the positive control), this EO was less toxic to bees than the control, encouraging further studies.

Chemical modification produces species-specific changes in cucurbitacin antifeedant effect.

Cucurbitacins are secondary metabolites that mediate insect plant interactions not only as allomones against generalists but also as kairomones for specialist herbivores. This study was undertaken to identify the potential of cucurbitacin derivatives as insect antifeedant agents. The antifeedant capacity against a Cucurbitaceae specialist [ Epilachna paenulata (Coleoptera: Coccinellidae)] and a polyphagous insect [ Pseudaletia adultera (Lepidoptera: Noctuidae)] was evaluated in preference tests in which the insects were given a choice between food plants either treated with the cucurbitacin derivatives or treated with the solvent. The activity was found not to be related to the basic cucurbitacin skeleton, as only 15 of the 28 tested cucurbitacin derivatives were active. Only one of the tested compounds was phagostimulant to the specialist insect (the hemissuccinate of 16-oxo-dihydrocucurbitacin B derivative), while all other active derivatives were deterrent against one of the insects (13 compounds) or both of them (3 compounds). Changes in ring A of the cucurbitacins, as well as in the side chain, modified the activity. As a general trend, when chemical modifications of the basic structure produced a change in activity, the response was opposite in both insects used as biodetectors, indicating that a selective variation in the activity may be achieved by chemical modifications of the cucurbitacin skeleton.

Bignoniaceae metabolites as semiochemicals.

Members of the family Bignoniaceae are mostly found in tropical and neo-tropical regions in America, Asia and Africa, although some of them are cultivated in other regions as ornamentals. Species belonging to this family have been extensively studied in regard to their pharmacological properties (as extracts and isolated compounds). The aim of this review is to summarize the reported scientific evidence about the chemical properties as well as that of the extracts and isolated compounds from species of this family, focusing mainly in insect-plant interactions. As it is known, this family is recognized for the presence of iridoids which are markers of oviposition and feeding preference to species which have became specialist feeders. Some herbivore species have also evolved to the point of been able to sequester iridoids and use them as defenses against their predators. However, iridoids also exhibit anti-insect properties, and therefore they may be good lead molecules to develop botanical pesticides. Other secondary metabolites, such as quinones, and whole extracts have also shown potential as anti-insect agents.

Formate analogs as antagonists of the sex pheromone of the honeydew moth, Cryptoblabes gnidiella: electrophysiological, behavioral and field evidence.

Cryptoblabes gnidiella Milliére (Lepidoptera: Pyralidae) is an economically important exotic pest of vineyards in Southern Brazil and Uruguay. The sex pheromone of C. gnidiella was identified as a mixture of (Z)-11-hexadecenal and (Z)-13-octadecenal, and has been used to monitor populations of this pest in Israel. The development of mating disruption for this species may be hampered by the chemical instability of the natural pheromone components. Therefore, studies on more stable pheromone analogs may provide tools for a control strategy based on behavior-modifying chemicals. We report here the electrophysiological and behavioral responses of C. gnidiella males to (Z)-9-tetradecenyl formate and (Z)-11-hexadecenyl formate, structural analogs of the pheromone components. In gas chromatography-electroantennogram detection (GC-EAD) studies, both analogs elicited responses from C. gnidiella male antennae. Pre-exposure to the formates did not affect the subsequent EAD responses to the natural pheromone components. The formates acted as pheromone antagonists in wind tunnel tests, inhibiting the responses of males toward both synthetic pheromone and calling females. In the field, captures of males in pheromone-baited traps decreased, in a dose-response pattern, when different amount of formates were added to the pheromone. These pheromone antagonists, thus, are potentially useful as mating disruptants for C. gnidiella in commercial vineyards.

Clytostoma callistegioides (Bignoniaceae) wax extract with activity on aphid settling.

A bioassay-guided fractionation of leaf extracts from Clytostoma callistegioides (Cham.) Bureau ex Griseb. (Bignoniaceae) led to isolation of a natural mixture of four fatty acids with anti-insect activity against aphids. The compounds were identified by GC-MS as palmitic, stearic, linoleic and linolenic acids and quantified as their methyl esters. The anti-aphid activity of the natural mixture was traced to linolenic and linoleic acids, as shown by the settling inhibition activity of synthetic samples. Interestingly, the saturated acids (palmitic and stearic) tested alone stimulated settling on one of the tested aphids (Myzus persicae), but not on the other tested species (Rhopalosiphum padi). Although ubiquitous, none of these free acids have been previously reported in this Bignoniaceae species. The leaf surface chemistry, which is likely involved in modulating aphid settling behavior, was further investigated for the occurrence of lipophilic substances by histochemical staining. Short, stalked glandular trichomes, previously undescribed for this species, stained with osmium tetroxide and Sudan III, suggesting that the secretion of the defensive acids is related to these surface trichomes.

Reproductive behaviour of Crocidosema (=Epinotia) aporema (Walsingham) (Lepidoptera: Tortricidae): temporal pattern of female calling and mating.

Crocidosema aporema (Walsingham) is a major pest of legumes in the southern cone of Latin America. The mating behaviour of two allopatric populations (Uruguay and Brazil) of C. aporema kept in captivity was studied by observing the posture of calling females, the temporal pattern of pheromone emission and mating, and the response of males to calling females in olfactometer tests. Female calling and mating was observed during the scotophase, from the first to the seventh night after adult emergence. Male response was evaluated at night using a single calling female in a Y-shaped olfactometer. Females adopted a characteristic calling posture, extruding the pheromone gland from the tip of the abdomen. Most females started calling during the second scotophase, and all females called from the third, between the fifth and seventh hours after the onset of the scotophase. Most of the couples mated once throughout the experiment, between the third and sixth night and during the middle of the dark phase. Males preferentially chose the female arm in olfactometer tests, considering both the first arm chosen and the number of visits during the observation period. Our results describe for the fi rst time the temporal pattern associated to the reproductive behaviour of C. aporema. We also provide evidence that this tortricid is monoandrous, and that pheromones are used in intersexual communication for mate finding. Our data will be used to optimize the collection of female sex pheromones for chemical characterization in order to develop a monitoring tool for this pest.

Reproductive behaviour of Crocidosema (=Epinotia) aporema (Walsingham) (Lepidoptera: Tortricidae): temporal pattern of female calling and mating

Crocidosema aporema (Walsingham) is a major pest of legumes in the southern cone of Latin America. The mating behaviour of two allopatric populations (Uruguay and Brazil) of C. aporema kept in captivity was studied by observing the posture of calling females, the temporal pattern of pheromone emission and mating, and the response of males to calling females in olfactometer tests. Female calling and mating was observed during the scotophase, from the first to the seventh night after adult emergence. Male response was evaluated at night using a single calling female in a Y-shaped olfactometer. Females adopted a characteristic calling posture, extruding the pheromone gland from the tip of the abdomen. Most females started calling during the second scotophase, and all females called from the third, between the fifth and seventh hours after the onset of the scotophase. Most of the couples mated once throughout the experiment, between the third and sixth night and during the middle of the dark phase. Males preferentially chose the female arm in olfactometer tests, considering both the first arm chosen and the number of visits during the observation period. Our results describe for the fi rst time the temporal pattern associated to the reproductive behaviour of C. aporema. We also provide evidence that this tortricid is monoandrous, and that pheromones are used in intersexual communication for mate finding. Our data will be used to optimize the collection of female sex pheromones for chemical characterization in order to develop a monitoring tool for this pest.

Sex pheromone of the bud borer Epinotia aporema: chemical identification and male behavioral response.

Epinotia aporema (Walsingham) is a Neotropical pest of legumes in southern South America. Its importance has increased during the last decade owing to the significant growth of soybean production in the region. Monitoring of E. aporema is difficult due to the cryptic behavior of the larvae, and hence, chemical control is carried out preventively. We analyzed the female-produced sex pheromone so as to develop monitoring traps and explore pheromone-based control methods. We analyzed pheromone gland extracts by combined chromatographic, spectrometric, and electrophysiological methods. Based on the comparison of retention indices, mass spectra, and electroantennogram (EAD) activity of the insect-produced compounds with those of synthetic standards, we identified two EAD-active compounds, (Z,Z)-7,9-dodecadienol and (Z,Z)-7,9-dodecadienyl acetate (15:1 ratio), as sex pheromone components of E. aporema. We also studied the behavior of males in wind tunnel tests using virgin females and different combinations of synthetic standards (15:1, 1:1, and 1:0 alcohol/acetate) as stimuli. A significantly greater percentage of males reached the chemical source with the 15:1 synthetic mixture than with any of the other treatments, indicating that these two compounds are pheromone components.

Screening of Uruguayan plants for deterrent activity against insects.

We evaluated the anti-insectan activity of extracts from different vegetative parts of ten plant species native to Uruguay. The selected plants belong to five families: Bignoniaceae: Clytostoma callistegioides, Dolichandra cynanchoides, Macfadyena unguis-cati; Sapindaceae: Dodonaea viscosa, Allophylus edulis, Serjania meridionalis; Lamiaceae: Salvia procurrens, Salvia guaranitica; Solanaceae: Lycium cestroides; and Phytolaccaceae: Phytolacca dioica. The extracts were evaluated in independent bioassays against four insect pests and one beneficial insect. Aphid settling inhibition was evaluated with a grass specialist, Rhopalosiphum padi, and a feeding generalist, Myzus persicae (both Hemiptera: Aphididae). Antifeedant activity was tested with adults of the specialist Epilachna paenulata (Coleoptera: Coccinellidae) and larvae of the generalist Spodoptera littoralis (Lepidoptera: Noctuidae). Finally, contact toxicity was assessed with honey bees, Apis mellifera (Hymenoptera: Apidae). Strong settling inhibition (SI) activity (expressed as %SI, where 100% means complete inhibition by the extract) was found only for the twig extracts of A. edulis (Sapindaceae) against M. persicae (% SI = 77 +/- 4). Antifeedant activity (expressed as % of feeding reduction (FR), where 100% means no consumption on extract-treated diet) against E. paenulata was significant for the leaf extracts of L. cestroides (Solanaceae) (% FR = 100 +/- 0) as well as of all Bignoniaceae and Sapindaceae species. No extracts were active against S. littoralis larvae, and most of them were innocuous to honey bees, with the exception of L. cestroides and S. meridionalis leaf extracts.

Biparental endowment of endogenous defensive alkaloids in Epilachna paenulata.

Coccinellid beetles contain a variety of defensive alkaloids that render them unpalatable to predators. Epilachna paenulata (Coleoptera: Coccinellidae) is a South American ladybird beetle that feeds on plants of the Cucurbitaceae family. The defensive chemistry of E. paenulata has been characterized as a mixture of systemic piperidine, homotropane, and pyrrolidine alkaloids. Whole body extracts of adult beetles contain four major alkaloids: 2-(2'-oxopropyl)-6-methylpiperidine (1); 1-(6-methyl-2,3,4,5-tetrahydro-pyridin-2-yl)-propan-2-one (2); 1-methyl-9-azabicyclo[3.3.1]nonan-3-one (3); and 1-(2''-hydroxyethyl)-2-(12'-aminotridecyl)-pyrrolidine (4). Comparative studies of the defensive chemistry of eggs, larvae, pupae, and adults showed differences in alkaloid composition and concentration among life stages. While adults contained mainly the homotropane 1-methyl-9-azabicyclo[3.3.1]nonan-3-one (3), eggs showed the highest concentration of the piperidine 2-(2'-oxopropyl)-6-methylpiperidine (1). We studied the origin of this alkaloid in the eggs by feeding newly emerged, virgin adult beetles with [2-(13)C]-labeled acetate, and by performing crosses between (13)C-fed and unlabeled males and females. GC-MS analysis of alkaloids from (13)C-fed males and females showed high incorporation of (13)C into the alkaloids, as evidenced from a 20-30% increase of isotopic peaks in diagnostic fragment ions, confirming the expected endogenous origin of these alkaloids. In addition, analyses of eggs from different crosses showed that labeled alkaloids from both parents are incorporated into eggs, indicating that E. paenulata males transfer alkaloids to the females at mating. Biparental endowment of chemical defenses into eggs has been shown previously in insects that acquire defensive compounds from dietary sources. To our knowledge, this is the first report of biparental egg endowment of endogenous defenses.

Enantiospecific synthesis and insect feeding activity of sulfur-containing cyclitols.

The first syntheses of two deoxythiocyanocyclitols (4-deoxy-4-thiocyano-L-chiro-inositol and deoxythiocyanoconduritol F) and two deoxysulfonylcyclitol acetals are reported by a chemoenzymatic enantioselective route. The compounds were prepared by a sequence of enzymatic and ruthenium-catalyzed dihydroxylations, and the results were studied regarding reaction conditions and co-catalyst for different derivatives. The new compounds were included in a minilibrary of deoxygenated cyclitols and evaluated for their capacity to influence the feeding behavior of Epilachna paenulata (Coleoptera: Coccinellidae), a common pest of the Curcubitaceae crops.

First record of L-quebrachitol in Allophylus edulis (Sapindaceae).

Allophylus edulis, commonly called 'Chal chal', is a member of the Sapindaceae occurring in the Uruguayan and Brazilian native flora. During the phytochemical analysis of two Chal chal specimens from two well-differentiated geographical zones (Assis, São Paulo, Brazil, and Santa Lucía, Canelones, Uruguay), considerable amounts of L-quebrachitol were isolated from both samples. The isolation was carried out from the ethanolic twig extracts obtained by maceration of both vegetal samples. White easily distinguishable crystals were mechanically separated, washed, and characterized by 1D and 2D NMR experiments and by MS data. Such techniques confirmed that the crystals isolated from sources collected in both countries resulted in the same compound, l-quebrachitol, a natural product not previously reported for this species and one that has been investigated as a sugar substitute for diabetics. Worthy of note, the content of L-quebrachitol in A. edulis may be the chemical basis to explain its ethnobotanical uses, since infusions of this plant are used to treat diabetes in the practice of local traditional medicine.

Chemical defense of an opilionid (Acanthopachylus aculeatus).

The opilionid Acanthopachylus aculeatus was shown to produce a defensive secretion containing quinones (2,3-dimethyl-1,4-benzoquinone, 2,5-dimethyl-1,4-benzoquinone and 2,3,5-trimethyl-1,4-benzoquinone), confirming the findings reported nearly a half century ago in a classic study. The mechanism by which the opilionid puts the secretion to use is described. When disturbed, the animal regurgitates enteric fluid, which it conveys by intercoxal clefts to the anterolateral corners of the carapace, where the two gland openings are situated. It then injects some of its quinonoid secretion into the fluid, and conveys the mixed liquid along the length of its flanks by way of two special channels. Such a discharge mechanism may be widespread among opilionids of the family Gonyleptidae (suborder Laniatores), to which A. aculeatus belongs. In a bioassay based on a scratch reflex in decapitated cockroaches (Periplaneta americana) the liquid effluent of A. aculeatus was shown to be potently irritating. Use of the effluent was demonstrated to protect the opilionid against ants (Formica exsectoides). Wolf spiders (Lycosa ceratiola) were shown to be minimally affected by the effluent (they showed little response when the fluid was added to their mouthparts as they fed on mealworms, their normal laboratory prey), although they proved to be aversive to mere contact with the opiliond itself, and to reject the animal without inducing it to discharge. A. aculeatus may therefore contain distasteful factors besides its glandular products.