B. Hölldobler, M. Möglich, and U. Maschwitz.
Myrmecophilic Relationship of Pella (Coleoptera: Staphylinidae) to Lasius fuliginosus (Hymenoptera: Formicidae).
Psyche 88:347-374, 1981.

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MYRMECOPHILIC RELATIONSHIP OF
(COLEOPTERA: STAPHYLINIDAE) TO
LASIUS FULIGINOSUS (HYMENOPTERA: FORMICIDAE) A large number of staphylinid beetles are closely associated with ants and termites (for review see Wilson 1971, Kistner 1979). Those species living with ants are commonly called myremcophiles. At least a few (Atemeles, Lomechusa) have "broken" the communica- tion code of their host species and are thereby able to become completely integrated in the social system of the ants (Holldobler 1967, 1970, 1971). In an attempt to understand the evolutionary pathways of this highly specialized social parasitic behavior, we studied closely related staphylinid species that do not live within the ant society but instead occupy the foraging trails and garbage dumps of an ant nest.
Many of such myrmecophilous staphylinids can be found with the formicine ant Lusius fuliginosus and most of them belong to the genus Pella. Apparently these beetles are not endowed with the behavioral repertory that would enable them to live within the ant colony, although they seem to have a close ecological association with ants (Holldobler 1972).
Kistner (1971) redefined the genus Zyras and raised the former subgenus Pella to generic rank. The first behavioral observations concerning PeZZa (=Zyras, Myrrnedonia) were published by Was- mann (1886, 1930). He stated that these beetles feed on dead or disabled ants, but that they also lie in wait near the entrance and hunt ants returning to the nest. Furthermore, Wasmann pointed out that because of their generalized and primitive structure these beetles can be regarded as close to the ancestral forms from which some of the more specialized staphylinid myrmecophiles were derived.
*Department of Biology, Harvard University, Cambridge, Mass., USA. **Present address: Am Lowentor 15, Darmstadt, W.-Germany. ***Fachbereich Biologic (Zoologic) der Universitat Frankfurt, W.-Germany. Manuscript received by the editor October 19, 1981.



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At our major study sites near Ochsenfurt, Riederau (both Bavaria, W.-Germany) and Gravenbruch (Hessen, W.-Germany) we found 12 staphylinid species associated with Lasius fuliginosus (Tab. 1). Our investigations concentrated on the genus Pella (mainly P. funesta, P. laticollis and P. cognata). Since the myrmecophilous behavior of these species was found to be very similar we will refer to the individual species only where necessary. In fact, when observing the beetles in the field it was usually not possible to identify the species precisely. We made additional observations with Pella humeralis, which can be found with L. fuliginosus, but which often also occurs near the nests of Formica polyctena (Wasmann 1920; Kolbe 1971).
The field observations were conducted throughout the years 1967-1969, and sporadically in 1970-1973. In an attempt to follow the life cycle of the beetles in the laboratory we set up a large colony of Lasius fuliginosus in a laboratory nest. The culture and mainte- nance of these ants over a longer period of time was particularly difficult, because L. fuliginosus constructs carton nests with the aid of a special symbiontic fungus (Cladosporium myrmecophilurn). A detailed description of the nest building behavior of L. fuliginosus and of the laboratory nest is given in Maschwitz and Holldobler (1970).
In order to measure quantitatively possible trophallactic feeding of the myrmecophiles by their host ants, tracer experiments were carried out using the radioisotope 32P mixed with honey-water. The quantity of marked food taken up by the ants was reflected in the counts per minute which were determined with a standard Geiger- Table 1
Staphylinids found near one nest of Lasius fuliginosus. Pella laticollis
Pella lugens
Pella cognata
Pella funesta
Pella humeralis
Oxypoda vittata
Rugilus rufipes
Thiasophila inquilina
Homoeusa acuminata
Sipalia circellaris
Atheta fungi
Atheta sodalis




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198 11 Holldoble~ Moglich, & Maschwitz - Pella 349 Miiller counter combined with an automatic sample changer (Philips). For further information concerning the tracer techniques applied in this study see Gosswald and Kloft 1958; Kloft 1959. For histological investigations live specimens were fixed in alco- holic Bouin (Dubosq Brasil) or Carnoy (Romeis 1948), embedded in Methyl Methacrylate, and sectioned 5-8p thick with a Jung Tet- rander microtome (Rathmayer 1962). The staining was Azan (Heidenhain).
For the chemical analysis of the defensive secretions of Pella, liquid material was collected with glass capillaries from the dissected glandular reservoirs or washed with water from the surface of the irritated beetles. The quinones were identified by thinlayer chroma- tography as 2,4 - dinitrophenylhydrazine in 2 N hydrochloric acid or by reduction with sulphurous acid. The dinitrophenylhydrazones were separated on alumina F 254 (Merck) with chloroform meth- anol (19:l) as mobile phase and on silica gel F 254 (Merck) with benzene-ethyl acetate (4:l) as mobile phase (Moore 1968). The hydroquinones were separated on silica gel F254 with benzene dioxane (3:l) as mobile phase and then sprayed with a solution of 0.5% hydrogen peroxide and a solution of peroxidase. The newly formed quinones were made visible by spraying with DNP and treating with ammonia vapour (Schildknecht and Kramer 1962). Hydrocarbons, terpenes and carbonic acids were analyzed by GLC. We used a Perkin Elmer chromatograph, model 300, equipped with a flame ionization detector. Columns: 1.8m X 2.7mm stainless steel, packed with a) 4% polypropylene glucol on Chromosorb G (100å C column temperature); b) 4% polyethyleneglycol 1500 on Chromo- sorb G (70å¡C) c) 25% diethylhexyl sebacinate plus sebacinic acid on Kieselgur 60-100 (140å¡C (30 ml Nz/min; FID). The life cycle of Pella funesta
The following description of the life cycle of Pella funesta is based on field observations and on data obtained from laboratory cul- tures. Pella laticollis appears to have a similar life cycle, but our observational data are not as complete as for P. funesta. In late March and early April a large number of P. funesta beetles were typically found in the excavation material on the base of the trunks of L. fuliginosus nest trees. At this time most of the beetles



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were lying motionless in the loose material and showed a kind of "dormance posture": the abdomen was bent over its back, with the legs and antennae folded tightly to the body. On warmer days, however, the beetles exhibited high locomotory and flight activity, and in the laboratory they showed a strong positive phototaxis. During this period we frequently observed beetles copulating in the laboratory nests. Toward the end of April the sexual behavior and flight activities ceased. In the laboratory as well as in the field the beetles were now active primarily during the night, while during the daytime they clustered under shelters near the Lasius fuliginosus nest. Only occasionally were we able to spot a beetle outside the shelters at daytime.
Also near the end of April we found the first beetle eggs in the "garbage dumps" of the laboratory nest of L. fuliginosus, and by early May the first Pella larvae had hatched. The larvae developed quite rapidly, so that in mid-May we found the first pupae in the "garbage dumps" of the ant nests, even though larvae could still be found throughout the months of June and July. In June the mortality of adult beetles in our laboratory nest increased markedly and in late July and August the first young beetles eclosed from their pupae. These beetles, as well as those collected in the field in early August, exhibited strong positive phototaxis and high flight activity for a few days. After this short period, however, the beetles were primarily active at night and during the day they stayed in shelters. Finally, in October, the number of beetles found outside the ant nest declined markedly and by November no more beetles could be found outside the nest. In December we excavated to L. fuliginosus nests. In both nests we found several Pella beetles in "dormance position" covered by loose nest material of the peripher- al nest chambers and on the ground inside the nest tree trunk. Presumably these beetles were overwintering within the Lasius fuliginosus nest until their activity period would start again in early spring the coming year.
From these observations we propose the following life cycle for Pella funesta: in early spring the adult beetles deposit eggs near the ants' "garbage dump" area. The larvae develop in the "garbage dump", pupate during the period from May to July and between July-August the adult beetles eclose. After eclosion the young beetles apparently migrate, as indicated by the short period of high diurnal locomotory and flight activity. After this period the beetles



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19811 Holldobler, Moglich, & Maschwitz - Pella 351 forage near the L. fuliginosus nest during the night and stay in shelters during the day. They overwinter in dormancy inside the L. fuliginosus nest. With the end of winter the beetles enter a second diurnal activity phase during which mating takes place. After repro- duction the beetles die, normally a few weeks before the new beetle generation ecloses in June.
The behavior of the larvae of Pella
The description of the behavior of the larvae is primarily based on observations in the laboratory. In the field and in the laboratory nest, the larvae were almost exclusively found near or in the "garbage dumps" of the L. fuliginosus nests. We frequently ob- served the larvae feeding on dead ants (Fig. 1B). During feeding the larvae always attempted to stay "out of sight" either by remaining entirely beneath the booty and devouring it from below or by crawling inside the dead ant's body. Occasionally 2-4 larvae could be observed feeding on the same ant cadaver. But when they became too crowded they frequently attacked each other, sometimes result- ing in one larvae eating the other (Fig. 1C). When ants encountered the larvae they usually attacked them. Almost invariably the larvae reacted with a typical defense beha- vior. They raised their abdominal tip towards the head of the ants. Usually the ant responded by stopping the attack and palpating the larva's tip (Fig. 1A). In most cases this short interruption was enough to allow the larvae to escape. We observed hundreds of such encounters between ants and Pella larvae; only a few ended fatally for the larvae.
Histological invesigations revealed that the Pella larvae have a complex dorsal glandular structure with a reservoir near the abdominal tip in the second last segment. In addition we found single cell glands positioned dorso-laterally in each segment. Similar glandular structures had previously been found in larvae of the myrmecophile staphylinids Atemeles and Lomechusa, and circum- stantial evidence strongly indicated that in these species the glands produce so-called pseudopheromones which release adoption beha- vior in the host ants (Holldobler 1967). We have no evidence to suggest that these glands have a similar function in Pella. However, it is possible that the more complex glandular structure at the abdominal tip, produces an appeasement secretion by which the aggressiveness of attacking ants can be briefly blunted.



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Fig. 1
Behavior of the larvae of Pella. A) Larva presenting abdominal tip to an attacking worker of Lasius fuliginosus. The ant interrupts attack and licks at the larva. B) Larva feeds on dead ant. C) Cannibatistic behavior of Fella larvae.



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Holldobler, M&lich, & Maschwitz - Pella
In any case, Pella larvae are able to come into close contact to workers of L. fuliginosus without being attacked, especially when the temperature is low (l4-17O C). Under those circumstances we have seen the beetle larvae licking the cuticle of live ants, including even the mandibles and mouthparts. This led us to the question of whether the beetle larvae occasionally solicit regurgitation in ants. In order to investigate this possibility ants were fed with honey- water labeled with the isotope 32P and then housed together with beetle larvae. For each sample we kept 30 radioactive ants with 5 beetle larvae in plastic containers (10 X 15 cm) with a moist gypsum bottom. One experimental series was conducted in a temperature range of 14.5-16Så¡C the other in 20-23O C. After 24 hours we measured the amount of radioactivity in each individual ant and larva. No significant amount of radioactivity had been transferred from the ants to the larvae, except in container 6, where one ant was found dead and obviously partly eaten by the larvae. Since the amount of radioactivity carried by some of the larvae was only very slightly above the background activity, we concluded that it was transferred by contamination. From this experiment it appears that the Pella larvae do not solicit regurgitation in ants. Their main food source seems to be dead ants or debris of the ants. In fact, they can easily be raised by keeping them entirely separated from living ants, just by feeding them regularly with dead ants. Predatory behavior of adult beetles
Since Wasmann's early observations (1886, 1920, 1925) very little has been reported concerning the biology of the myrmecophilous Pella. Wasmann reported that all species he had studied (P. humeralis, P. funesta, P. cognata, P. similis, P. lugens and P. laticollis) live with Lasius fuliginosus, and only P. humeralis can also be found with species of the Formica rufa group. According to Wasmann all these Pella species prey on ants, concentrating espe- cially on disabled ants. In addition Wasmann observed that the beetles are active primarily during the night. In a more recent publication Kolbe (1971) failed to find a predatorial behavior in P. humeralis and concluded that this species primarily feeds on dead ants. Similar observations were made with Pella japonicus, which lives with Lasius spathepus (= L. fuliginosus var. spathepus Wheeler) (Yasumatsu 1937; Kistner 1971). Kistner also observed that these beetles "ate small insects that are being transported by the



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354 Psyche [Vol. 88
ants". However, he could not "see the Pella eating live ants or fighting any of the ants on the trail".
Our observations of Pella funesta, P. laticollis and P. humeralis confirmed that these species live as scavengers, feeding on dead or disabled ants and debris discarded by the ants. However, we also observed these beetles acting as very effective predators on the ants. Most of the following studies were made with P. laticollis and P. funesta.
During the main foraging season from May to October Lasius fuliginosus is active day and night. Foragers travel along well established trunk trails to feeding sites which are sometimes more than 40 m distant. At daytime we only occasionally saw Pella beetles moving along or nearby the trail. However, when we watched the trunk trails with a flash light at night many Pella were seen running along the ants' foraging routes. Although most beetles were found within a range of 5 m from the nest tree of L. fuliginosus, we also found beetles on the trunk trail as far as 22 m away from the nest. On 6 different occasions we witnessed Pella beetles hunting L. fuliginosus workers at night on the foraging trail. When an ant was killed it was dragged a few centimeters away from the trail and eaten under a shelter, sometimes by several beetles simultaneously. More detailed observations on the behavioral interactions of Pella and L. fuliginosus were made in the laboratory. As long as enough dead ants were available at the ants' nest midden, the beetles showed no predatory behavior at all, limiting themselves to a diet of ant cadavers (Fig. 2A). But when the beetles were starved for a few days and then placed together with ants in an observation arena, the predation by Pella became strikingly prominent, although the time of onset was often very unpredictable. We saw the beetles hunting during the daytime, but we observed such activity most frequently in the evening or at night. The beetles chased after individual ants and pursued them through approximately 2-6 cm (very rarely through longer distances than that). When the beetle moved directly behind the ant it attempted to mount it and insert its head between the ant's head and thorax. When attacked the ant usually reacted by suddenly stopping and pressing the femur rapidly and tightly to its body (Fig. 5). Often this reaction threw the beetle off the back of the ant, allowing the ant to escape. In one series of observations we counted 178 beetle onslaughts on L. fuliginosus workers within a period of 3 hours; of these, only 9 attempts (5%) were successful.



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Fig. 2 A) Pella beetle feeding on dead L. fuliginosus worker. Frequently the beetles lick first the mouth parts of the ants, before tearing the cadavers apart. They might be attracted to mouthparts by sweet remainders of honeydew. B) Cluster of Pella beetles around a prey object.




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Fig. 3
Photograph and drawing of Pella attacking live worker of L futiginosus.



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19811 Holldobler, Moglich, & Masch~yitz - Pella 357 The hunting behavior of the beetles was always the same in P. laticollis, P. funesta and P. humeralis: the beetle attacked from behind and always attempted to insert its head between the head and thorax of the ant. We inspected several ants which had just been immobilized by an attack of a beetle and found that in most cases the pronotum was widely separated from the head and usually the oesophagus and connectives of the nervous system were cut. Occasionally we observed 2-3 beetles chasing behind one ant (Fig. 4). Once the ant was caught by a beetle the other beetles joined in subduing and killing the ant. Although individual beetles often tried to drag the prey away from the rest of the "hunting pack", usually several beetles fed on the prey simultaneously. No aggres- sion among the beetles was observed in this situation. However, when the beetles were starved for several days and were kept without ants, they occasionally chased each other, jumping on each other's back as they normally did when hunting ants. But we never saw cannibalistic behavior among the adult beetles, even when the beetles were densely crowded around a prey object (Fig. 2B). Defense and appeasement behavior in adult beetles Defense with tergal gland secretion:
Usually the Pella beetles run around with their abdomen curved slightly upwards. When encountering an ant, the beetles flex the abdomen even more strongly. This is a typical and frequently described behavior of many staphylinid myrmecophiles and is commonly considered a defense response (Wasmann 1886, 1920; Jordan 1913; Patrizi 1948; Koblick and Kistner 1965; Pasteels 1968; Holldobler 1970, 1972; Kolbe 1971). It has been suggested that during this abdominal flexing the beetles discharge secretions from their tergal gland (Jordan 1913; Kistner and Blum 1971). The tergal gland is located between the sixth and seventh abdominal tergites (Fig. 6), and is unique to the subfamily Aleo- charinae (Jordan 191 3; Pasteels 1968). The chemistry of the tergal gland secretions of several species has been investigated and found to be extraordinarily diverse (Blum et al. 1971; Brand et al. 1973; Kolbe and Proske 1973).
Kistner and Blum (1971) suggested that Pella japonicus and possibly also P. comes, both of which live with Lasius spathepus, produce citronella1 in their tergal glands. This substance is also a



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Psych POL 8t
F- 4 Sequence of groyp hunting behavior of Pella. A) Two beetles chase forager of L futighww. One of the beetles is jumping on the back of the ant. B) T ant b been captured and subdud by both beetles. C) A third beetle is joining t hunting group-




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19811
Holldobler, Moglich, & Maschwilz - Pella Fig. 5
A) Pella in '6death feigning" position. B) A "death feigning" beetle is carried around by a L. fuliginosus worker. C) Pella presents abdominal tip to attacking L. fuliginosus worker. The ant licks at the abdominal tip.



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360 Psyche [Vol, 88
major compound of the mandibular gland secretions of their host ants, for which it functions as an alarm pheromone. Although no Pella tergal gland contents were available for chemical analysis, because irritated beetles seemed to smell like the ants' mandibular gland secretion, Kistner and Blum speculated that Pella produce in their tergal glands citronella1 and thereby mimic the alarm phero- mone of their host ants. They suggested that in this way the beetles can "cause the ants to reverse their direction; a reaction which allows the myrmecophiles to escape".
Our investigations of the defensive strategy employed by the European Pella towards their host ants Lasius fu1iginosus led to Fig. 6 Schematical drawing of a Pella beetle indicating the position of the exocrine glandular complexes. TG: tergal gland; AG: appeasement gland complex.



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Table 2
GLC analysis of the secretion of Pella laticollis + = present; - = absent; ? = not tested
isovaleric n-butyric
acid (3- n-valeric
n-decane methylbu- isobutyric 2-methylbu- (main peak) n-undecane n-tridecane tyric acid) acid tyric acid citronella1 column a + + + + +
- -
column b + + + ? ? ?
-
column c + + + + +
-
9




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Holldobler, Moglich, & Maschwitz - Pella chained fatty acids in the secretion of P. laticollis (Tab. 2). However, in none of the species could we find citronellal. The common presence of quinones in the tergal gland secretions of Pella and the related aleocharine species agree with the previous findings by Blum et a1 (1971), who found the tergal gland secretion of Lomechusa strumosa to contain benzoquinone, methyl-benzo- quinone, ethyl-benzoquinone and n-tridecane, the latter substance accounting for more than 80% of the volatiles detected in the secretion. In addition Brand et al. (1973) analyzed the tergal gland secretion of Drusilla canaliculata, also an aleocharine beetle, finding quinones and hydroquinones together with alkanes, saturated and unsaturated aliphatic aldehydes. Pasteels (1968) demonstrated that D. canaliculata effectively employs the tergal gland secretion as a repellent-defense weapon against ants in a similar fashion as we described it for Pella.
Although we could not find any resemblance of the Pella tergal gland secretions to the mandibular gland secretions of Lasius fuliginosus, it was noteworthy that the Pella secretions contained undecane, a hydrocarbon commonly found in the Dufour's glands of formicine ants (for review see Blum and Hermann 1978) and considered to be an alarm pheromone in L. fuliginosus (Dumpert 1972). However, isolated tergal gland secretions of P. laticollis elicited a repellent reaction rather than an alarm response in L.