Toxic associations : A review of the predatory behaviors of millipede assassin bugs ( Hemiptera : Reduviidae : Ectrichodiinae )

Millipedes (Diplopoda) are a diverse group of arthropods that include 16 orders represented by approximately 12,000 species in 145 families (Sierwald & Bond, 2007). In 11 of these orders, millipedes are protected from predators by chemical defenses produced in glands that vary in number among orders and are located laterally or mid-dorsally in the diplosegments (Eisner et al., 1978; Hopkin & Read, 1992). The major defense components of these secretions seem to exhibit a phylogenetic pattern: Julida, Spirostreptida, and Spirobolida (Superorder Juliformia) produce benzoquinones; Callipodida and Chordeumatida produce phenols; Glomerida and Polyzoniida produce alkaloids and quinazolinones; Polydesmida produce cyanogenic compounds; and Platydesmida, Siphonocryptida, and Siphonophorida produce terpenoids (Eisner et al., 1978; Hopkin & Read, 1992; Sierwald & Bond, 2007). Despite such defenses, many vertebrates prey on millipedes (Sierwald & Bond, 2007). In addition, some invertebrates are specialized predators or parasitoids on juliform millipedes (Banks, 1911; Picard, 1930; Lawrence, 1984; Eisner et al., 1998; Dejean et al., 2001; Brunke et al., 2009; Larsen et al., 2009). None of these groups of millipede specialists are particularly speciose. This is dramatically different in the Ectrichodiinae, the fifth largest subfamily of Reduviidae (Hemiptera: Heteroptera), or assassin bugs, that appears to be specialized on millipedes (Green, 1925; Haridass & Ananthakrishnan, 1980; Haridass, 1985). The circumtropical millipede assassin bugs comprise >660 species in 123 genera (Maldonado, 1990; Dougherty, 1995; Carpintero & Maldonado, 1996; Weirauch et al., 2009; Chlond, 2010). Species have been collected while feeding on millipedes, but details of the predatorprey relationships, including prey specificity and point of mouthpart insertion are largely undocumented. Thus, it is unknown if Ectrichodiinae target the millipede’s nervous system to maximize the impact of their toxic saliva. It is also unknown if Ectrichodiinae target the body regions that possess defensive glands to sequester their own defensive compounds, or if they avoid the millipedes’ defense glands. In addition, predation in Ectrichodiinae may be a communal activity (Haridass & Ananthakrishnan, 1980; Haridass, 1985) rather than a solitary behavior as usually observed in Reduviidae (Readio, 1927; McMahan, 1983; Li et al., 2010), but details on this unusual subsocial behavior are scarce. Here, we compile published data and evaluate images available on the Internet for prey specificity, point of stylet insertion, and communal predation. In addition, data for Rhiginia cinctiventris (Stål, 1872) are presented based on observations in the field and laboratory at one site in Costa Rica; feeding observations have never been reported for this genus before. Literature and original data were analyzed in response to three questions: (1) Does the literature data suggest a pattern of Ectrichodiinae prey preference for species in particular millipedes orders? (2) Assuming that Ectrichodiinae either target or circumvent the millipede’s defense glands, where do they insert their mandibular and maxillary stylets to inject toxic saliva and digestive enzymes? (3) Are communal predation strategies commonly observed in nymphs and adults? REVIEW Eur. J. Entomol. 109: 147–153, 2012 http://www.eje.cz/scripts/viewabstract.php?abstract=1691 ISSN 1210-5759 (print), 1802-8829 (online)

The circumtropical millipede assassin bugs comprise >660 species in 123 genera (Maldonado, 1990;Doug-herty, 1995;Carpintero & Maldonado, 1996;Weirauch et al., 2009;Chlond, 2010).Species have been collected while feeding on millipedes, but details of the predatorprey relationships, including prey specificity and point of mouthpart insertion are largely undocumented.Thus, it is unknown if Ectrichodiinae target the millipede's nervous system to maximize the impact of their toxic saliva.It is also unknown if Ectrichodiinae target the body regions that possess defensive glands to sequester their own defensive compounds, or if they avoid the millipedes' defense glands.In addition, predation in Ectrichodiinae may be a communal activity (Haridass & Ananthakrishnan, 1980;Haridass, 1985) rather than a solitary behavior as usually observed in Reduviidae (Readio, 1927;McMahan, 1983;Li et al., 2010), but details on this unusual subsocial behavior are scarce.
Here, we compile published data and evaluate images available on the Internet for prey specificity, point of stylet insertion, and communal predation.In addition, data for Rhiginia cinctiventris (Stål, 1872) are presented based on observations in the field and laboratory at one site in Costa Rica; feeding observations have never been reported for this genus before.Literature and original data were analyzed in response to three questions: (1) Does the literature data suggest a pattern of Ectrichodiinae prey preference for species in particular millipedes orders?(2) Assuming that Ectrichodiinae either target or circumvent the millipede's defense glands, where do they insert their mandibular and maxillary stylets to inject toxic saliva and digestive enzymes?(3) Are communal predation strategies commonly observed in nymphs and adults?

LITERATURE REVIEW AND ANALYSIS OF ONLINE IMAGE MATERIAL
Publications and online images containing information on Ectrichodiinae feeding behavior were evaluated for information regarding ectrichodiine and millipede taxa, ectrichodiine developmental stage, stylet insertion, and communal predation (Table 1).Even though some references identified millipedes to genus or species (Cachan, 1952;Miller, 1953;Haridass & Ananthakrishnan, 1980;Lawrence, 1984;Giliomee, 1985;Ambrose, 1999), the validity of these names have changed over the years and identification without voucher material is tentative.Thus, we focus on establishing a pattern based on the prey order level instead.(http://www.whatsthatbug.com/2008/08/22/millipede-assassin-bugs-from-south-africa/; accessed 18 Oct. 2011) took images used in the analysis and gave permission to publish the data and images (Fig. 1).Two images did not specify the photographer or provide contact information; these images were found on the following websites: http://www.flickr.com/photos/12639178@N07/5631578778/ (accessed 18 Oct. 2011) and http://www.whatsthatbug.com/2008/08/22/millipedeassassin-bugs-from-south-africa/(accessed 18 Oct. 2011).

FIELD AND LABORATORY PROCEDURES
Specimens of Rhiginia cinctiventris were collected at La Selva Biological Research Station, Costa Rica (10.430862°N 84.006467°W) from August 9-15, 2010.Ectrichodiinae and millipedes were placed individually into petri dishes or larger containers with moist tissue.Immature ectrichodiines were determined to be the same species as adults based on coloration and proximity to adults.Sex and developmental stages were determined prior to feeding observations.Individual specimens or groups of individuals were exposed to millipedes representing the orders Spirobolida and Polydesmida, resulting in 25 trials.For each trial, specimens were removed after 20 min if the ectrichodiine(s) did not approach, grip, or probe the millipede.If individuals interacted with the millipede, observations continued until the individual(s) had ceased these interactions for more than 15 min or until after feeding.All ectrichodiine specimens and one representative of each millipede species were preserved in 100% ethanol, barcode labeled, databased, and deposited at the Entomological Research Museum at the University of California, Riverside (UCR).

ECTRICHODIINAE PREY SPECIFICITY
Representatives of 12 Ectrichodiinae genera have been documented in the literature to feed on millipedes in 42 separate observations (Table 1).Seventeen observations involved millipedes in the order Spirostreptida, six in Spirobolida, and one in Polydesmida.Six additional observations included millipedes that could only be identified to the superorder Juliformia, based on identifications made by Rowland Shelley (some online images) and identification keys (Loomis, 1968; Sierwald, http://archive.fieldmuseum.org/millipeet/milli_key.html; accessed 18 Oct. 2011).Two of the 25 laboratory observations and one field observation made by the senior author indicate that Rhiginia cinctiventris preys on Spirobolida and Polydesmida.Although feeding was not observed directly in the field, observation of an adult female and two late instar (3-5) nymphs with swollen abdomens in close proximity to a dead spirobolid millipede (Fig. 2A) indicate a recent meal.
The observation that only three of the 16 millipede orders have been reported as prey of Ectrichodiinae may be, in part, due to a lack of data or may be based on the fact that these orders have high species diversity (Sierwald & Bond, 2007).However, this observation may also hint to a potential prey preference for the benzoquinoneproducing Juliformia over cyanogenic Polydesmida.The relative toxicity of benzoquinones and cyanogenic compounds to ectrichodiines is unknown.Based on material safety data sheets, benzoquinone has an LD50 of 25 mg/kg (mouse; oral), whereas hydrogen cyanide has an LD50 of 3.7 mg/kg (mouse; oral).The degree of toxicity between the two compounds may be relatively similar for ectrichodiines.

POINT OF STYLET INSERTION
From the literature, only 18 observations made reference to the millipede's body region attacked by ectrichodiines, all of which were on the trunk.Fourteen of these stated that the ventral or ventro-lateral area was targeted (Figs 1C, 1D, 1F, 1J), with 8 of these attacks at intersegmental membranes (Fig. 1I).Four less specific observations by Miller (1953) indicated that ectrichodiines targeted trunk intersegmental membranes, and Lawrence (1984) mentioned intersegmental membranes without reference to body region.Haridass (1985) stated that ectrichodiines also insert stylets at the head-collum intersegmental membrane.In our two laboratory trials, specimens of R. cinctiventris inserted stylets at the headcollum intersegmental membrane and/or at intersegmental membranes along the ventral, ventro-lateral, and/or dorsal trunk (Fig. 2B).Overall, the data suggest Ectrichodiinae primarily target the millipede's ventral or ventro-lateral trunk, with some exceptions.
to toxic salivary secretions.The millipede's ventral or head-collum region may be selected to avoid inserting stylets into laterally or dorsally located defensive glands along the trunk during millipede immobilization and consumption.It remains to be shown if, once immobilized, the millipede's defense gland reservoirs are targeted by ectrichodiines to sequester toxic secondary metabolites.At least half of the genera of Ectrichodiinae exhibit aposematic coloration to a larger extent than seen in most other reduviid subfamilies.Although ectrichodiines are presumably well protected from vertebrate predation due to their generally powerful bites and defensive secretions (Louis, 1974;Ambrose, 1999), a millipede diet may provide ectrichodiines with additional defensive benefits in the form of toxic secondary metabolites.Secondary defense metabolites have been investigated in other insects, such as aposematically colored Lepidoptera that sequester many chemicals from their host plants and biochemically alter them for defense purposes (Bowers, 1990;Nishida, 2002).Ectrichodiinae may potentially employ a similar strategy by targeting the defense gland reservoirs.

COMMUNAL PREDATORY STRATEGIES
Evidence for communal predation comprises 13 observations.Of these, two involved groups of conspecific nymphs in the presence of a conspecific adult, whereas eight involved groups of conspecific nymphs.Conspecific adults engaging in communal behaviors have only been documented in Cleptria cinctiventris Stål (1855) [Lawrence, 1984].Evidence of immatures engaging in solitary predation has only been documented for a Haematorrhophus species (Green, 1925).Communal predation was observed in only one of our laboratory trials with R. cinctiventris (Fig. 2C): early instar (1-2) nymphs, in the presence of an adult female and two later instar nymphs, frequently attempted predation on a millipede without immobilizing it.Overall, the data may indicate that communal, conspecific feeding among immatures or immatures and adults is the rule but rather an exception among adults.One potential explanation is that the salivary secretions produced by a single nymph may not be sufficiently toxic to immobilize and kill a millipede.Thus, the attack of multiple individuals may be necessary to increase the toxic effects, especially to subdue large-bodied prey items.

CONCLUSION
The predator-prey association between Ectrichodiinae and millipedes was first documented almost a century ago (Green, 1925), but has since received little attention.Judging from published data, it is obvious that the feeding behavior of Ectrichodiinae is still poorly understood.About 2% of ectrichodiine species have been documented to prey on millipedes, leaving ample room to further test the claim that Ectrichodiinae, as a group, specialize on millipedes.Based on our analysis, Ectrichodiinae may be exclusive millipede specialists, with a potential preference for the benzoquinone-producing Juliformia.Milli-pedes were usually not identified below the order level in the literature and online images.Thus, future investigations should attempt accurate genus or species determination of millipedes.Furthermore, the millipede's ventral trunk area and intersegmental membranes, including between the head and collum, are typically targeted.Whether these areas are attacked in relation to the location of the millipede nervous system or defense glands remains to be investigated.Lastly, immature and adult Ectrichodiinae exhibit both solitary and communal predation.The majority of observations suggest that immatures typically exhibit communal predation, whereas adults usually predate solitarily.Given that these conclusions are general and preliminary, it is imperative that future studies provide detailed documentation regarding these aspects of ectrichodiine predation.Other relevant investigations that will contribute to understanding this phenomenon include examining the chemical composition of ectrichodiine salivary secretions and the ability of ectrichodiines to sequester secondary metabolites from diplopods for defense.
Data extracted from published literature and Internet images for analysis of Ectrichodiinae predatory behavior.

Fig. 2 .
Fig. 2. Field and laboratory images of Rhiginia cinctiventris predation on millipedes at La Selva Biological Station, Costa Rica.A -Field image of an adult and two immature Rhiginia cinctiventris near a spirobolid millipede carcass at La Selva Biological Station, Costa Rica.B -An adult female feeds on a small polydesmid millipede.C -An adult female, two early instar nymphs, and two later instar nymphs in the presence of a spirobolid millipede.