DIVERSITY OF SCOLYTINAE (COLEOPTERA:...

Kendra et al.: Ambrosia Beetle Diversity 123

DIVERSITY OF SCOLYTINAE (COLEOPTERA: CURCULIONIDAE) ATTRACTED TO AVOCADO, LYCHEE, AND ESSENTIAL OIL LURES

P

AUL

E. K

ENDRA

1

*, J

ORGE

S. S

ANCHEZ

1

, W

AYNE

S. M

ONTGOMERY

1

, K

ATHERINE

E. O

KINS

2

, J

EROME

N

IOGRET

1

,J

ORGE

E. P

EÑA

3

, N

ANCY

D. E

PSKY

1

AND

R

OBERT

R. H

EATH

1

1

USDA-ARS, Subtropical Horticulture Research Station, Miami, FL 33158

2

Florida Department of Agriculture and Consumer Services, DPI, CAPS, Gainesville, FL 32608

3

University of Florida, Tropical Research and Education Center, Homestead, FL 33031

*Corresponding author; E-mail: [email protected]

A

BSTRACT

The redbay ambrosia beetle,

Xyleborus glabratus

Eichhoff (Coleoptera: Curculionidae: Sco-lytinae), is an exotic wood-boring insect that vectors laurel wilt, a lethal vascular disease oftrees in the Lauraceae, including avocado (

Persea

americana

) and native

Persea

species (red-bay, swampbay). As part of research to identify host-based attractants for

X. glabratus,

wediscovered that a diverse array of non-target ambrosia beetles was attracted to the samesubstrates as

X. glabratus

. During Sep-Dec 2009, several field tests were conducted in northFlorida (in woodlands with advanced stages of laurel wilt) with traps baited with commer-cial lures of the essential oils, manuka and phoebe, and with freshly-cut wood bolts of avo-cado (a known host) and lychee (

Litchi chinensis

, a non-host high in the sesquiterpene

α

-copaene, a putative host attractant). In addition, manuka-baited traps were deployed in av-ocado groves in south Florida to monitor for potential spread of

X. glabratus.

The combinedtrapping results indicated that none of these substrates was specific in attraction of

X. gla-bratus

. Numerous non-target ambrosia beetles were captured, including 17 species repre-sentative of 4 tribes within the subfamily Scolytinae. This report provides photo-documentation and data on the species diversity and relative abundance for a group ofpoorly-studied beetles, the scolytine community in Florida

Persea

habitats.

Key Words: ambrosia beetles,

Persea americana

,

Litchi chinensis

, manuka oil, phoebe oil

R

ESUMEN

El escarabajo de la ambrosía del laurel rojo (redbay),

Xyleborus glabratus

Eichhoff (Coleop-tera: Curculionidae: Scolytinae), es un insecto exótico barrenador de madera que transmite lamarchitez del laurel, una enfermedad vascular mortal de árboles de la familia Lauraceae, loscuales incluyen el aguacate (

Persea americana

) y las especies nativas del género

Persea

(re-dbay, swampbay). Como parte de la investigación para identificar las substancias químicasque atraen a

X. glabratus

, fue descubierto que un arsenal diverso de otros escarabajos de laambrosía que no eran de interés económico fue atraído a las mismas substancias. Entre sep-tiembre y diciembre del 2009, se realizaron varias pruebas en el norte de la Florida (en arbo-ledas con etapas avanzadas de la marchitez del laurel) usando trampas con cebos comercialesde los aceites esenciales manuka y de phoebe, y con madera de aguacate recien cortada (unhuésped conocido) y del lychee (

Litchi chinensis

, que no es huésped, pero es alto en el sesqui-terpeno

α

-copaene, una substancia química atractiva). Además, trampas con manuka fuerondesplegadas en arboledas de aguacate en el sur de la Florida para supervisar la extensión po-tenciál del

X. glabratus

. Los resultados de las capturas combinados indicaron que ninguna deestas substancias eran específicas en la atracción del

X. glabratus

. Numerosos escarabajos dela ambrosía fueron capturados, incluyendo 17 especies que representan cuatro tribus dentrode la subfamilia Scolytinae. Este informe proporciona la foto-documentación y datos en la di-versidad de la especie y la abundancia relativa para un grupo de escarabajos poco estudiado,la comunidad del Scolytinae en los hábitats de

Persea

de la Florida.

Translation provided by the authors.

The redbay ambrosia beetle,

Xyleborus glabra-tus

Eichhoff (Coleoptera: Curculionidae: Scolyti-nae), is an exotic wood-boring insect that vectorslaurel wilt, a lethal vascular disease of trees inthe Lauraceae (Fraedrich et al. 2008). Haploid

males are flightless and remain within the hosttree, while diploid females (typically sibling-mated) disperse to colonize new hosts. Unlikemost ambrosia beetles, female

X. glabratus

areprimary colonizers, capable of attacking healthy

124

Florida Entomologist

94(2) June 2011

unstressed trees. During gallery excavation, fe-males introduce spores of a symbiotic fungus,

Raffaelea lauricola

T.C. Harr., Fraedrich &Aghayeva (Harrington et al. 2008), carried in my-cangial pouches located at the base of the mandi-bles (Fraedrich et al. 2008). The fungus providesfood for both larvae and adults, but it also invadesthe host vascular system and results in systemicwilt and ultimately tree death. Native to south-eastern Asia,

X. glabratus

was first detected inthe U.S. in 2002 near Port Wentworth, Georgia(Rabaglia et al. 2006). Since then, the vector-dis-ease complex has spread along the coastal plaininto South Carolina and Florida, and has been re-ported from a single county in Mississippi(USDA-FS 2010). In northern Florida, high mor-tality has occurred in native

Persea

species, in-cluding redbay (

P. borbonia

(L.) Spreng.) andswampbay (

P. palustris

(Raf.) Sarg.), and therapid southward spread of the pest complex cur-rently threatens commercial groves of avocado (

P.americana

Mill.), a confirmed susceptible host(Mayfield et al. 2008). Florida’s avocado produc-tion, centered in Miami-Dade County, is worth$13 million annually (USDA-NASS 2010), and re-placement costs of all avocado trees (commercialand backyard) in Miami-Dade, Broward, PalmBeach, and Lee Counties have been estimated at$429 million (Evans & Crane 2008).

Due to the serious economic threat posed by

X.glabratus

, there is a critical need for effective at-tractants to detect, monitor, and control thespread of this invasive pest. Preliminary researchprovided no evidence of an aggregation phero-mone and no strong attraction to its fungal sym-biont, to its frass, or to ethanol (a standard attrac-tant for ambrosia beetles); suggesting that hosttree volatiles are the primary attractants for dis-persing females (Hanula et al. 2008). Additionalstudies identified manuka and phoebe oils (essen-tial oil extracts from the tea tree,

Leptospermumscoparium

Forst. & Forst., and the Brazilian wal-nut,

Phoebe porosa

Mez., respectively) as effectivebaits for field monitoring of

X. glabratus

in SouthCarolina (Hanula & Sullivan 2008). Based oncomparisons of volatile chemicals emitted fromchipped redbay wood, manuka oil, and phoebe oil,Hanula & Sullivan (2008) hypothesized that 2sesquiterpenes,

α

-copaene and calamenene, werelikely the primary host attractants.

While conducting research to evaluate attrac-tion of female

X. glabratus

to wood volatiles andessential oil lures, we discovered that a diversenumber of non-target ambrosia beetles (both en-demics and exotics established in Florida) wereattracted to the same substrates as

X. glabratus

.Several field tests were conducted in north-cen-tral Florida (Alachua and Marion Counties) innatural stands of redbay and swampbay withknown infestations of

X. glabratus

and visiblesigns of laurel wilt disease. We used 4-funnel

Lindgren traps and/or sticky panels baited withcommercially available essential oil lures(manuka and phoebe) or with freshly-cut woodbolts of avocado (a confirmed host) and lychee(

Litchi chinensis

Sonn., a presumed non-host).Lychee is in the family Sapindacaeae; it lacks thetypical aromatic laurel volatiles, but it has a highcontent of

α

-copaene (Niogret et al. unpublished).During that same period, monitoring traps(Lindgren traps baited with manuka lures) weredeployed in avocado groves in south Florida (Mi-ami-Dade County). This report summarizes andillustrates the ambrosia beetles captured over a4-month period (Sep-Dec 2009) in Florida to (1)provide a tool for action agencies and field scien-tists to facilitate identification of non-target spe-cies captured in

X. glabratus

monitoring traps, (2)document the species diversity and relative abun-dance for the scolytine community in

Persea

hab-itats, and (3) identify potential secondary coloniz-ers of

Persea

hosts subsequent to initial attack by

X. glabratus.

M

ATERIALS

AND

M

ETHODS

Field test 1 was conducted in Citra, MarionCounty, FL at the University of Florida Agricul-tural Experiment Station (PSREU). The backedge of the station bordered an upland woodedarea dominated by mature live oak (

Quercus vir-giniana

Mill.) with an understory that includedredbay trees symptomatic for laurel wilt. Test 1was run from 27 Aug-22 Oct 2009 and consisted of6 treatments: a commercial manuka lure (Syn-ergy Semiochemicals, Burnaby, BC), wood boltsfrom 3 avocado cultivars representative of the 3horticultural races (‘Simmonds’, West Indianrace; ‘Brooks Late’, Guatemalan race; and ‘Seed-less Mexican’, Mexican race), bolts from lychee(cv. ‘Hanging Green’), and an unbaited control.Wood bolts were collected from the USDA-ARSgermplasm collection at the Subtropical Horticul-ture Research Station (SHRS), Miami, FL 1 dprior to test deployment. The ends of the boltswere coated with wax to prevent desiccation, andthen both ends re-cut when used as baits at thestart of the test. All baits were deployed in four-funnel Lindgren traps (BioQuip, RanchoDominguez, CA) with 300 mL of an aqueous solu-tion of 10% propylene glycol (Low-Tox antifreeze;Prestone, Danbury, CT) added to the collectioncup. For the manuka treatment, a single lure washung from the trap lid by a wire twist tie. For thewood substrates, 2 freshly-cut bolts (5 cm diam

×

15 cm length) were suspended with wire from thelid on opposite sides of the trap. Experimental de-sign was a randomized complete block, with 5 rep-licate blocks arranged in a linear array along thefence at the back of the research station. Within ablock, traps were spaced 10 m apart, 1.5 m abovethe ground, and spacing was 50 m between repli-


cate blocks. Traps were checked every 2 weeks fora total of 8 weeks. At each sampling date, the re-tention solutions (with insect captures) were col-lected, a thin layer was sawed from the lower endof each bolt (to “renew” release of wood volatiles),the collection cups were refilled, and trap posi-tions were rotated sequentially within each blockto minimize potential positional effects on beetlecapture.

Field tests 2 and 3 were conducted in CrossCreek, Alachua County, FL at the Lochloosa Wild-life Conservation Area (St. John’s River WaterManagement District). The study site consisted ofmesic flatwoods composed of an overstory of slashpine (

Pinus elliottii

Englem) with a mixed under-story that included numerous swampbay trees ex-hibiting advanced stages of laurel wilt. Test 2 wasconducted from 7 Oct-2 Dec 2009 and evaluatedthe same 6 treatments described above. Test 3was conducted from 5 Nov-29 Dec (at a site adja-cent to test 2) and contained a commercial phoebelure (Synergy Semiochemicals) in addition to the6 treatments above. However, with tests 2 and 3there were differences in trap type and trap lay-out. The essential oil lures were still deployed infour-funnel Lindgren traps, but the wood boltswere paired and hung vertically with 2 whitesticky panels (23 cm

×

28 cm, Sentry wing trapbottoms; Great Lakes IPM, Vestaburg, MI) sta-pled back-to-back at the bottom of the bolts.Sticky panels were further secured with severalbinder clips around the edges. Tests 2 and 3 fol-lowed randomized complete block design, with 5replicate blocks arranged in a rectangular grid.Each block consisted of a row of traps hung ~2 mhigh in non-host trees, with a minimum of 10 mspacing between adjacent traps in a row, and with50 m spacing between rows. Both tests were 8weeks in duration and checked every 2 weeks. Ateach check, the retention solutions and stickypanels were collected, a thin layer was sawedfrom the bottom of each bolt, the solutions/panelswere replaced, and the trap positions were ro-tated sequentially within each row.

In addition to the field tests conducted in northFlorida, monitoring/survey traps were deployedin several avocado groves in Miami-Dade County,FL during the fall of 2009. All monitoring trapsconsisted of four-funnel Lindgren traps baitedwith manuka lures, which were hung ~2 m aboveground within the canopy of avocado trees. Trapswere checked at 2-week intervals and sites in-cluded the SHRS avocado germplasm collection inMiami and 3 commercial groves in Homestead.

All sample collections (from monitoring trapsand field tests) were sorted in the laboratory atSHRS, and scolytine species were counted, photo-graphed, and stored in 70% ethanol. Specimensremoved from sticky panels were soaked over-night in histological clearing agent (Histo-clearII; National Diagnostics, Atlanta, GA) prior to

storage in alcohol. Beetle identifications wereconfirmed at FDACS-DPI (Gainesville, FL) by K.E. Okins, and voucher specimens were depositedat both DPI and SHRS.

R

ESULTS

The combined trapping results from field testsand monitoring traps totaled 659 ambrosia bee-tles, consisting of 17 species from 4 tribes withinthe subfamily Scolytinae (Table 1). More than90% of the captures were from the tribe Xyle-borini, and only 1 specimen (

Coccotrypes distinc-tus

(Motshulsky)) was representative of the tribeDryocoetini. With the exception of

X. glabratus

(Fig. 1), most beetles were captured in fairly lownumbers, with many species represented by onlya single capture, despite significant trapping ef-forts with a variety of host-based attractants.

Xy-leborus glabratus

comprised the majority of cap-tures in north Florida, as expected due to its inva-sive pest status, but the percentages varied bysite (15% in Marion County with test 1; 75.4%and 86.2% in Alachua County with tests 2 and 3,respectively). Four other species of

Xyleborus

were captured (Fig. 2), with

X. ferrugineus

(Fabr-icius) (Fig. 2A) and

X. affinis

(Eichhoff) (Fig. 2B)the 2 most abundant species after

X. glabratus

.There were several other representatives withinthe tribe Xyleborini (Fig. 3), with

Ambrosiodmusobliquus

(LeConte) (Fig. 3A) a dominant speciesat both the Alachua site and in the Miami-Dadeavocado groves.

The tribe Cryphalini was represented by

Hy-pothenemus dissimilis

(Zimmerman) and severalother

Hypothenemus

species

difficult to discern tospecies level

(Fig. 4).

Hypothenemus

beetles weremajor components at the Marion site and in Mi-ami-Dade County. Within the tribe Corthylini, 5species were captured, of which 4 are presented inFig. 5. Two of those ambrosia beetles had distinc-tive morphological features. Females of

Corthyluspapulans

Eichhoff (Fig. 5C) have greatly enlargedterminal antennal segments which bear severallong, recurved setae (Fig 5E); males of

C. papu-lans

lack the characteristic setae. In

Pityoboruscomatus

(Zimmerman) (Fig. 5D), females areunique in that the mycangia are located on thepronotum, and consist of a pair of large shallowdepressions covered with dense pubescence (Fig.5F; Furniss et al. 1987).

D

ISCUSSION

The subfamily Scolytinae contains 2 function-ally distinct groups of beetles - bark beetles whichfeed on phloem from the inner bark of host trees,and ambrosia beetles which cultivate and feed onsymbiotic fungi within the xylem layers (Rabaglia2002). Among the bark beetles, there are majorforest pests which have been well studied, includ-

126


94(2) June 2011

ing the southern pine beetle,

Dendroctonus fron-talis

Zimmerman (Chellman & Wilkinson 1980),the western pine engraver,

Ips

pini

(Say) (Kegleyet al. 1997), and several other

Ips

spp. found inthe southeastern U.S. (Conner & Wilkinson1998). In contrast, the ambrosia beetles are gen-erally not of economic importance and conse-quently have received less attention. They areminute beetles, spend the majority of their lifeconcealed within host trees, and typically attackstressed or dying trees. Despite the large numberof described species (e.g., >500 currently recog-nized

Xyleborus

spp. worldwide, Rabaglia et al.2006), much is unknown regarding the basic biol-ogy, ecology, host range, fungal symbionts, andpopulation dynamics of many endemic ambrosiabeetles. Far less is known about exotic invasivespecies, which are not pests in their native landsbut may acquire pest status when introduced intonew environments, as is the case with

X. glabra-tus

in the U.S.Although our research was focused on identifi-

cation of attractants specifically for detection andcontrol of

X. glabratus

, information was obtained

concurrently on the species diversity and relativeabundance for the ambrosia beetles found in na-tive

Persea

habitats in north-central Florida. Insouth Florida the trapping effort was less inten-sive, but preliminary data was also obtained forthe species composition in avocado groves. Thissummary report identifies the species of Scolyti-nae most likely to be encountered while monitor-ing for

X. glabratus

, and the photo-documenta-tion provides fellow researchers (non-taxono-mists) and action agency personnel with a conve-nient tool for preliminary identification of non-target captures.

Some of the non-target beetles identifiedherein are species that may potentially functionas secondary vectors of the laurel wilt pathogen.Once healthy trees are attacked by

X. glabratus,the stressed trees are susceptible to further at-tack by secondary colonizers that can contributeto the rapid mortality seen in laurel hosts. Obser-vations made on dead swampbay trees at theLochloosa Conservation Area (Kendra et al. un-published) indicated that multiple wood-boringspecies attacked those Persea trees, as evidenced

TABLE 1. AMBROSIA BEETLES (CURCULIONIDAE: SCOLYTINAE) CAPTURED IN MARION, ALACHUA, AND MIAMI-DADECOUNTIES, FL FROM SEP-DEC 2009, ARRANGED ACCORDING TO LAWRENCE & NEWTON (1995).

Marion Co. Alachua Co. Miami-Dade Co

Test 1a Test 2b Test 3b Monitoringc

Tribe DryocoetiniCoccotrypes distinctus (Motschulsky) 1

Tribe XyleboriniAmbrosiodmus lecontei Hopkins 1Ambrosiodmus obliquus (LeConte) 6 14 22Premnobius cavipennis Eichhoff 1Theoborus ricini (Eggers) 1Xyleborus affinis (Eichhoff) 2 11 4Xyleborus californicus Wood 2 1Xyleborus ferrugineus (Fabricius) 3 34 22 1Xyleborus glabratus Eichhoff 3 193 287Xyleborus volvulus (Fabricius) 7 3

Tribe CryphaliniHypothenemus dissimilis (Zimmerman) 1 1Hypothenemus spp. 9 1 1 22

Tribe Corthylini

Subtribe CorthylinaCorthylus papulans Eichhoff 1Monarthrum mali (Fitch) 1

Subtribe PityophthorinaPityoborus comatus (Zimmerman) 1Pseudopityophthorus minutissimus (Zimmerman) 1Pseudopityophthorus pruinosus (Eichhoff) 1

a8-wk field test in redbay; Lindgren traps baited with wood bolts (avocado, lychee) or manuka oil lures.b8-wk field test in swampbay; Sticky traps baited with wood bolts (avocado, lychee); Lindgren traps baited with manuka/phoebe

oil lures.cMonitoring in avocado groves; Lindgren traps baited with manuka oil lures.


Fig. 1. The redbay ambrosia beetle Xyleborus glabratus Eichhoff, vector of a lethal wilt fungus (Raffaellea lau-ricola) causing high mortality of trees in the Lauraceae in the southeastern U.S. A. Female. B. Male. (Note: Malesof X. glabratus are flightless; this specimen was obtained from host wood, not from a flight trap.)

Fig. 2. Four species of Xyleborus not of economic importance. A. X. ferrugineus (Fabricius). B. X. affinis (Eich-hoff). C. X. volvulus (Fabricius). D. X. californicus Wood. (All specimens female.)

128 Florida Entomologist 94(2) June 2011

by bore holes of various diameters. These second-ary colonizers may potentially pick up Raffaeleafrom the host xylem and transport it to new trees,accelerating the spread of laurel wilt. In northFlorida, X. ferrugineus, X. affinis, and A. obliquuswere the most abundant species in native Perseahabitats; in avocado A. obliquus and Hypothene-mus spp. were dominant. In South Carolina, Ha-nula et al. (2008) found that Xylosandrus cras-siusculus (Motschulsky) was attracted towounded redbay trees. Further research shouldevaluate these additional beetle species to (a) de-termine if stressed (diseased) trees in the Lau-raceae can serve as hosts, and if so, then (b) deter-mine if Raffaelea spores can be recovered fromthe mycangia of ambrosia beetles that developedwithin Raffaelea-infected hosts. In other systems,there is evidence that exchange or “cross contam-ination” of symbiotic fungi may occur among am-brosia beetle species that occupy a common breed-ing site (Gebhardt et al. 2004). Alternatively, Raf-faelea spores may potentially be transported pas-

sively by the setae and/or cuticular asperities(protuberances) commonly found on the anteriorslope of the female pronotum, as has been demon-strated for Hypothenemus hampei (Ferrari) andspores of Fusarium solani (Martius) (Morales-Ra-mos et al. 2000).

The commercial lures currently available forX. glabratus are non-specific in attraction, sohigh numbers of non-target captures are likelyto be encountered with the monitoring system(manuka-baited Lindgren traps) employed bythe State of Florida. Manuka and phoebe oillures were originally developed for field moni-toring of another (phylogenetically distant)wood-boring beetle, the emerald ash borer Agri-lus planipennis Fairmaire (Coleoptera: Bupres-tidae) (Crook et al. 2008). Preliminary research(Kendra et al. unpublished) indicated that theseessential oil lures were not only non-specific, butmay have limited field life for attraction of X.glabratus. With the data set presented here, ap-proximately 30% of the captures were non-tar-get species of Scolytinae. Development of effec-tive strategies for early detection and control(i.e., attract-and-kill systems) of X. glabratus iscontingent on identification of specific attracta-nts. In the absence of species-specific phero-mones or food-based attractants for X. glabratus(Hanula et al. 2008), this will be a difficult chal-lenge.

CONCLUSIONS

Multiple trapping studies targeting the redbayambrosia beetle, Xyleborus glabratus, effectivelygenerated a survey of the overall scolytine com-munity resident in Florida Persea habitats. Theseambrosia beetle species that co-occur with X. gla-bratus, are attracted to the same host-based vola-tile chemicals; and they are the non-target spe-cies likely to be encountered in traps set out tomonitor for X. glabratus. Those species that canfunction as secondary colonizers of Persea hostsshould be evaluated as potential secondary vec-tors for transmission of the laurel wilt pathogen,Raffaelea lauricola.

ACKNOWLEDGMENTS

We gratefully acknowledge David Long, Mike Win-terstein (USDA-ARS; Miami, FL), Rita Duncan (Univ.Florida; Homestead, FL), Gurpreet Brar, and StephenMcLean (Univ. Florida; Gainesville, FL) for technicalassistance; Patti Anderson (FDACS-DPI, Gainesville,FL) for Persea identifications; Bud Mayfield (USDA-Forest Service; Asheville, NC) for advice on field trap-ping; Ray Schnell (USDA-ARS; Miami, FL) for adviceon avocado germplasm samples; David Jenkins(USDA-ARS; Mayagüez, PR) and 2 anonymous re-viewers for suggestions with the manuscript; PansyVázquez-Kendra and Elena Schnell for translation ofthe abstract; and Connie Rightmire (St. John’s River

Fig. 3. Ambrosia beetles within the tribe Xyleborini.A. Ambrosiodmus obliquus (LeConte). B. Theoborus ri-cini (Eggers). C. Premnobius cavipennis Eichhoff. (Allspecimens female.)


Water Management District) for assistance in obtain-ing a special use permit for the Lochloosa WildlifeConservation Area. This work was supported in partby the USDA-ARS National Plant Disease Recovery

System and the Florida Avocado Administrative Com-mittee. This report presents the results of researchonly; mention of a proprietary product does not con-stitute an endorsement by the USDA.

Fig. 4. Ambrosia beetles within the tribe Cryphalini. A. Hypothenemus dissimilis (Zimmerman). B. Hypothene-mus sp. (Both specimens female.)

Fig. 5. Ambrosia beetles within the tribe Corthylini. A. Monarthrum mali (Fitch). B. Pseudopityophthorus pru-inosus (Eichhoff). C. Corthylus papulans Eichhoff. D. Pityoborus comatus (Zimmerman). E. Detail of C. papulans(anterior end; ventral view) showing enlarged terminal antennal segments bearing long recurved setae. F. Detailof P. comatus (anterior end; dorsal view on left, lateral view on right) showing pronotal mycangia (oval pits coveredwith dense setae), the storage site for symbiotic fungal spores. (All specimens female.)


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LAWRENCE, J. F., AND NEWTON JR., A. F. 1995. Familiesand subfamilies of Coleoptera (with selected genera,notes, references and data on family-group names),pp. 779-1006 In J. Pakalud and S. A. Slipinski [eds.],Biology, Phylogeny, and Classification of Coleoptera:Papers Celebrating the 80th Birthday of Roy A.Crowson. Muzeum I Instytyt Zoologii PAN, Wasza-wa.

MAYFIELD III, A. E., PEÑA, J. E., CRANE, J. H., SMITH, J.A., BRANCH, C. L., OTTOSON, E. D., AND HUGHES, M.2008. Ability of the redbay ambrosia beetle (Co-leoptera: Curculionidae: Scolytinae) to bore intoyoung avocado (Lauraceae) plants and transmit thelaurel wilt pathogen (Raffaelea sp.). Florida Ento-mol. 91: 485-487.

MORALES-RAMOS, J. A., ROJAS, M. G., SITTERTZ-BHAT-KAR, H., AND SALDAÑA, G. 2000. Symbiotic relation-ship between Hypothenemus hampei (Coleoptera:Scolytidae) and Fusarium solani (Moniliales: Tuber-culariaceae). Ann. Entomol. Soc. America 93: 541-547.

RABAGLIA, R. J. 2002. XVII. Scolytinae Latreille 1807,pp. 792-805 In R. H. Arnett, Jr., M. C. Thomas, P. E.Skelley, and J. H. Frank [eds.], American Beetles,vol. 2, Polyphaga: Scarabaeoidea through Curculion-oidea. CRC Press, Boca Raton, FL.

RABAGLIA, R. J., DOLE, S. A., AND COGNATA, A. I. 2006.Review of American Xyleborina (Coleoptera: Curcu-lionidae: Scolytinae) occurring north of Mexico, withan illustrated key. Ann. Entomol. Soc. America 99:1034-1056.

USDA-FS. 2010. Laurel Wilt Distribution. UnitedStates Department of Agriculture, Forest Service,Forest Health Protection, Southern Region.

USDA-NASS. 2010. Noncitrus Fruits and Nuts: 2009preliminary summary, Fr Nt 1-3 (10) a. UnitedStates Department of Agriculture, National Agricul-tural Statistic Service.

Ferreira & Scheffrahn: Light Attraction Behavior of a Drywood Termite 131

LIGHT ATTRACTION AND SUBSEQUENT COLONIZATION BEHAVIORS OF ALATES AND DEALATES OF THE WEST INDIAN DRYWOOD TERMITE

(ISOPTERA: KALOTERMITIDAE)

M

ARIA

T

ERESA

F

ERREIRA

1,2

AND

R

UDOLF

H. S

CHEFFRAHN

1,2

1

Fort Lauderdale Research and Education Center, University of Florida,3205 College Avenue, Davie, FL 33314

2

Azorean Biodiversity Group (CITA-A), Departamento de Ciências Agrárias, Universidade dos Açores,Pico da Urze, Angra do Heroísmo, Portugal

A

BSTRACT

Laboratory studies were conducted during the 2007 and 2008 dispersal seasons of the WestIndian drywood termite

Cryptotermes brevis

(Walker), a serious urban pest of wooden struc-tures. Attraction to light and subsequent colonization of this species were studied by observ-ing the response of alates to lit and dark chambers. Several intensities of light were testedto determine if light intensity had a role in the alates’ attraction to light and subsequent col-onization. A bioassay was conducted with semi-shaded wood blocks to quantify negative pho-totaxis for the dealates. We found that the alates of

C. brevis

preferred flying into lit areasfor colonization, and that the number of colonizations was highest in the high light intensitytreatments. Negative phototaxis of the dealates was observed because these preferred to col-onize in the dark habitat treatments. This information is important when deciding what con-trol methods may be used to prevent

C. brevis

from colonizing wood structures. Traps witha high intensity light to attract

C. brevis

alates and to prevent infestation may be a way tomonitor and control this urban pest.

Key Words:

Cryptotermes brevis,

phototaxis, colonization, alates, dealates

R

ESUMO

Estudos num laboratório foram realizados durante a época de voo de dispersão de 2007 e2008 para a térmita da madeira seca

Cryptotermes brevis

(Walker), uma praga urbana de es-truturas de madeira séria. A atracção à luz e subsequente colonização desta espécie foi es-tudada, observando a resposta dos alados a câmaras de preferência de luz. Váriasintensidades de luz foram testadas para determinar se a intensidade da luz tinha um papelna atracção dos alados pela luz e subsequente colonização. Um ensaio usando blocos de ma-deira semi-cobertos para quantificar o comportamento fototáctico negativo dos dealados foiconduzido. Nós observámos que os alados de

C. brevis

preferem áreas com maior iluminaçãopara colonizarem, e que o número de colonizações era maior no tratamento com maior inten-sidade de luz. O comportamento fototáctico negativo dos dealados foi observado porque osdealados preferem colonizar nos tratamentos de habitats escuros. Esta informação é impor-tante quando se tem de decidir que métodos de controlo podem ser usados para prevenir atérmita

C. brevis

de colonizar estruturas de madeira. Usar armadilhas com uma intensidadeluminosa elevada para atrair alados de

C. brevis

e prevenir uma infestação poderá ser umaforma de monitorizar e controlar esta praga.

Translation provided by the authors.

The West Indian drywood termite

Cryptoter-mes brevis

(Walker) (WIDT) is a serious urbanpest that causes significant levels of damage towooden structures. This termite was first de-scribed in Jamaica in 1853 and has a tropicopoli-tan urban distribution except in Asia (Scheffrahnet al. 2008). Like most Kalotermitidae the WIDTnests in its food source, wood, where it spendsmost of its life cycle. A colony of drywood termitescan vary in size from hundreds to a few thousandtermites (Nutting 1970), and several colonies canbe found inside a single piece of wood. Myles et al.(2007), for example, found as many as 30 coloniesof WIDT in a single floor board.

Drywood termites are major pests, accountingfor about 20% of the budget spent on termite con-trol in the United States (Su & Scheffrahn 1990).One of the main methods for controlling thesepests has been the use of fumigation to eliminateexisting colonies. Fumigation, however, does notprevent new infestations, and therefore it is ben-eficial to combine fumigation with additional pre-ventative control methods. Preventing this spe-cies from founding a new colony during the dis-persal flight season is an important technique forthe control of this pest. This study aims to developa better understanding of the behavior of theWIDT during the dispersal flight season, the time

132


94(2) June 2011

when WIDT is most accessible to physical, chem-ical, or behavioral management efforts.

The life cycle of WIDT includes a dispersalflight where the mature winged forms (alates)leave their previous colony to form new colonies.The dispersal flights are the only occasion whenthis species is found outside of wood (Kofoid 1934)because it never leaves the nest to forage for newfood sources (Korb & Katrantzis 2004). After fly-ing, the alates shed their wings and associate aspairs of female and male dealates. These pairswill crawl on the substrata in search for a suitableplace to start a new colony (Snyder 1926; Wilkin-son 1962; Nutting 1969; Minnick 1973).

According to Light (1934a) most termitescastes are negatively phototactic, but the alates,attracted to light, seek to emerge into openingsand fly toward light. However, there are no datacorrelating colonization sites with lighting condi-tions. Positive phototaxis of

C. brevis

and conge-nus alates is followed by negative phototaxis ofthe dealates (Wilkinson 1962; Minnick1973), al-though no data have been produced to confirmthis observation.

The present study quantified the phototacticcolony site selection of

C. brevis

alates anddealates. The first hypothesis tested was that fa-vorable colonization sites in lighted areas aremore likely to be selected by alates of

C.brevis

,and that colonization will be higher at higherlight intensities. The second hypothesis testedwas that dealates search for darker areas on thesubstrate to colonize.

M

ATERIALS

AND

M

ETHODS

Termites

Experiments were conducted at the Universityof Florida, Fort Lauderdale Research and Educa-tion Center (FLREC), Davie, Florida, in a roompartially filled with wood infested by

C. brevis

that originated from several sites around SouthFlorida. The wood was stored in a dark room atambient temperature (average 25.6ºC) and ambi-ent relative humidity (average 73.5%). The ter-mite alates used in the experiments dispersednaturally from the infested wood. The room re-mained dark except for the time the experimentswere conducted, and the alates were free to flyanywhere in the room. The first experiments tookplace between Apr and Jul 2007, and the secondexperiments between Apr and Jun 2008.

Light Intensity Experiments

Twenty four transparent plastic boxes (36

×

23

×

28 cm) served as light preference chambers. Theboxes were wrapped in aluminum foil to isolatethe light box from adjacent boxes. The boxes wereplaced with the open side facing the infested wood

and a hole was cut in the side of each box in orderto fit (Fig. 1) an electrical cord for a set of whiteLight Emitting Diodes (LED) (HolidayLEDS™model No TS-70) strung in a series of 5 singlelight bulbs attached with tape and hung throughthe hole. The LEDs were all connected to eachother in a continuous string of lights mounted ina series and connected to a single power source.Electrical tape was used to position the lightbulbs in place as well as prevent light from dis-persing through the cut hole, so that the onlylight source was inside each box. The LED setswere randomly distributed among 12 lit boxesand 12 dark boxes. The lit boxes had a light inten-sity of approximately 40 lux as measured by alight meter (Extech Instruments model No403125) and the dark boxes had approximately0.11 lux (due to some contaminating light fromnearby experiments occurring at the same time).A cube of wood (5

×

5

×

5 cm) with 6 drilled holeswas placed in the center of each box (Fig. 1). Each2.3 mm diam hole was 1.5 cm deep, and there wasa single hole per face of the block. Four thumbpush pins were placed on the underside of theblock to allow for enough space (3 mm) for the ter-mite to access the hole on the underside.

The difference in colonization between differ-ent light intensities was analyzed with the same24 boxes previously described. The LED lightswere also used but this time there were 4 differ-ent set ups for the different light intensities with6 replicates per light intensity (measured by thelight meter): 6 of the boxes were dark with (

≈

0.11lux); 6 boxes had 1 LED light bulb with an inten-sity of approximately 11 lux; 6 boxes had 5 LEDstogether (

≈

40 lux); 6 boxes had 10 LEDs attachedwith an intensity of approximately 480 lux. Theboxes with the different light intensities wererandomly distributed. A block of wood (15x2x9cm) with 24 holes (12 on top and 12 on the bottom,and each 1.5 cm deep and 2.3 mm diam) wasplaced in the center of each box. After 3 months,the blocks were collected and the number of colo-nized holes per block was counted. A hole was con-sidered to be colonized when a complete fecal seal(covering of the hole with hardened fecal materialfrom the termites) was present.

Negative Phototaxis Experiment

The negative phototaxis of the dealates wasstudied with a white PVC pipe (51 cm

×

7 cm in-side diam) wrapped with electrical tape andclosed on one end. A 102

×

2

×

5 cm board with 40holes, each 2.3-mm diam and drilled 2.5 cm apart,was placed inside the pipe. The outermost holeswere 1 cm from the edge of the board. Of the 40holes, 20 were always exposed to light (regularfluorescent indoor lighting) and 20 were exposedto decreasing levels of light toward the closed endof the PVC pipe (Fig. 2). The board was visually


divided into 10-cm sections (excluding 1 cm ateach end). Each section had 4 holes available forcolonization. A board with the same dimensionsand number of holes was used as a control andwas completely exposed to the same light inten-sity. The 10-cm sections were lettered from A to Jwith sections A, B, C, D, and E inside the PVC pipeand sections F, G, H, I, and J outside (Table 1). Thisexperimental protocol was replicated 4 times. Lightmeasurements were made inside and outside thePVC pipe (Table 1). After dispersal flight seasonwas over the number of colonized holes was countedfor each 10-cm section based on the previously de-scribed colonization criteria.

Statistical Analysis

The data for the lit versus dark boxes were an-alyzed by a non-parametric Wilcoxon MatchedPairs test (SAS Institute 2003) to test whetherthe numbers of colonizations in the dark and lit

areas were different. Colonization differences be-tween the light intensities were tested with Stu-dent’s

t

-test (SAS Institute 2003).A Chi-squared test for independence (SAS In-

stitute 2003) was used to test whether the distri-bution of colonizations was dependent on the lightintensity. A Student’s

t-

test for dependent sam-ples was used to determine whether the differ-ences between the numbers of colonizations ineach 10-cm section were significant, (SAS Insti-tute 2003).

R

ESULTS

Light Intensity Experiments

Lit vs dark boxes.

A total of 43 holes were col-onized. There were significantly (

t-t

est = 4.01E-05,

P

< 0.0001) more holes colonized in the lightedboxes (2.8 ± 0.3, mean ± SEM) than in the dark ar-eas (0.8 ± 0.2 (mean ± SEM)).

Fig. 1. Arena for light intensity experiments. Aluminum foil isolated the box and the LED lights were placedabove the block of wood.

134


94(2) June 2011

Light intensity

.

A total of 76 holes were colo-nized in the light intensity experiment. A highernumber of colonizations was recorded in highlight intensity boxes. There were significant (

t-

test,

P

< 0.05) differences in colonizations amongthe various light intensities (Fig. 3).

Negative Phototaxis Experiment

A total of 175 holes were colonized during thenegative phototaxis experiment. There were nosignificant differences in number of colonizationsbetween the 10-cm sections (

P

≥

0.29) in the con-trol boards. The distribution of number of coloni-zations was independent of the section where thecolonizations occurred (Chi-squared = 5.9541,

P

=0.75; n.s.) with no section of the control board pre-ferred over any other section.

In the boards placed in the PVC pipes coloniza-tion distribution was not independent of the sec-tion where it occurred (Chi-squared = 33.3939,

P

=0.001) with a significantly higher number of colo-nizations occurring in the dark sections (Fig. 3).Sections A, B, C, and D inside the PVC pipe had asignificantly higher number of colonizations thansections G, H, I, and J outside the PVC pipe (

P

<0.05). Section E (inside the PVC pipe) was not sig-nificantly different (

P

≥

0.16) from sections G-J(outside the PVC pipe); and section F (outside thePVC pipe) was not significantly different (

P

≥

0.08)from sections A-D (inside the PVC pipe) (Fig. 4).

D

ISCUSSION

The results of the light versus dark experi-ment confirmed the hypothesis that colonizationof the alates occur more frequently in lit areas.The termite

C. brevis

in South Florida fliesmainly between 1:00 and 2:00

AM

(unpublisheddata) when it is very dark. These results showedthat colonization sites located in areas that are litduring the night may be more susceptible to infes-tation by

C. brevis

during dispersal flights. Thepresence of artificial lights may cause a change ofbehavior for some species of animals (Longcore &Rich 2004), but artificial lights may be beneficialto structure infesting termites like

C. brevis.

Theattraction of these termites to artificial lightsputs structures that have a continuous nighttimelight source at higher risk of being infested thanstructures near by that are not lit.

The light intensity experiment showed that in-creasing light intensities increased the number oftermite colonizations (Fig. 3). Minnick (1973) re-ported differences in the wavelength of light pre-ferred by

C. brevis,

and Guerreiro et al. (2007) re-ported differences in color preference for thealates. Neither of them reported results based onlight intensity. The fact that we found that 11 luxof light intensity was significantly different from480 lux shows that the increase in light intensitycaused an increase in colonization of the woodblocks by the termites.

Cryptotermes brevis

has

Fig. 2. Arena for negative phototaxis experiment. PVC pipe wrapped in black tape covered half of the wood block.Controls had no PVC pipe cover.

T

ABLE

1. D

ISTANCES

OF

10-

CM

WOOD

SECTIONS

FROM

THE

CLOSED

END

OF

51-

CM

PVC

PIPE

AND

THE

LIGHT

INTENSITYAT

THE

CENTER

OF

EACH

SECTION

.

Section

Inside of PVC pipe Outside of PVC pipe

A B C D E A B C D E

Distance from closed end of PVC pipe (cm) 10 20 30 40 50 60 70 80 90 100Light Intensity (lux) 0.01 0.04 0.08 0.20 0.70 600 600 600 600 600


been observed to have as many as 2 founded colo-nies in a total of 22 nuptial chambers (Scheffrahnet al. 2001) which means that there is a 9% suc-cess rate of colonization. This shows that invest-ing in preventing

C. brevis

alates from foundingcolonies is important because their success rate ishigh enough to make prevention methods neces-sary. The attraction to light by alates can be usedto create light traps as a form of preventing infes-tations and re-infestations by

C. brevis

alates andas a means of partial control of this species. Suchlight traps inside structures that are already in-fested may minimize the spread of the infestation,and the results of this study showed that more in-tense the light used the more alates will be at-tracted; thereby making the use of light a possiblealternative to use of chemicals for prevention. Fur-ther studies with different light wavelengths canhelp improve light traps as an alternative methodto prevent the founding of colonies by

C. brevis

.Previous experiments with

C. havilandi

(Sjöst-edt) (Wilkinson 1962) and

C. brevis

(Minnick1973) showed negative phototaxis in dealates ofthese species. After landing, the dealates searchand colonize dark areas. Due to the nature ofwood structures, it could be argued that this be-havior is not really negative phototaxis but thatbecause the cracks and holes are usually hiddenand in dark areas the dealates end up colonizingthere. If so, the behavior would be dependent noton light intensity but on the locations of a good

places to colonize. However, the present studyshowed that negative phototaxis of dealates didoccur. Independent distributions of colonizationsoccurred in the controls, but independent coloni-zations did not occur in the semi-shaded blocks;this confirmed the negative phototaxis hypothe-sis. The lighter segment of the wood block in-serted in the PVC pipe had significantly less colo-nizations than the darker segment. However, thenumbers of colonizations in section F (immedi-ately outside the PVC pipe) were not significantlydifferent from the numbers in the darker areasinside the PVC pipe.

Colonization of section F might have occurredbecause the termites colonizing that area hadsearched for colonizing sites in the dark area in-side the PVC pipe where earlier colonizers had al-ready taken all suitable sites, i.e., a site satura-tion. Also they may have landed near the PVCpipe cueing in on the darker area nearby and col-onizing the sites near that dark area. However,further studies on this are needed to understandwhy the section closest to the PVC pipe on thelight side was significantly more colonized thanthe section immediately inside the dark PVCpipe; where it would be expected considering thenegative phototaxis behavior. One way to ap-proach this might be to use a higher density of col-onizing holes or fewer termites, so that the num-ber of holes is not a limiting factor.

In termites, both the alates and dealates havewell developed compound eyes (Light 1934b) andtheir behavior during flight season has shownthat they do respond to light while in flight (posi-tive phototaxis) and to have negative phototaxisafter landing. Further studies on the behavior of

C. brevis

during the flight season can help im-prove different methods to prevent colonizationand subsequent infestation by this species.

This study has shown that alates fly to and col-onize more in higher light intensity areas, whilethe dealates have an opposite behavior colonizingmore in darker areas. This knowledge is useful toimprove light traps as a control method againstcolony foundation from

C. brevis

.

A

CKNOWLEDGMENTS

We thank the late Boudanath Maharajh for techni-cal support, and Roxanne Connelly, Jonathan F. Day,and Paulo A. V. Borges for reviewing an early version ofthis manuscript. We also thank Dr. James L. Nation andanonymous reviews whose invaluable critical commentshelped improve this manuscript. Financial support forthis research was provided in part by the University ofFlorida and the Portugal Foundation for Science andTechnology (FCT-SFRH/BD/29840/2006).

R

EFERENCES

C

ITED

G

UERREIRO

, O., M

YLES

, T. G., F

ERREIRA

, M., B

ORGES,A., AND BORGES, P. A. V. 2007. Voo e fundação de

Fig. 3. Average number of holes colonized by C.brevis per light intensity (n = 43). Different letters rep-resent significant differences at P < 0.05.

Fig. 4. Average number of C. brevis colonized holesper section (n = 175). Sections A, B, C, D, and E were in-side the PVC pipe and sections F, G, H, I, and J wereoutside the PVC pipe. Bars with the same letter werenot significantly different at P < 0.05.


colónias pelas térmitas dos Açores, com ênfase naCryptotermes brevis, pp. 29-46 In P. Borges and T.Myles [eds.], Térmitas dos Açores. Principia, Estoril,127 pp.

KOFOID, C. A. 1934. Biological backgrounds of the ter-mite problem, pp. 1-12 In C. A. Kofoid, S. F. Light, A.C. Horner, M. Randall, W. B. Herms, and E. E. Bowe[eds.], Termites and Termite Control. Univ. Califor-nia Press, Berkeley, California, 795 pp.

KORB, J., AND KATRANTZIS, S. 2004. Influence of envi-ronmental conditions on the expression of the sexualdispersal phenotype in a lower termite: implicationsfor the evolution of workers in termites. Evol. Devel-op. 6: 342-352.

LIGHT, S. F. 1934a. The constitution and development ofthe termite colony, pp. 22-41 In C. A. Kofoid, S. F.Light, A. C. Horner, M. Randall, W. B. Herms, and E.E. Bowe [eds.], Termites and Termite Control. Univ.California Press, Berkeley, California, 795 pp.

LIGHT, S. F. 1934b. The external anatomy of termites,pp. 50-57 In C. A. Kofoid, S. F. Light, A. C. Horner, M.Randall, W. B. Herms, and E. E. Bowe [eds.], Ter-mites and Termite Control. Univ. California Press,Berkeley, California, 795 pp.

LONGCORE, T., AND RICH, C. 2004. Ecological light pol-lution. Front. Ecol. Environ. 2: 191-198.

MINNICK, D. R. 1973. The flight and courtship behaviorof the drywood termite, Cryptotermes brevis. J. Envi-ron. Entomol. 2: 587-591.

MYLES, T. G., BORGES, P. A. V., FERREIRA, M., GUER-REIRO O. BORGES, A., AND RODRIGUES, C. 2007. Filo-

genia, biogeografia e ecologia das térmitas dosAçores, pp. 15-28 In P. Borges and T. Myles [eds.],Térmitas dos Açores. Principia, Estoril, 127 pp.

NUTTING, W. L. 1969. Flight and colony foundation, pp.233-282 In K. Krishna and F. M. Weesner [eds.], Bi-ology of Termites.Volume I. Academic Press, Londonand New York, 598 pp.

NUTTING, W. L. 1970. Composition and size of some ter-mite colonies in Arizona and Mexico. Ann. Entomol.Soc. America 63: 1105-1110.

SAS. 2003. Version 9.1. SAS Institute, Cary, NorthCarolina.

SCHEFFRAHN, R. H., BUSEY, P., EDWARDS, J. K., KRE-CEK, J., MAHARAJH, B., AND SU, N-Y. 2001. Chemicalprevention of colony foundation by Cryptotermesbrevis (Isoptera: Kalotermitidae) in attic modules. J.Econ. Entom. 94: 915-919.

SCHEFFRAHN, R. H., KRECEK, J., RIPA, R., AND LUPPI-CHINI, P. 2008. Endemic origin and vast anthropo-genic dispersal of the West Indian drywood termite.Biol. Invasions. http://www.springerlink.com/content/914p8530t781632n/fulltext.html. Accessed July 2008.

SNYDER, T. E. 1926. The biology of the termite castes.Quart. Rev. Biol. 1: 522-552.

SU, N.-Y., AND SCHEFFRAHN, R. H. 1990. Economicallyimportant termites in the United States and theircontrol. Sociobiology 17: 77-94.

WILKINSON, W. 1962. Dispersal of alates and establish-ment of new colonies in Cryptotermes havilandi(Sjöstedt) (Isoptera, Kalotermitidae). Bull. Entomol.Res. 53: 265-288.

Pereira et al.: Male

A. suspensa

Lipid and Protein Content 137

INFLUENCE OF METHOPRENE AND DIETARY PROTEIN ON MALE

ANASTREPHA SUSPENSA

(DIPTERA: TEPHRITIDAE) LIPID AND PROTEIN CONTENT

R

UI

P

EREIRA

1,2

, J

OHN

S

IVINSKI

2

, J

EFFREY

P. S

HAPIRO

2

AND

P

ETER

E. A. T

EAL

2

1

Entomology and Nematology Department, University of Florida, Gainesville, Florida

2

Center for Medical, Agricultural and Veterinary Entomology, USDA-ARS, Gainesville, Florida

A

BSTRACT

Because both the application of a juvenile hormone analog, methoprene, and the addition ofprotein to the adult diet increased the sexual success of male Caribbean fruit fly,

Anastrephasuspensa

(Loew) (Diptera: Tephritidae), it was hypothesized that both might also impactmale nutritional status. Total content of lipid and of protein in

A. suspensa

males were mea-sured to discover if there was an effect of these treatments alone or in combination on thecontent of each of these subtstances. In the first 24 hours following adult emergence, 6 dif-ferent treatments were applied (all possible combinations of methoprene in acetone solutionor acetone alone, and protein-diet enrichment). Adult weight was determined for all treat-ments at 5, 10, 15, 20, 25, 30 and 35 d post-emergence. Dietary protein had a positive effecton the weight and total lipid and protein contents during the first 35 d of adult male life.There were minimal negative impacts from methoprene applications. Even though maleswere more active sexually, there was no significant change in weight or protein content dur-ing the study period. However, total lipid content decreased with age. The usefulness of me-thoprene to enhance the sexual performance of mass-reared tephritids destined for sterilerelease appears to outweigh any physiological costs/limitations that such treatment mightconfer.

Key Words: adult age, adult weight, Caribbean fruit fly, hydrolyzed yeast, juvenile hormone,sexual maturation

R

ESUMEN

Debido a que tanto la aplicación de un análogo de la hormona juvenil, metopreno, y la adi-ción de proteínas a la dieta del adulto aumento el éxito sexual del macho de la mosca de lafruta de Caribe,

Anastrepha suspensa

(Loew) (Diptera: Tephritidae), se planteó la hipótesisde que ambos también podrían afectar el estatus nutricional masculino. Se midió el conte-nido total de lípidos y de proteínas en los machos de

A. suspensa

para descubrir si había unefecto de estos tratamientos solos o en combinación sobre el contenido de cada una de estassustancias. En las primeras 24 horas después de la emergencia de adultos, se aplicaron 6 di-ferentes tratamientos (todas las combinaciones posibles de metopreno en solución de ace-tona en solución o solo acetona y con el enriquecimiento de proteínas en la dieta). Sedeterminó el peso adulto para todos los tratamientos a los 5, 10, 15, 20, 25, 30 y 35 días des-pués de la emergencia. Las proteínas dietéticas tuvo un efecto positivo sobre el peso y el totalde lípidos y proteínas durante los primeros 35 días de vida de los machos adultos. Hubo unmínimo de impactos negativos de las aplicaciones de metopreno. A pesar de que los machoseran más activos sexualmente, no hubo ningún cambio significativo en el peso o el contenidode proteína durante el período de estudio. Sin embargo, el contenido de lípidos totales dis-minuyeron con la edad. La utilidad de metopreno para mejorar el desempeño sexual de mos-cas tefrítidas criadas en masa destinadas para programas que liberan los machos estériles

parece superar los costos fisiológicos y las limitaciones que dicho tratamiento puede conferir.

Topical application of the juvenile hormone an-alog, methoprene, on the dorsal surface of adultmale Caribbean fruit flies,

Anastrepha suspensa

(Loew) (Diptera: Tephritidae), increases malesexual success (Pereira et al. 2009), apparentlybecause it increases the production of male sexpheromone. In addition it accelerates sexual mat-uration by several days (Teal & Gomez-Simuta2002). The addition of protein to the adult diethas a similar effect on sexual performance, but

the underlying cause(s) has yet to be investigatedexperimentally. When methoprene and proteinare combined there is an additive increase inmale sexual performance, and males are ~ 4 timesmore likely to mate than males not exposed to me-thoprene nor given access to protein (Pereira etal. 2010).

Presumably, increased pheromone productionoccurring at an earlier age, as well as acceleratedsexual activity, is energetically demanding and

138


94(2) June 2011

may affect the balance of the metabolic com-pounds (Teal et al. 2000). One hypothesis fre-quently mentioned for the relatively long, some-times more than 2 weeks, pre-reproductive periodfound in many adult frugivorous Tephritidae isthat the time is used to acquire resources neededfor reproduction (Sivinski et al. 2000). Decreasesin resource foraging time due to accelerated sex-ual maturation and increases in body nutrientsresource expenditure might be expected to resultin substantive changes in a fruit fly’s nutritionalstatus that could affect longevity and long-termsexual performance. We further supposed thatthese expenses could be particularly difficult toincur in the absence of a protein enriched adultdiet.

As a result, we hypothesized that the nutri-tional effects of methoprene and diet, both aloneand in combination, might have an important ef-fect on sexual performance of male flies reared forsterile insect technique (SIT) programs. It is fromthis perspective that we address the followingquestions: (1) What influence does methoprenetreatment have on male

A. suspensa

weight andlipid/protein nutrient stores over a period of 35 d;~ 33% of males survive to this age under labora-tory conditions (Sivinski 1993); and (2) do proteinenriched and protein deprived diets affect maleweight and lipid/protein content, and is there aninteraction between diet and methoprene treat-ment?

Considering the potential importance of adultdiet to SIT, relatively little nutritional work hasbeen done with

A. suspensa

. The rate and thetemporal patterns of consumption of carbohy-drates, proteins, and amino acids by adults(Sharp & Chambers 1984; Landolt & Davis-Her-nandez 1993), as well as the role of food availabil-ity and quality on male pheromone productionhave been studied to some extent (Epsky & Heath1993; Teal et al. 2000; Teal & Gomez-Simuta2002). A positive influence of sucrose on malepheromone calling (Landolt & Sivinski 1992) andsurvival (Teal et al. 2004) has been reported.However, no work has been done specifically onthe nutritional impact of protein incorporationinto adult diets. This is the first study of male te-phritid nutritional balance challenged by artifi-cially elevated “juvenile hormone” titers to im-prove sexual performance.

M

ATERIAL

AND

M

ETHODS

Insects

The Caribbean fruit flies used in this studywere obtained from laboratory colony at the Cen-ter for Medical, Agricultural and Veterinary Ento-mology (CMAVE) USDA-ARS, at Gainesville, FL.At the time of the study, the colony was 3 yearsold and had been produced according to the con-

ventional mass rearing protocols (FDACS 1995).Pupae were collected from the colony and sortedby size in a pupal sorting machine (FAO/IAEA/USDA 2003). Pupal size was homogenized to re-duce male size and weight variability; large maleshave been shown to have a sexual advantage oversmaller males (Burk & Webb 1983; Burk 1984;Webb et al. 1984; Sivinski & Dodson 1992; Sivin-ski 1993). Males used for this experiment werefrom pupal size class of 10.9 0.71 mg (

n

= 30) inweight. This is considered a mid-size pupalweight for field collected

A. suspensa

males in in-fested guava fruits (Hendrichs 1986). Throughoutthe experiment flies were maintained in a labora-tory room with a photoperiod of 13L:11D (lightfrom 0700 to 2000 h), a light intensity of 550 50lux, a temperature of 25 1°C and a relative hu-midity of 55 5%.

Diet and Hormonal Treatments

Following emergence, males were subjected to1 of the following 6 diet and hormonal treat-ments:

•

M

+

P

+

: topical methoprene in acetone;access to sugar and hydrolyzed yeast

•

M

+

P

-

: topical methoprene in acetone;access to sugar

•

M

-

P

+

: topical acetone; access to sugar andhydrolyzed yeast

•

M

-

P

-

: topical acetone; access to sugar

•

P

+

: no topical application; access to sugarand hydrolyzed yeast

•

P

-

: no topical application; access to sugar.

Methoprene (5 µg in 1 µL acetone) was appliedtopically within the first 24h after emergence.Controls consisted of application of 1 µL acetoneonly (M

-

) or no topical application (P

+

and P

-

). Inorder to conduct the topical application, maleswere immobilized in a net bag, and the solutionwas applied through the mesh on the dorsal sur-face of the thorax from a micro-pipette. No anaes-thesia was used to immobilize the flies. Precau-tions were taken to avoid cross contaminationsamong experimental subjects. Male flies exposedto the different treatments were maintained inscreen cages (30 cm by 30 cm by 30 cm), with amaximum male density of 200 flies/cage. Flieswere allowed free access to food (according toabove treatments) and water. In protein-deprivedtreatments (P

-

) flies were only provided withsugar. Protein was provided to the flies in theform of hydrolyzed yeast mixed with sugar (1:3parts, respectively). This mixture is considered ahigh quality diet for

Anastrepha

species (Jácomeet al. 1995; Aluja et al. 2001).

Experimental cages were maintained for up to35 d. For weight and chemical analysis, male flies

Pereira et al.: Male

A. suspensa

Lipid and Protein Content 139

were sampled at the following ages: 5, 10, 15, 20,25, 30, and 35 d of adult age. For each age andtreatment, 5 flies were randomly sampled andstored at -84ºC until used for analysis. In order toobtain the base line information after emergence,5 newly emerged (without access to any food orwater) and untreated flies were collected as well.Because lipid content in

Ceratitis capitata

(Wied.)has been found to vary according to the time ofthe day, due to the different activities in whichmales were engaged (Warburg & Yuval 1997), wesampled males at the same time each day (16:30h, immediately before the beginning of the callingperiod). Flies were weighed individually prior tohomogenization for lipid and protein determina-tion.

Quantification of Lipids and Proteins

Individual male flies were homogenized in asolution of PBS buffer at pH 7.25 (8.77 g of 0.15 MNaCl and 7.1 g of 50 mM Na

2

HPO

4

in 1 L of wa-ter). The homogenate was then brought up to 4.0mL with PBS. Lipids were extracted from the ho-mogenate by adding 40 mg of Na

2

SO

4

to half of theinitial volume, and 3.75 mL of chloroform: meth-anol (1:2) (Bligh & Dyer 1959) was used to sepa-rate polar and non-polar constituents of the ho-mogenate. An additional 1.25 mL of chloroformwas added to the homogenate and vortexed for 4min at 4,000 rpm. The non-polar chloroformphase was collected. The remaining solution wasre-extracted with chloroform (1.875 mL), vor-texed and collected. Chloroform was evaporatedin a Speed Vac device (Thermo Savant, San Jose,CA).

Lipid contents were determined by the vanillinreagent method (Van Handel 1985; Warburg &Yuval 1996), with triolein being used as a stan-dard. Quantification of lipids was done by react-ing 10 µL of sample with 190 µL of vanillin re-agent. Lipid content was determined colorimetri-cally at 530 nm in a spectrophotometer (Bio-tekInstruments, Winooski, VT).

Protein determination was done according thePierce BCA protein assay (Pierce, Rockford, IL).One mL of the polar fraction of the homogenatewas centrifuged for 1 min at 14,000 rpm. Half thevolume was mixed with 100 µL of sodium deoxy-choate reagent (0.15 w/v) and 100 µL of 72% (w/v)tricloroacetic acid (TCA) to precipitate the pro-teins. After incubation at room temperature for 10min and centrifugation for 10 min at 14,000 rpmthe supernatant was discarded. The precipitatewas dissolved and reacted with 50 µL of 5% (w/v)sodium dodecyl sulfate (SDS) and 1 mL of Piercemicro BCA

TM

protein assay reagent (Pierce, 1999).After incubation in a water bath at 37ºC for 30min, proteins in samples and standards were de-termined colorimetrically at 562 nm in a spectro-photometer (Bio-Tek Instruments, Winooski, VT).

Statistical Analyses

Data were analyzed by two-way analysis ofvariance (ANOVA) to detect the interactions be-tween age and treatment for the parametersstudied, independently (weight, lipid content, andprotein content). These analyses were followed byan ANOVA to detect differences between meansin the treatments. Tukey’s test was used to sepa-rate means (Ott & Longnecker 2001). Statisticalanalyses were performed with R software (ver-sion 2.1.0, www.r-project.org).

R

ESULTS

Male weight

Average adult weight varied between 5.8 mgand 11.6 mg (Fig. 1). There was no interaction be-tween treatment and adult age (

F

35,192

= 1.16,

P

=0.256), and no effect of age (

F

7,192

= 1.59,

P

= 0.140;Table 1) on adult weight. There was, however, asignificant effect of treatment (

F

5,192

= 24.46,

P

<0.05). Protein-fed males generally had signifi-cantly higher fresh weights than sugar fed males(Fig. 1, Table 2).

Lipid content

Significant effects of treatment (

F

5,192

= 131.37,

P

< 0.001), adult age (

F

7,192

= 83.14,

P

< 0.001), andthe interaction of adult age and treatment (

F

35,192

=6.34,

P

< 0.001) were found. Male lipid contentper treatment per age (Fig. 2) differed bothamong ages and for different treatments (Table 1)and among treatments for different ages(Table 2). In protein-deprived males, lipid con-tents dropped at 5 d after emergence, while pro-tein-fed males maintained stable lipid levels dur-ing the first 10 d of adult life (Fig. 2). Afterwards,lipids dropped to lower levels. Methoprene treat-ment did not affect lipid levels in either protein-fed or protein-deprived male flies.

Protein Content

Significant effects of treatment (

F

5,192

= 44.63,

P

< 0.001), adult age (

F

7,192

= 15.00,

P

< 0.05), and theinteraction of adult age and treatment (

F

35,192

=4.77,

P

< 0.001) were found. Significant differ-ences in protein content among the different ageswere found within each treatment except fortreatment M

-

P

+

(Fig. 3, Table 1), and among treat-ments at all ages (Table 2). Protein-fed malesmaintained higher protein levels than protein-de-prived males (Fig. 3). In protein-fed males, pro-tein content steadily increased through time,while in protein-deprived males, protein levelsdeclined. Methoprene did not affect the level ofprotein in either protein-fed or protein-deprivedmales.

140


94(2) June 2011

D

ISCUSSION

In male

A. suspensa

there was a clear effect of aprotein-enriched diet on weight, total lipid, and to-tal protein content over the first 35 d of adult life.In contrast, there was no effect of methoprene oracetone application on the studied parameters. Re-gardless of diet type, weight and total protein con-tent were relatively stable during adult life. In con-trast, total lipid content steadily decreased withage. This decline began later, however, in flies fed aprotein-enriched diet (10 d after emergence).

In all the treatments, consumption of proteinresulted in insects able to regulate their weight,protein levels, and lipid content at higher levelthan insects without a protein food source. This isbroadly consistent with what has been observedin Tephritidae in general and other

Anastrepha

spp. in particular (Aluja et al. 2001). In nature,adult tephritids feed on a variety of carbohy-drates and proteins derived from fruit juices, hon-eydew, and bird feces (Hendrichs et al. 1991; War-burg & Yuval 1997; Yuval & Hendrichs 2000).Protein enhances reproductive performance in

C.

Fig 1. Mean (±SD) adult weight (n = 5) of male Caribbean fruit flies at different adult ages among the 6 treat-ments featuring methoprene (M) and protein (P) and their various combinations.

T

ABLE

1. A

NALYSIS

OF

VARIANCE

(ANOVA)

FOR

MALE

CARIBBEAN

FRUIT

FLY

WEIGHT

,

TOTAL

LIPIDS

,

AND

TOTAL

PRO-TEINS

AMONG DIFFERENT AGES IN 6 DIFFERENT TREATMENTS FEATURING METHOPRENE (M) AND PROTEIN (P)AND THEIR VARIOUS COMBINATIONS (NS, NON SIGNIFICANT DIFFERENCES, P > 0.05; * 0.01< P < 0.05; *** P< 0.001).

Treatments Male weight Total lipids Total proteins

M+P+ F7,32 = 1.1559 (ns) F7,32 = 7.5932 *** F7,32 = 3.2711 *M+P- F7,32 = 1.9367 (ns) F7,32 = 32.76 *** F7,32 = 8.6007 ***M-P+ F7,32 = 1.8041 (ns) F7,32 = 11.124 *** F7,32 = 2.0186 (ns)M-P- F7,32 = 2.0277 (ns) F7,32 = 35.906 *** F7,32 = 7.7853 ***P+ F7,32 = 1.0071 (ns) F7,32 = 12.504 *** F7,32 = 2.9685 *P- F7,32 = 0.1291 (ns) F7,32 = 20.805 *** F7,32 = 4.8732 ***

Pereira et al.: Male A. suspensa Lipid and Protein Content 141

capitata (Warburg & Yuval 1997; Kaspi et al.2000; Shelly & Kennelly 2002; Shelly et al. 2002;Yuval et al. 2002), and protein-fed males start tocall earlier in life (Papadopoulos et al. 1998). Pro-tein-fed males are more competitive in terms ofpost copulatory sexual selection as well (Taylor &Yuval 1999). In Bactrocera dorsalis (Hendel), in-corporation of protein into adult diet significantlyincreases survival and mating success (Shelly elal. 2005). Among Anastrepha species, Aluja et al.(2001) evaluated the effects of different adult nu-trients, including protein and sugar, on male sex-

ual performance in adults of 4 species, (A. ludens(Loew), A. obliqua (Macquart), A. serpentina(Wied.), A. striata Schiner). Overall, protein-fedmales were more sexually successful than pro-tein-deprived, except for A. ludens where no dif-ferences were found. Neither did male diet influ-ence A. ludens female reproductive potential fol-lowing trophalaxis (Mangan 2003). However, in amore recent study protein did improve A. ludenssexual performance (Aluja et al. 2008).

Thus, perhaps not surprisingly, protein-en-hanced diets typically, but not always, enhance

Fig. 2. Mean (±SD) total lipid content (n = 5) of male Caribbean fruit flies at different adult ages among the 6treatments featuring methoprene (M) and protein (P) and their various combinations.

TABLE 2. ANALYSIS OF VARIANCE (ANOVA) FOR MALE CARIBBEAN FRUIT FLY WEIGHT, TOTAL LIPIDS, AND TOTAL PRO-TEINS AMONG TREATMENTS FOR DIFFERENT AGES FEATURING JUVENILE HORMONE (JH) AND PROTEIN (P)AND THEIR VARIOUS COMBINATIONS (* 0.01< P < 0.05; ** 0.001< P < 0.01; *** P < 0.001).

Adult age (days) Male weight Total lipids Total proteins

5 F5,24 = 4.546 ** F5,24 = 26.285 *** F5,24 = 3.5202 *10 F5,24 = 4.566 ** F5,24 = 28.881 *** F5,24 = 26.629 ***15 F5,24 = 5.521 ** F5,24 = 12.548 *** F5,24 = 10.277 ***20 F5,24 = 2.624 * F5,24 = 13.873 *** F5,24 = 8.9948 ***25 F5,24 = 4.370 ** F5,24 = 50.275 *** F5,24 = 4.2223 **30 F5,24 = 4.429 ** F5,24 = 21.339 *** F5,24 = 8.9493 ***35 F5,24 = 5.120 ** F5,24 = 36.197 *** F5,24 = 9.5048 ***


sexual success. However, there are perhaps re-vealing differences in physiology and foragingtactics for food and mates among males of differ-ent species with different diets and activities. Forinstance, unlike A. suspensa, protein-fed C. capi-tata males have lower lipid content than thosethat are protein-deprived (Kaspi et al. 2000). Inaddition, while lekking males are heavier andcontain significantly more protein and sugar thanresting males, they do not contain more lipids(Yuval et al. 1998). Lipids (fatty acids, phospho-lipids, and sterols) have a nutritional role distinctfrom carbohydrates and proteins. Yuval et al.(1994) described lipids metaphorically as an ener-getic trust fund, whereas carbohydrates are com-parable to a readily accessible cash account. Per-haps the difference between A. suspensa and C.capitata in their use of protein for lipogenesis re-flects a difference in energy use patterns, with A.suspensa putting more reserves in “long-term” ac-counts for future use. This in turn might reflectmore predictably encountered food sources for C.capitata or a lower daily chance of mortality for A.suspensa that leads in turn to “planning” for thefuture.

Many kinds of fatty acids and phospholipidsare synthesized by insects, but all insects requiresterols in their diet (Chapman 1998). Reduction

of total lipid content with age in A. suspensa canbe the result of somatic activities, since lipids rep-resent stored energy, even if some restoration oflipid reserves occurs by lipogenesis (Warburg &Yuval 1996). In male A. suspensa, at least in thefirst 10 days of adult life of protein-fed males,there is a slight increase in lipid content. Thesame phenomenon occurs in C. capitata (Warburg& Yuval 1996) and A. serpentina (Jácome et al.1995). Total lipid content declined following maleA. suspensa sexual maturation. Sharp decreasesindicate that males started to utilize their meta-bolic reserves, and this seems likely to correspondto the energetic requirements of any number ofsexual and agonistic behaviors and processes(e.g., Sivinski et al. 2000). One of these potentialexpenditures that can be indirectly examinedwith the present data is pheromone production.

Nestel et al. (1986) suggested that lipid re-serves in male C. capitata may play an importantrole in the regulation and production of sex pher-omone. Nestel et al. (2005) found a decrease inlipid body content after sexual maturation (as thepresent data reveal for A. suspensa), but later onthe content displayed a harmonic pattern wheretotal lipid content increased and decreased at aperiodicity of 10 days. In A. suspensa, male pher-omone production increases when methoprene is

Fig. 3. Mean (±SD) total protein content (n = 5) of male Caribbean fruit flies at different adult ages among the6 treatments featuring methoprene (M) and protein (P) and their various combinations.

Pereira et al.: Male A. suspensa Lipid and Protein Content 143

applied (Teal et al. 2000). However, we found thatapplication of methoprene did not affect lipid con-tent of flies maintained on different diet treat-ments.

The differences in total protein content be-tween protein-fed and protein-deprived malesmay be influenced by ingested protein in the gut.However, the gradual increase in total proteincontent over time in both protein-deprived (after10 d as adult) and protein-fed males is both diffi-cult to explain and inconsistent with artificial-diet protein alone accounting for the difference.Tephritids are known to feed on animal excre-ment (Prokopy et al. 1993; Epsky et al. 1997), andperhaps the consumption of bacteria from theirown feces or bacteria growing on dead flies or onfood sources, inadvertently provided them with aprotein source. Regardless of the origin of this ad-ditional protein, the difference in protein contentson the different diets suggests it was not suffi-cient to completely satisfy nutritional require-ments.

The findings of this study have implications forSIT programs. Among the most important is thatwhile the addition of methoprene has male sexualadvantages it appears to have no immediate nu-tritional detriments. Thus it is a relatively “cost-free” means of improving the performance ofmass-reared and released flies. The incorporationof dietary protein has a positive effect on adultweight and lipid and protein content all of whichare plausibly related to performance as well(Yuval et al. 1998). Due to these effects, the incor-poration of protein in adult diet for SIT programsis also recommended.

ACKNOWLEDGMENTS

We thank David Nestel (IPP-The Volcani Center,Beit-Dagan, Israel), Nikos Papadopoulos (University ofThessaly, Magnisia, Greece), and Steve Ferkovich(CMAVE, USDA-ARS, Gainesville-FL, USA) for criticalreviews of an earlier version of this manuscript. Thisproject was funded in part by the International AtomicEnergy Agency (Research Contract 12863). Financialsupport was provided to RP by the Centro de Ciência eTecnologia da Madeira through the Ph.D. grant BD I/2002-004.

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Lee & Thomas: Taxonomic Status of

Cucujus clavipes

145

CLARIFICATION OF THE TAXONOMIC STATUS OF

CUCUJUS CLAVIPES

WITH DESCRIPTIONS OF THE LARVAE OF

C. C. CLAVIPES

AND

DIVERSITY OF SCOLYTINAE (COLEOPTERA:...

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