BEHAVIOR

Biolmninescence and Biological Aspects of BrazilianRailroad-Worms (Coleoptera: Phengodidae)

VADIM R. VIVIANI AND ETELVINO J. H. BECHARA

Departamento de Bioquimica, Instituto de Quimica, Universidade de Sao Paulo,CP 26077-CEP: 05599-970, Sao Paulo, SP, Brasil

Ann. Entomol. Soc. Am. 90(3): 389̂ 398 (1997)ABSTRACT Seventeen Brazilian railroad-worm species in 8 genera (Phrixothrix, Steno-phrixothrix, Mastinocerus, Mastinomorphus, Taximastinocerus, Brasilocenis, Euryopa, Pseu-dophengodes) collected in southeastern and west central Brazil, near the Parque Nacional dasEmas, were studied. The studied species generally inhabit pluvial tropical forests, but Masti-nomorphus sp., was found in open marshy areas of west central cerrados. The life history ofBrazilian phengodids resembles in many aspects that of the North American Zarhipis inte-gripennis (LeConte). We detail here the life cycle of Mastinomorphus sp.) In larvae and inlarviform females of the species studied, the bioluminescence ranges from yellow-green to red(Am.LX = 562-638 nm) for the head and from green to orange (Amax = 535-592 nm) for thelateral lanterns. In adult males, the emission color varies from green to yellow (Amax = 549-580 nm), except Euryopa spp., who emit orange light (Amax = 592-598 nm). Luminescencewas observed among all life stages of the species studied, in contrast to the North AmericanPhengodini, whose adult males do not glow. Bioluminescence from the larval and female headlanterns may serve for illumination of the nearby environment, whereas the lateral lanterns oflarvae and adult males and females seem to be important for self defense.

KEY WORDS railroad-worms, bioluminescence, Mastinocerini, Phengodini

THE FAMILY PHENGODIDAE includes «170 de-scribed species (W. Wittmer, personal communi-cation) found mainly in the Neotropical Region.Forty-nine species from Brazil have beendescribed (W. Wittmer, personal communication),but probably many more remain to be discovered.Wittmer (1976) reviewed the group and cataloged27 American genera in the tribes Phengodini, Mas-tinocerini, and Pennicilloporini (Phengodinae).Subsequent systematic work has been principallyon North Central American species (Linsdale1964; Paulus 1975; Wittmer 1976, 1986, 1988,1993; Zaragoza Caballero 1978, 1981, 1984 a-d,1986, 1988, 1989; Zaragoza Caballero and Herrera1986; Zaragoza Caballero and Wittmer 1986).Studies focusing on the biology of this group werecarried out on the North American species Zar-hipis integripennis (LeConte) (Tiemann 1967) andthe South American railroad-worm Phrixothrix tie-manni Wittmer (Tiemann 1970). Costa et al.(1989) have also described an unidentified larva ofPhrixothrix.

Herring (1987) reported the occurrence of bio-luminescence in the genera Phengodes, Phrixoth-rix, Stenophrixothrix, Mastinocerus, Zarhipis, Cen-ophengus, Cydiscus, Falsophrixothrix, Dioptoma,and Diplocadon. Three new species of phengodidswere discovered in west-central Brazil near theParque Nacional das Emas (State of Goids) by our

group and described as Phrixothrix vivianii Witt-mer (Wittmer 1993), Euryopa clarindae Wittmer,and Euryopa laurae Wittmer (Wittmer 1996). Wealso reported the spectral distribution of 10 Bra-zilian species of phengodids and an approximatephysical-chemical characterization of their lucif-erin-luciferase system (Viviani and Bechara 1993,1995).

Here we report biological information obtainedfrom field and laboratory observations and biolu-minescence spectra for other phengodid species,which include members of the genera Phrixothrix,Stenophrixothrix, Mastinocerus, Mastinomorphus,Taximastinocerus, Euryopa, and Pseudophengodes.The life cycle of Mastinomorphus sp.j, whose larvaand larviform female previously were placed inMastinocerus nigricollis (Viviani and Bechara1993), also is described.

Materials and Methods

Reagents. Adenosine triphosphate (ATP), D-lu-ciferin, ethylenediaminetetracetic acid (EDTA), di-thiothreitol (DTT), tm(hydroxymethylamino)methane (Tris), magnesium sulfate (MgSO4), andTriton X-100 were obtained from Sigma (St. Louis,MO). Sodium phosphate was purchased fromMerck (Darmstadt, Germany). All solutions wereprepared with bidistilled, MilliQ deionized water.

0013-8746/97/0389-0398$02.00/0 © 1997 Entomological Society of America

390 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 90, no. 3

Insects. Collection and field observations ofphengodids were made during 1990-1995 in thefollowing 5 localities: (1) Fazenda Santana-Sousasin the municipality of Campinas, state of Sao Pau-lo: Phrixothrix hirtus (Olivier, 1909), Mastinocerussp.2, and Mastinocerus sp.4. This locality is coveredlargely by =100 ha of remnant mesophil tropicalforest, surrounded by open fields and transition ar-eas and bounded by the Atibaia River at 700 maltitude. (2a) Fazenda Santa Cruz in the munici-pality of Costa Rica, state of Mato Grosso do Sul:Phrixothrix vivianii Wittmer, Phrixothrix heydeniOlivier, Stenophrixothrix brasiliensis (Pic), Mastin-ocerus nigricollis (Pic), Taximastinocerus hickeri(Pic), Mastinomorphus sp.1; Mastinomorphus sp.2,Brasilocerus minasensis Wittmer, Euryopa clarin-dae Wittmer, and E. laurae Wittmer. This regionis located in the central west Brazilian cerradosmorphoclimatic domain, which contains 4 mainformations: cerrado, gallery forest, marshy areas,and pasture; (2b) Parque Nacional das Emas, stateof Goi£s: E. clarindae, E. laurae, and Mastinomor-phus sp.j. This region includes the area of cerradoat the eastern border of Rio Formoso and its gal-lery forest. It is located 60 km north of the localitydescribed in 2a. (3) Estacao Biologica de Borac6iain the municipality of Sales6polis, state of Sao Pau-lo: Stenophrixothrix pollens (Berg), Brasilocerusimpressicollis (Wittmer), Phrixothrix hirtus, andPseudophengodes brasiliensis Wittmer. This area isa typical Atlantic forest reserve located near theseashore at 850 m altitude, 110 km northeast ofSao Paulo. (4) Sitio Cambar& in the municipalityof Capao Bonito, state of Sao Paulo: Brasilocerussp. (impressicollis?). This ranch contains areas ofvirgin Atlantic forest, secondary forest, eucalyptusbelts, and open fields.

Adult males were attracted by light traps withwhite (300 W), blue (8 W), and UV (8 W) bulbs(Zaragoza Caballero 1989) just after sunset (in De-cember at 1900 hours) and into the night. Larvaeand females were sought on the soil surface anddug from the soil of their habitats. In the labora-tory, larvae and females were reared in circularplastic jars (11 cm high, 7 cm diameter) and aquar-ia (20 by 13 by 13 cm) with soil taken from theirrespective habitats. They were fed chicken liver ex-tract and living and dead Diplopoda and Isoptera.Adults also were kept alive in circular jars with wetabsorbent paper. All insects were reared at 21°Cunder seasonal photoperiods.

Adult males were identified by W. Wittmer (Na-turhistoriscb.es Museum Basel, Switzerland). Lar-vae of Mastinomorphus sp.1; which were incor-rectly identified as M. nigricollis by Viviani andBechara (1993), and Mastinocerus sp.2 were fur-ther identified by rearing them in the laboratory toadult males. Larvae of Phrixothrix vivianii, P. hir-tus, and Mastinomorphus sp.2 were associated withthe adult males found in the respective localities.The species previously discussed as Stenophrixoth-rix sp.2 and Phrixothrix sp.4 (Viviani and Bechara

1993) were further identified as B. impressicollisand E. clarindae, respectively. Unidentified larvae,as well as undetermined adult males, were placedin the probable genus followed by a specific num-ber. In some cases the probable species is cited inparentheses. Adult females were identified as suchonly by oviposition; adult females also are knownto be much larger than the respective adult andlarval males. Vouchers of all species studied arehoused in the collection of the Museu de Zoologiaof the Universidade de Sao Paulo.

Bioluminescence Spectra. Bioluminescencespectra were recorded on a SPEX model Fluorologspectrofluorometer (Edison, NJ), operating withonly the sample photomultiplier switched on. Thespectra were corrected for the spectral photosen-sitivity of the equipment by an internal program(MCorrect). Previously, Viviani and Bechara (1993)measured the bioluminescence spectra in a Perkin-Elmer model LS-5 spectrofluorometer (OakBrook, IL) and corrected them for the spectralphotosensitivity of the equipment, using the fluo-rescent standards quinine sulfate and rubrene. Thecorrection of the in vivo spectra obtained for ni-gricollis and vivianii adult males and impressicollislarvae and also for the in vitro luciferase standardreactions gave essentially the same result with bothsets of equipment. Live last instars and adult fe-males were immobilized in glass capillaries, withthe anterior or lateral lantern portion of the glassmasked with black paper (to obtain independentspectra) and placed in a spectrophotometric cu-vette in the sample compartment of the equip-ment. The spectra were scanned ^5 min after be-ing manipulated, when movement had nearlyceased and the insect was emitting a constant low-level glow. In our experience, the best conditionfor obtaining reproducible larval bioluminescencespectra is with die insect near ecdysis, because itis less active, avoiding distortions caused by suddenmovement. Adult male phengodids were stimulat-ed to glow with an injection of 5 /xl of commercialepinephrine (1 mg/ml) as described previously (Vi-viani and Bechara 1993). Adult males were im-mobilized with transparent tape on glass laminaeand placed in front of the emission window. In allcases, the lantern whose spectrum had to bescanned was positioned in front of the middle ofthe emission window to avoid distortion caused bythe different geometries of detection and self-ab-sorption. Each spectrum represents an average ofat least 3 independent scans for each specimen ofa given species. Because of the scarcity of mostspecies studied here, the number of specimensvaried between 1 and 5.

In vitro bioluminescence spectra were traced inthe same equipment as described above. Crude ex-tracts of phengodid lanterns were prepared as pre-viously described (Viviani and Bechara 1993,1995). Lanterns from 4-5 individuals of a givenspecies were excised and homogenized in 3 ml ofcold 0.10 M phosphate buffer, pH 7.5, containing

May 1997 VlVIANI AND BECHARA: BIOLOGY OF BRAZILIAN RAILROAD-WORMS 391

1 mM EDTA, 1 mM DTT, and 1% Triton X-100.The extracts were centrifuged at 14,000 Xg for 20min at 4°C. The supematants were assayed forlight production. The bioluminescent reaction wasstarted by injecting 10 /xl of crude extract into 990/xl standard solution (containing 1 mM D-luciferin,2 mM ATP, 4 mM MgSO4, and 1 mM EDTA, dis-solved in 0.10 M Tris-HCl at pH 7.8) into a quartzcuvette placed in front of the spectrofluorometeremission window. After 3 min, when the biolumi-nescence intensity decayed to a constant level, thespectrum was scanned.

Photographs. Photographs of glowing phengo-did larvae were taken using a Minolta XG-1 cam-era equipped with a 50-mm macrometric lens. Forthis purpose, Ektakrome films 400 and 100 ASAwere used. All photographs were obtained with theinsect glowing in the dark with maximal slit andexposure times varying from 30 s to 2 min de-pending on the film and insect brightness.

Results

Life History Observations. Habitats. We havecataloged 10 species in central cerrados and 7 spe-cies in the southeastern Atlantic Forest domain ofSao Paulo State (Table 1). Based on the collectionof adult males and larvae, southeastern phengodidsare found mainly in the tropical forest. However,it is not clear which are the actual habitats of westcentral species because adult males may be attract-ed to light traps from both the gallery forest andsurrounding open fields, including virgin cerradosand pastures, and our data on larvae are still in-complete. Mastinomorphus sp.j is the only specieswe studied which inhabits open marshy environ-ments.

Oviposition and Eggs. We observed oppositionof 22 Mastinomorphus sp.j females. From Decem-ber to January, each female laid a compact massof eggs during a single oviposition in fissures in thesoil or in soil chambers they constructed, then theyencircled the eggs until eclosion. During this pe-riod, the females were aggressive and attacked in-trusive objects when disturbed by the fingertip ora pencil. The females died within 1 wk after theireggs hatched. A Phrixothrix sp. female behavedsimilarly; however, it did not construct soil cham-bers and died before the larval eclosion. The num-ber of eggs laid by Mastinomorphus sp.! femalesvaried from 4 to 50, with 14 ± 4 eggs (mean ±SD) (n = 22). The eggs are orange-brownish,round, and hard, and have an average diameter of1 mm. The incubation time observed in the labo-ratory, at 21°C under seasonal photoperiods, variedfrom 33 to 51 d, (42 ± 7 d, n = 22).

Larvae. Phengodid larvae are generally found intropical forests on dark soil beneath decaying logsand leaves, a typical habitat for millipedes. P. hirtuslarvae were found in soil with a strong odor ofvegetable matter in decomposition and rich in fun-gi. A larval female was found in the soft soil under

a decaying log, together with passalid and elateridlarvae and millipedes. Larvae of B. impressicollisalso commonly occurred in rain forests on moss-rich soil. P. vivianii larvae often were found prey-ing on small black millipedes at the base of termitemounds inhabited by larvae of Pyrearinus termi-tilluminans (Elateridae) (Bechara 1988). Larvae ofMastinomorphus sp.i were found on dark soil inmarshy areas with low grass. Finally, larvae of 2unidentified species, probably Stenophrixothrix,are arboreal and were found active at heights upto 1.7 m on leaves and branches of bushes in theAtlantic forest.

Phengodid larvae were active during the rainyseason, concomitantly with millipedes. Larvae of P.hirtus and B. impressicollis were found from Oc-tober through April, whereas the west central cer-rado species, P. vivianii and P. heydeni, were col-lected from October to February. In the winter,larvae of the southeastern species were inactiveand buried in the soil. P. hirtus and Mastinocerussp.2 larvae could be found down to 30 cm, themaximum depth sampled.

Larvae of all species were nocturnal, and weremore active on humid nights. However, it is notknown to what extent larvae are active in the fieldduring other periods of the day. During several oc-casions, in the laboratory, Phrixothrix spp. larvaewere seen eating millipedes during the daytime. P.hirtus specimens were found at temperatures from13 to 25°C. In December, larvae of P. vivianii wereseen to start their activity at 2100 hours, but onwarm (24°C) and dry nights they began to appearonly later at 2300 hours. In a normal pattern ofactivity, larvae walked above ground, sometimesburying themselves and later reemerging on thesurface.

Phengodid larvae are specialist predators of mil-lipedes but will feed on other prey. In the labo-ratory, Phrixothrix and Brasilocerus spp. were fedthe wood millipede Julus sp. among other species.In the field, P. hirtus larvae were found attackingthese millipedes. A P. laticollis larva, kindly givenby J. Sivinsky (University of Florida), fed on Bra-zilian species of millipedes. In the laboratory, lar-vae of B. impressicollis also were observed to feedon living wood termites. They injected a dark liq-uid into these Isoptera and then sucked it back,suggesting extracorporeal predigestion such as oc-curs in lampyrid and elaterid larvae (Colepicolo etal. 1987). In the laboratory, Mastinomorphus sp.ilarvae were fed chicken liver extract.

Pupae. Pupae were found buried in soil rich indecaying leaves in the forest from July to August.Two male pupae of Mastinomorphus sp.i were ob-tained in March; their pupal stage lasted 21 d un-der laboratory conditions. Another pupa was ob-tained in December and lasted 14 d. In thelaboratory, the pupal stage of Mastinocerus sp.2(July) and P. heydeni (February) males lasted 20and 22 d, respectively, at 21°C. Comparatively, the

392 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 90, no. 3

Table 1. Geographical occurrence, habitats, and collection dates of phengodids

Species Habitat Locality

MastinoceriniPhrixothrix

hirtus(Adult males n = 2)(Associated larvae n = 19)

heijdeni(Adult male ;i = 1)(Larvae n = 19)

vivianii(Adult males n = 25)(Associated larvae n = 49)

sp-5(Larva n = 1)

Stenoph rixoth rix

brasiliensis(Adult males ;i = 4)

pallens(Adult males n = 1)

Mastinocenis

sp-4(Larvae n - 6)

sp.2

(Larvae n = 6)nigricollis

(Adult males n = 83)

Tfl.vino;»irts(mocen<s

hickeri(Adult males n = 5)

Mastinomorphus

sp-1(Adult males n = 2)(Adult females n = 22)(Larvae n = 400)

sp-2(Adult males n = 2)(Associated larvae n = 2)

£«n/opa

clarindae(Adult male n = 8)

/an me(Adult male n = 10)

Brasilocenis

impressicollis(Adult male n = 1)(Associated larvae n = 67)

sp.3 (impressicollis ?)(Larvae n = 50)

mi/iose/isis(Adult male n = 1)

Phengodini

Pseudophengodes

brasiliensis(Adult males n = A)

1,2

3 ,4

4

2

3 ,42

2

1

3 , 4

3, 4

5

3 ,4

3, 4

3 ,4

1

1

3 ,4

1

Fazenda Santana-Sousas-Campinas-SPVIII-1990, X-199209-11-91, 29-1-92, 06-11-92, 10-111-92Fazenda Santa Cruz-Costa Rica-MSXII-1991, XII-1993, XII-1994XII-1991, XII-1992, XII-1993, XII-1994Fazenda Santa Cruz-Costa Rica-MSX-1989, XII-1990, XII-1991, XII-1992, XII-1993, XII-1994XII-1990, XII-1991, XII-1993, XII-1994, XII-1996Sao Paulo-SPX-1994

Fazenda Santa Cruz-Costa Rica-MSX-1989, XII-1990, XII-1991, XII-1992Jardim Bonfiglioli, Sao Paulo-SPX-1994

Fazenda Santana-Sousas-Campinas-SPVIII/IX-1990, 23-VI-91, 30-VI-91, VII-1991Fazenda Santana-Sousas-Campinas-SPVI/VII-1991Fazenda Santa Cruz-Costa Rica-MSX-1989, XII-1990, XII-1991, 11-1992, XII-1992, XII-1993, XII-1994

Fazenda Santa Cruz-Costa Rica-MSXII-1990, XII-1991, XII-1992

Fazenda Santa Cruz-Costa Rica-MSLaboratory XII-1993, HI-1994X-1989, XII-1990, XII-1991, XII-1992, XII-1993, XII-1994X-1989, XII-1990, XII-1991, XII-1992, XII-1993, XII-1994Fazenda Santa Cruz-Costa Rica-MSXII-1990, XII-1993, XII-1995

Fazenda Satna Cruz-Costa Rica-MSXII-1991, XII-1992Fazenda Santa Cruz-Costa Rica-MSXII-1992, XII-1993

Estacao Biol6gica de Borac6ia-Sales6polis-SPX-1990, III-1996III-1991, XI-1991 XI-1192. Sao Paulo-SP X-1994, IV-1996Sitio Cambara-Capao Bonito-SPXII-1990, I/HI-1991Fazenda Santa Cruz-Costa Rica-MSXII-1990, XII-1992

Estacao Biologica de Borac6ia-Sales6polis-SPll-XI-93, XII-1993, 13-1-94, ll-XI-95

1, woods in Atlantic forest ecosystem; 2, open fields in Atlantic forest ecosystem; 3, gallery woods in cerrado ecosystem; 4, openfields and pastures in cerrado ecosystem; 5, marshy open field in cerrado ecosystem.

pupal North American Zarhipis lasted 20-35 d(Tiemann 1967).

Adults. Adult behavioral observations were car-ried out mainly with west central cerrado species.Adult males were collected from October throughJanuary. Some M. nigricollis also were collected inFebruary. Adults were very sensitive to the climaticconditions, being attracted only on windless, warm

(19-26°C) and humid nights (85-95% RH). Adultmales appeared mainly between 2030 and 2100hours attracted to white lights in December (Fig.1) (sunset at 1900 hours on 6 December). UV andblue light traps also were effective in attractingadult males, the blue light alone being most effec-tive. In the Campinas region, 2 Phrixothrix hirtusmales were collected during August and Novem-

May 1997 VlVIANI AND BECHARA: BIOLOGY OF BRAZILIAN RAILROAD-WORMS 393

2?- 40

30

20

10

19:00 19:30 20:00 20.30 21:00

TIME

21:30 22:00 22:30 23.00

Fig. 1. Frequency of M. nigricollis adult males (n =57) attracted to white-light trap in 1st hours of Decembernights (sunset at 1900 hours) near Parque Nacional dasEm as (State of Goias).

ber. An adult male of B. impressicollis, the prey ofa reduvid, was collected on the grass during anApril night at the Biological Station, Borac6ia.

In the laboratory, the longevity of adult malephengodids is very short, in general no longer than1 wk even when maintained in humidified vials andfed sugar water. The lifespan observed for adultmales of Mastinomorphus sp.x and P. heydeni thatemerged in the laboratory was 7 d. Mastinomor-phus sp.! females were tested unsuccessfully withfield-collected males for mating activity, both un-der laboratory and field conditions. However, wedo not know if the females were receptive.

Very little is known about adult females, whichoften are mistaken for larvae. Adult females ofMastinomorphus sp.i were recognized when theyoviposited. They are much larger than the respec-tive males. The female behavior is described inmore detail in the oviposition section.

Bioluminescence. Eggs. Very weak, invisible lu-minescence was detected from embryos after theday 15, using a photon-counting apparatus. Thespectral distribution of this luminescence is notknown. Viviani and Bechara (1993) reported atemperature dependence of the bioluminescenceintensity of fertilized phengodids eggs, with an ap-parent activation energy of 58 kj-mol"1. Tiemann(1967) reported the appearance of a weak glow inZ. integripennis eggs after 25 d of incubation.

Larvae. Three different patterns of biolumines-cence distribution were found along the larvalbody of the different species, as follows: (1) Bras-ilocerus spp. (Fig. 2A): larvae displayed 11 pairs ofdorsolateral lanterns, emitting green or yellow-green color (Amax = 550-557 nm), and 2 large an-terior lanterns of yellow-green color (Amax = 565-570 nm; Table 2). (2) Mastinocerus spp. (Fig. 2B):Larvae displayed 11 pairs of dorsolateral lanternsemitting yellow light (Amax = 578-580 nm), and asmall cephalic yellow-orange lantern (Amax = 580-597 nm) (Table 2). In this case, the cephalic lan-tern may be seen mainly frontally by its fight beam,in contrast to the other types where the light can

be seen clearly dorsally (Table 2). An unidentifiedlarva, probably Mastinomorphus sp.2 (Fig. 2C), had2 well-defined anterior lanterns (cephalic and post-cephalic) emitting orange light (AITULX = 597 nm)and lateral lanterns also emitting orange light (Amax= 586 nm) (Table 2). (3) Phrixothrix spp. (Fig.2D): Larvae displayed 11 pairs of dorsolateral lan-terns, emitting green or yellow-green light (Amax =535-568 nm), and 2 anterior (cephalic and postce-phalic) lanterns, appearing visually as a single lan-tern that emitted reddish light (Amax = 600-638nm) (Table 2); P. heydeni larvae emitted yellow-green light from the lateral lanterns (Amax = 568nm) (Fig. 3) and, together with Phrixothrix sp.(emitted the furthest red light among phengodidsstudied so far), by the head lantern (Amax = 636nm) (Fig. 3).

At night, phengodid larvae could be locatedmost often by the head lantern glow, the laterallanterns remaining dark. Eventually, larvae wereseen with all lanterns switched on, making themeasier to find at greater distances. Under these cir-cumstances, larval behavior may include searchingfor prey.

In all cases, fine neural control of the lateral lan-terns could be observed by following the stimula-bility and duration of luminescence (Halverson etal. 1973). In the field, larvae of Phrixothrix andBrasilocerus spp. sometimes were found with alllanterns glowing. Upon mechanical stimulation, allMastinocerini larvae switched on their lateral lan-terns. In almost all cases, the lateral lanterns wereobserved to switch on only when larvae were at-tacked by ants or were attacking millipedes. Ini-tially, the terminal abdominal segments wereswitched on; depending on the intensity of thestimulation, the other abdominal lanterns also maybe switched on. Occasionally larval Mastinocerusspp. and Mastinomorphus spp. kept penultimatesegment lanterns glowing for a longer time thanthe others.

The absorbance of the larval Phrixothrix spp.dorsal cuticle increases exponentially (5 times)from the red (700 nm) to the near UV (300 nm)region of the spectrum (results not shown), result-ing in filtration of the light emitted.

Pupae. In general, male pupae lack the head lan-tern, but bright lateral lanterns are retained. A dif-fuse glow can be observed from the cephalic re-gion only in the first 2 d of the pupal stage; it thendisappears. The color of the remaining lateral lan-tern luminescence appears to be identical to thatof adult and larval lateral lanterns, indicated by vi-sual observation for Mastinomorphus sp.! and invivo bioluminescence spectra in the case of P. hey-deni (Amax = 568 nm) (Fig. 3; Table 2).

Adults. The lanterns of adult males of the Mas-tinocerini we studied are located dorsolaterallyalong the abdominal and thoracic segments, theprothoracic segment having a dorsomedian Ughtorgan. In E. laurae, we observed a very small light

394 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 90, no. 3

Fig. 2. Phengodids glowing: (A) Mature larval B. impressicollis (2-3 cm long). (B) Larviform female Mastinomor-phus sp.| (1.5-2 cm). (C) Larval Mastinomorphus sp.2 (1.5-2 cm). (D) Mature larval P. heydeni (4-5 cm). Picturestaken using Ektakrome 400 ASA film (D) and 100 ASA film (A, B, C).

organ frontally on the head between the eyes,emitting the same orange color as lateral lanterns.

Luminescence could be observed throughoutthe lifetime of the male adult stage in the phen-godid species studied here, being displayed onlywhen the insect is handled or disturbed, like thelateral lanterns of larvae and pupae. In nature,adults rarely were seen glowing. An adult male ofS. brasiliensis was observed glowing when caughtby a spider web. Also, adult P. brasiliensis maleswere found emitting a greenish glow (Fig. 4A) ininner woody areas during flight; they were attract-ed by a spotlight partly covered with the hand.This species has lampyrid-like light organs on theventral side of the penultimate segment.

In general, the color of adult male biolumines-cence ranges from green to yellow (Table 2). How-ever, we found adult males of E. clarindae (Fig.4C), E. laurae, and Mastinomorphus sp.2 emittingorange and reddish light. In Mastinomorphus sp.!adult males, the bioluminescence color of lateraland thoracic lanterns is the same as that of larval

and female lateral lanterns (Amax = 578 nm) (Table2) and appears to be identical to that of male pu-pae. The same appears to be the case of Phrixoth-rix heydeni (Amax = 568 nm) (Fig. 3; Table 2).

In Vitro Spectra. In vitro bioluminescence spec-tra of phengodid extracts are very similar to therespective in vivo ones; however, generally they arebroader. In addition, in the case of P. heydeni lar-vae, in vitro spectra are slightly shifted to the blueregion relative to in vivo ones (Fig. 3).

Discussion

Life History. The high incidence of phengodidsin woody habitats is expected, given the abundanceof millipedes, their preferred prey. The morphol-ogy of adult males, with short elytra and soft in-tegument appropriate to slow and low flight, ap-pears to be adapted to a habitat free of wind andwith relatively constant temperature, such as thatof forests.

May 1 9 9 7 VIVIANI AND BECHARA: B I O L O G Y O F BRAZILIAN R A I L R O A D - W O R M S 3 9 5

Table 2 . Bioluminescence colors of larval and adult phengodids

Taxa Lantern location

In vivo \ m a x > nm»

In vitro X m a x , nmb Reference c

Mastinocerini Brasilocerus

impressicollis Larval head 565 560 1 impressicollis 569 (n = 1) — 2

Larval lateral 550 557 1 Adult male Green — 2

sp.3 (impressicollis?) Larval head 565 — 1 sp.3 (impressicollis?) Larval lateral 5 5 0 — 1

Euryopa clarindae Adult 6 598 (n = 5) — 2

600 — 1 laurae Adult S 592 (n = 3) — 2 Mastinocems

sp-4 Larval head 597 — 1 sp-4 Larval lateral 577 — 1

nigricollis Adult male 549 556 1 nigricollis 550 (n = 2) — 2

Mastinomorphus sp i Female head 578 (n = 5) 577 2 sp i

Female lateral 580 (n = 5) 580 2 Adult male 580 (n = 2) — 2

sp-2 Larval head 597 (n = 1) — 2 sp-2 Larval lateral 5 8 6 (n = 1) — 2 Adult male Orange — 2

Phrixothrix hirtus Larval head 6 0 9 604 1

Larval lateral 563 572 1 Adult male Yellow-green — 2

vivianii Larval head 620 608 1 620 (n = 1) — 2

Larval lateral 542 542 1 Adult male 5 6 0 — 1

560 556 2 heydeni Larval head 636 (n = 4) 628 2 heydeni

Larval lateral 568 (n = 4) 548 2 Male pupa 568 (n = 1) — 2 Adult male 568 (n = 1) — 2

sp-2 Larval head 600 — 1 sp-2 Larval lateral 5 4 0 — 1

sp-5 Larval head 574 (n = 1) — 2 sp-5 Larval lateral 638 (n = 1) — 2

Stenophrixothrix brailiensis Adult male 583 — 1 pattern Adult male 575 (n = 1) — 2

Taxinomastinocerus hickeri Adult male 562 (n = 1) — 2

Phengodini Pseudophengodes

brasiliensis Adult male 542 (n = 3) — 2 Phengodes

laticollis Larva 535 — 1

" In cases where bioluminescence spectra were not recorded, apparent color is given. Maximum wavelengths reported are the mean of 3 - 4 values obtained for each specimen. Estimated error, ± 3 nm. n, number of specimens analyzed.

b In vitro spectra were measured as described in Materials and Methods using samples consisting of lanterns extracted from 4--5 individuals. Estimated error, ± 3 nm.

c 1, Viviani and Bechara 1993; 2, this article.

The life history of Brazilian Mastinocerini re­sembles that of North American Phengodini. The duration of the entire life cycle is unknown, but the appearance of adults once a year suggests that it is at least 1 yr. Oviposition is performed in a way similar to that of Z. integripennis (Tiemann 1967) but, in addition, Mastinomorphus sp.i females con­struct an inner soil chamber and surround their

eggs. Females die before eclosion of their eggs and their bodies may serve as a food source for the young larvae, but it is not clear if the larvae actu­ally feed on dead bodies or the associated micro-fauna. The incubation time for Mastinomorphus s P i e g g s ( 35 -50 d) is a little shorter than that ob­served for Zarhipis (50-60 d) (Tiemann 1967), probably because of the tropical climate. Phengo-

396 ANNALS O F T H E E N T O M O L O G I C A L S O C I E T Y O F A M E R I C A Vol. 90, no. 3

1 ^f~-m , , , j

450 500 550 600 650 700 \ max(nm)

Fig. 3 . Bioluminescence spectra of P. heydeni. . . . , in vivo spectrum of male pupa lateral lanterns (n = 1); , in vivo spectrum of larval lateral lanterns (n = 4); in vitro spectrum of larval lateral lanterns crude extract;

, in vivo spectrum of larval head lantern (n = 4); , in vitro spectrum of larval head lantern crude extract. Spectra were recorded in a Spex model Fluorolog spec-trofluorometer and corrected for photoresponse of equip­ment.

did larvae of almost all known Brazilian species are specialist predators of millipedes. In the laboratory, B. impressicollis larvae also were avid predators of termites; however, it is not known whether pre-dation on Isoptera occurs in nature. Larvae of Mastinomorphus sp.! probably feed on the micro-fauna in their habitat, but they were never seen preying on living millipedes.

In the laboratory, the Brazilian Mastinocerini we studied survive no more than 1 wk, similar to adult males of Z. integripennis (9 d) (Tiemann 1967). Adult male activity appears to be affected by cli­matic conditions: they usually fly on warm, humid, and windless nights.

Bioluminescence. The continuous glow of the head lanterns when the larvae are walking suggests an illumination function. The circumstances under which the lateral lanterns are switched on suggest a defense function. In fact, a sudden flash can re­pel potential predators. Initial luminous intensity quickly decreases, possibly reducing the risk of be­ing targeted by predators. Also, the dorsolateral lo­cation of the lanterns in an insect walking on the ground suggests that these are to be viewed by organisms above them. On grass, for example, there may be spiders, frogs, or other potential predators that approach larvae dorsally when they are walking on the ground. Aposematism associ­ated with distasteful properties also is a possible function for lateral lantern bioluminescence, such as occurs in the case of lampyrid larvae in which the luminous signal may be used to advertise their unpalatability (Sivinsky 1981). Phrixothrix larvae often eject a possibly distasteful corrosive reddish liquid from the anus when they are handled (Tie­mann 1970).

In our experience, collective warning seems im­probable because known phengodid larvae live sol­itarily and were never found in high population

0.01 I I I I L _

450 500 550 600 650 * ( M l )

Fig. 4 . In vivo bioluminescence spectra of adult male phengodids. (A) P. brasiliensis (n = 3) . (B) T. hickeri (n = 1). (C) E. clarindae (n = 5) . Spectra were recorded in a Spex model Fluorolog spectrofluorometer and correct­ed for photoresponse of equipment.

densities. This density was estimated by their glow, however, so the ones not shining were not taken into account. However, a large number of Phen-godes larvae confined by inundations in the earth was found by Wing (1984). Larvae of Mastinomor­phus sp.i, the most abundant species found up to now, were not observed to live clustered in small areas. This is in contrast with larvae of several lam­pyrid species (Sivinsky 1981), which live at high densities confined to small areas and may use si­multaneous emission to frighten potential enemies or to advise fecund females about overcrowding and competition for food sources.

As in the case of lampyrids and elaterids (Wood 1995), the color of phengodid bioluminescence is determined primarily by luciferase molecular structures (Viviani and Bechara 1993, 1995). This is attested by distinct in vitro bioluminescence spectra and by the different physical-chemical properties of these enzymes. However, in the case of Phrixothrix spp. larvae and probably other spe­cies, the cuticle may exert a slight filtering effect in the blue region.

The biological significance of the wide range of bioluminescence colors observed in phengodids is not clear. In fireflies, the bioluminescence colors are species-specific adaptations to optimize detec­tion in the different photic environments in which the species evolved (Seliger et al. 1982). The role of red fights (A m a x = 6 0 0 - 6 3 8 nm) in Phrixothrix larvae is not well understood. Sivinsky (1981) sug­gested an adaptative advantage i f the larvae them­selves are able to see the red fight, for the great majority of invertebrates are blind in this spectral region (Menzel 1975). Preliminary studies on the electroretinograms of Phrixothrix larva, performed by A. Lall (Howard University, Washington, DC) , showed that it has a broad visual spectral sensitivity shifted to the red, suggesting they can see red light (V.R.V., E .J .H.B. , D. Ventura, and A. Lall, unpub-

May 1997 VIVIANI AND B E C H A R A : B I O L O G Y O F BRAZILIAN R A I L R O A D - W O R M S 397

lished data). I f this were the case, the larvae could search for a prey using a visual channel not existing in other species. At the same time, the red-shifted colors of the head lanterns relative to the lateral lanterns in larval phenogodids may be an adapta­tion that decreases the visibility of the head lantern during continuous glowing, avoiding targeting and thus potential attacks. It is notable that Phrixothrix larvae were found in localities with reddish brown soil, which reflects best the frontal red light. Ad­ditionally, Brasilocerus larvae, which emit yellow-green light (A m a x = 5 6 5 - 5 7 0 nm) (Table 2) from their anterior lanterns, were found mainly in yel­lowish soil. However, in the case of Mastinocems spp. that emit yellow-orange colors from these lan­terns (A m a x = 580 nm), we could not find a clear relationship with soil color. The illumination func­tion hypothesis is thus attractive but still specula­tive, the color being optimized for the reflectance of the background soils. In general, green to yellow colors are emitted by the lateral lanterns of the species studied (A m a x = 535 -580 nm) (Table 2) . Orange light emission by the lateral lanterns of an unidentified (Mastinomorphus sp.2?) larva (Table 2) is unique among the larvae described in the lit­erature (Fig. 2) .

Unlike known adult males of Phengodini with vestigial luminescence 1 or 2 d after emergence, Mastinocerini luminescence appears to be func­tional. Continuance of the same bioluminescence color in the lateral lanterns of larval, pupal, and adult stages of Mastinomorphus sp.x and P. heydeni suggests conservation of the same luciferase iso-form throughout its fife cycle. This contrasts with lampyrids and elaterids in which the luminescence color changes in ontogenic succession of different luciferase isoenzymes (Strause and DeLuca 1981, Colepicolo et al. 1986). Visual observations indi­cate that, as in the case of P. tiemanni male pupa and larvae (Tiemann 1970), the lateral biolumines­cence color of adult males and larvae of P. hirtus and B. impressicollis are similar. This seems not to be the case in P. vivianii, which displays different lateral light colors in the adult and larval stages, although they were identified only by association.

The function of luminescence in adult phengo-dids currently is not well understood. Tiemann (1970) observed that adult males and females of P. tiemanni display a pyrotechnic show during (but not before) mating, suggesting that biolumines­cence does not play an important role in sexual attraction. Sexual attraction in phengodids appears to be mediated mainly by pheromones (Knaus 1907, Lloyd 1978). Currently, the stimulability and perhaps color (A m a x 5 4 9 - 5 8 0 nm) (Table 2) of the adult male phengodids we studied support a de­fensive function. Illumination of the nearby envi­ronment may be a function of P. brasiliensis adult male bioluminescence because adults fly into the dense vegetation of the forest (rich in potential predators), emitting a continuous glow, such as do some lampyrids when landing (Lloyd 1968). The

emission o f orange light by adult males o f the ge­nus Euryopa (Fig. 4 E ) and of Mastinomorphus sp.2, however, is contrary to a defensive hypothesis. This orange emission may be only a characteristic inherited from the larval stage, as suggested above. The smaller antennae of adult males of the genus Euryopa, in relation to other genera, is consistent with a decreased importance of sexual attraction through pheromones. It is possible that these spe­cies are active during twilight, when the reddish colors would contrast better with the green photic environment of the grass where these insects are active. In this case, luminescence would be impor­tant for sexual attraction.

In conclusion, luminescence occurs in all of the following life stages of the Brazilian phengodids we studied: Phrixothrix spp., Stenophrixothrix spp., Mastinocems spp., Mastinomorphus spp., Taximas-tinocems spp., Brasilocems spp., Euryopa spp., and Pseudophengodes spp. Phengodids exhibit the widest range of bioluminescence colors among bi-oluminescent Coleoptera (A m a x 5 3 5 - 6 3 8 nm). Their larvae and larviform females emit in the green-or­ange ( A m a x = 5 3 5 - 5 8 6 nm) and yellow-green-red ( A m a x

= 5 6 0 - 6 3 8 nm) region of the spectrum by the lateral lanterns and cephalic lanterns, respec­tively. Adult males generally emit in the green-yel­low (A m a x = 540—583 nm) region of the spectrum, except that Euryopa spp. and Mastinomorphus sp.2

emit orange-red light (A m a x = 5 9 2 - 6 0 0 nm). Pupae of Mastinomorphus sp.i and P. heydeni emit in the same region as the respective adult males and lar­val lateral lanterns. Larval head lantern lumines­cence appears to be important for illumination of the nearby environment, whereas lateral lantern luminescence seems to be adapted for self-defense and aposematism. In P. brasiliensis, the continuous glow is consistent with illumination and intersexual communication. The duration of Brazilian phen-godid life stages is generally shorter than that of North American species, but, apart from biolumi­nescence, other biological aspects such as larval habitats and prey selection are quite similar.

Acknowledgments

This article is dedicated on the occasion of his 60th birthday to Waldemar Adam (Chemistry, Wiirzburg Uni­versity), who was the first to synthesize a dioxetanone— the hypothetical energy-rich intermediate of beetle bio­luminescence. Special thanks go to Walter Wittmer (Na-turhistorhisches Museum, Basel) for the identification of the phengodid specimens, to Cleide Costa (Museu de Zoologia of the Universidade de Sao Paulo), to Sergio A. Vanin (Instituto de Biociencias, Universidade de Sao Pau­lo), to John Makenson (International University of Flor­ida), and to Frank Quina (Instituto de Quimica of the Universidade de Sao Paulo) for critical revision and sug­gestions of the manuscript. We also are indebted to Jose Sanchez, Nicolas Albuquerque, and Ana Claudia Toledo Prado dos Santos for helping us collect the phengodids, and to Carlos Jose Paz for rearing the insects. We also thank Augusto Dalia, owner of Fazenda Santana in Sou-

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sas-Campinas, and Aldo Campanha, owner of Sitio Cam-bara in Capao Bonito, for permitting us to collect phen-ogodids on their properties. This work was supported by grants from the Fundacao de Amparo a Pesquisa do Es-tado de Sao Paulo (FAPESP), the Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq), the Programa de Apoio ao Desenvolvimento Cientifico e Tec­nologico (PADCT), and the Alexander von Humboldt Foundation (from the 1993 Humboldt Award to G. Ci-lento). E.J .H.B. is the recipient of a fellowship from the Guggenheim Foundation.

References Cited

Bechara , E . J . H . 1 9 8 8 . Luminescent elaterid beetles: biochemical, biological and ecological aspects, pp. 123-177. In A. L. Baumstark [ed.], Advances in oxy­genated processes. JAI Press, London.

Colepicolo, P. N., C. Costa, and E . J . H . B e c h a r a . 1 9 8 6 . Brazilian species of luminescent Elateridae. Luciferin identification and bioluminescence spectra. Insect Biochem. 16: 803-810 .

Colepicolo, P. N., E . J . H . Bechara , C. F e r r e i r a , and W. Terra . 1 9 8 7 . Digestive enzymes in close and distant genera of the same family: properties of mid­gut hydrolases from luminescent Pyrophorus diver-gens (Coleoptera: Elateridae) larvae. Comp. Biochem. Physiol. B 87: 755-759 .

Costa, C , S. A. Vanin, and S. Casari-Chen. 1 9 8 9 . Larvas de Coleopteros do Brasil. Museu de Zoologia (Universidade de Sao Paulo), Sao Paulo.

Halverson, C. R . , J . F. Case, J . Buck, and D. Thie­mann. 1 9 7 3 . Control of luminescence in phengodid beetles. J . Insect Physiol. 19: 1327-1339.

Knaus, W. 1 9 0 7 . Phengodes Illiger—a note on lumi­nous females and larvae. Entomol. News 18: 3 1 8 - 3 1 9 .

Herring, P. J . 1 9 8 7 . Systematic distribution of biolu­minescence in living organisms. J . Biolumin. Chem-ilumin. 3: 147-163 .

Linsdale, D. D. 1 9 6 4 . A revision of the genus 7/irhipis LeConte (Coleoptera: Phengodidae). Wasmann J. Biol. 22: 225 -260 .

Lloyd, J . E . 1 9 6 8 . Illumination, another function of firefly flashes? Entomol. News 10: 2 6 5 - 2 6 8 .

1 9 7 8 . Insect bioluminescence, pp. 241 -272 . In P. J . Herring [ed.], Bioluminescence in action. Academic, New York.

Menzel, R. 1 9 7 5 . Colour receptor in insects, pp. 1 2 1 -153. In G. A. Horridge [ed.], The compound eye and vision in insects. Claredon, Oxford.

Paulus, H. F. 1 9 7 5 . Penicillophorus ctenotarsus n. gen. et n. sp. aus Kolumbien, mit einer Beschreibung einer neuen Tribus Penicillophorini der Phengodidae (Co­leoptera, Polyphaga, Cantharoidea). Z. Anbgem. Oes-terr. Entomol. 25: 69 -80 .

Seliger, H. H. , A. B . Lall, J . E . Lloyd, and W. H. Biggley. 1 9 8 2 . The colors of firefly biolumines­cence—II. Experimental evidence for the optimiza­tion model. Photochem. Photobiol. 36: 3 6 1 - 6 8 8 .

Sivinsky, J . 1 9 8 1 . The nature and possible functions of luminescence in Coleoptera larvae. Coleopt. Bull. 35: 167-180.

Strause, L. G., and M. DeLuca. 1 9 8 1 . Characteristics of luciferase from a variety of firefly species: evidence for the presence of luciferase isoenzymes. Insect Bio­chem. 11: 417-422 .

Tiemann, D. 1 9 6 7 . Observation on the natural history of the western banded glow-worm Xarhipis integri-pennis. Proc. Calif. Acad. Sci. 35: 2 3 5 - 2 6 4 .

1 9 7 0 . Nature's toy train, the railroad-worm. Natl. Geogr. Mag. 138(1): 56 -67 .

Viviani, R. V., and E . J . H . B e c h a r a . 1 9 9 3 . Biophysical and biochemical aspects of phengodid biolumines­cence. Photochem. Photobiol. 58: 615 -622 .

1 9 9 5 . Bioluminescence of Brazilian fireflies (Coleop­tera: Lampyridae): spectral distribution and pH effect on luciferase-elicited colors. Comparison with elaterid and phengodid luciferases. Photochem. Photobiol. 62: 4 9 0 - 4 9 5 .

Wing, S. 1 9 8 4 . A spate of glow-worms (Coleoptera: Phengodidae). Entomol. News 95:55-57.

Wittmer, W. 1 9 7 6 . Arbeiten zur einer Revision der Familie Phengodidae. Entomol. Arb. Mus. Frey 27: 4 1 5 - 5 2 4 .

1 9 8 6 . Ein Beitrag zur Kenntnis der Phengodidae (Co­leoptera). An. Inst. Biol. Univ. Nac. Auton. Mex. 56: 159-176 .

1 9 8 8 . Die Familie Phengodidae (Coleoptera) von Cer-ro de la Neblina, Venezuela. (38. Beitrag zur Kenntnis der Neoptropischen fauna). Mitt. Entomol. Ges. 38: 72 -94 .

1 9 9 3 . Zwei neue Phengodidae (Coleoptera) aus Bra-silien. Mitt. Schweiz. Entomol. Ges. 42: 130-132 .

1 9 9 6 . Ein weiterer beitrag zur kenntnis der Phengo­didae (Coleoptera). Rev. Bras. Entomol. 40: 125-129 .

Wood, K. V. 1 9 9 5 . The chemical mechanism and evo­lutionary development of beetle bioluminescence. Photobiology 52: 6 6 2 - 6 7 3 .

Zaragoza Caballero, S. 1 9 7 8 . Una nueva especie de Phengodes Illiger San Angel, Mexico, D. F. An. Inst. Biol. Univ. Nac. Auton. Mex. 46: 6 9 - 7 4 .

1 9 8 1 . Description de dos nuevas especies de Phengo­des (Coleoptera: Phengodidae). An. Inst. Biol. Univ. Nac. Auton. Mex. 51: 377 -382 .

1 9 8 4 a . Nota sobre una interesante estructura de Dis-tremocephalus Wittmer (Coleoptera: Phengodidae: Mastinocerini). An. Inst. Biol. Univ. Nac. Auton. Mex. 55: 315^318.

1 9 8 4 b . Descripcion de dos especies nuevas e nuevos registros de Cenophengus (Coleoptera: Phengodidae). An. Inst. Biol. Univ. Nac. Auton. Mex. 55: 195-202 .

1 9 8 4 c . Descripcion de un genero e una nueva especie de Mastinocerini (Coleoptera: Phengodidae). An. Inst. Biol. Univ. Nac. Auton. Mex. 55: 2 0 3 - 2 0 8 .

1 9 8 4 d . Catalogo de la familia Phengodidae. An. Inst. Biol. Univ. Nac. Auton. Mex. 55: 3 0 7 - 3 2 4 .

1 9 8 6 . El genero Distremocephalus Wittmer en Mexico (Coleoptera: Phengodidae). An. Inst. Biol. Univ. Nac. Auton. Mex. 56: 189-202 .

1 9 8 8 . Una especie nueva de Cenophengus de Mexico (Coleoptera: Phengodidae). An. Inst. Biol. Univ. Nac. Auton. Mex. 58: 6 5 1 - 6 5 4 .

1 9 8 9 . La familia Phengodidae en "Los Tuxtlas" Vera Cruz, Mexico. An. Inst. Biol. Univ. Nac. Auton. Mex. 59: 7 7 - 9 8 .

Zaragoza Caballero, C , and C. G. H e r r e r a . 1 9 8 6 . Una nueva especie de Mastinowittmerus (Coleoptera: Phengodidae: Mastinocerini). An. Inst. Biol. Univ. Nac. Auton. Mex. 56: 2 0 3 - 2 0 6 .

Zaragoza Caballero, S., and W. Wittmer. 1 9 8 6 . Nue­vas especies de Phengodes Illiger (Coleoptera: Phen­godidae) de Mexico. An. Inst. Biol. Univ. Nac. Auton. Mex. 56: 177-188 .

Received for publication 8 August 1996; accepted 17 December 1996.