Journal of the American Mosquito Control Association, 15(4):493-499, 1999

Copyright @ 1999 by the American Mosquito Control Association, Inc.

ERYNGIUM SPP (UMBELLIFERAE) AS PHYTOTELMATA AND THEIRCULEX rcULEN INHABITANTS IN TEMPERATE ARGENTINA

R. E. CAMPOST nNr L. P LOUNIBOS'�

ABSTRACT The host-plant fidelity, seasonality, and population dynamics of 3 species of Culex (Culex) were

examined from monthly samples of mosquito immatures extracted from the leaf axils of Eryngium pandanifolium

and Eryngium cabrerae growing sympatrically in the Punta Lara region of Buenos Aires Province, Argentina.

Culex hepperi was collected only from E. pandanifulium, and abundance peaks of its larvae and pupae were

asynchronous in forest and field habitats. However, the proportion of plants positive for Cx. hepperl, as well as

larval densities and developmental success, did not differ between forest and field. Culex renatoi was collected

only from E. cabrerae in the field habitat. The monthly numbers of Cx. hepperi and Cx. renatoi were not

significantly correlated with the proportion of their host plants that held water. The stage-specific distributionsof Cx. hepperi and Cx. renatoi numbers were highly skewed towards lst instars and did not differ betweenspecies or, for Cx. hepperi, between forest and field habitats. Culex castroi was collected regularly from theaxils of both species of Eryngium, but usually in lesser numbers than either Cx. hepperi or Cx. renatoi. Asso-ciation analyses indicated significant positive associations between Cx. castroi and the other 2 culicine speciesin host plants. Culex renatoi occurred preferentially in small E. cabrerae, but Cx. castroi did not discriminateplants based on their size. These 3 species of Culex are known only from Eryngium spp. phytotelmata. Wesuggest that their host-plant specificity is maintained by oviposition preferences, which are more highly selectivefor Cx. hepperi or Cx. renatoi than for Cx. castroi.

KEY WORDS Phytotelmata, Eryngium spp., Culex (Culex) spp., seasonality, specificity

INTRODUCTION

Phytotelmata, the water-holding catchments ofterrestrial plants, are important habitats for somegroups of aquatic insects and, in particular, for mos-quitoes (Frank and Lounibos 1983). Four hundredspecies of Culicidae have been estimated as inhab-iting phytotelmata (Fish 1983), with many endemicor highly specialized species probably awaiting dis-covery. [n a survey of mosquitoes inhabiting phy-totelmata in a Venezuelan rain forest, Machado-Al-lison et al. (1985) recognized 4 of 2l species asrestricted to 1 species of plant and 5 other culicidspecies as occurring in plants belonging to only 1family, such as the Bromeliaceae or Araceae.

Eryngium (Umbelliferae) is a cosmopolitan ge-nus of perennial, rhizomatous herbs that includesmore than 220 species in temperate and tropicalregions (Cabrera 1965). TWenty-flve species of thegenus ,re known from Argentina, 17 of which oc-cur in Buenos Aires Province (Cabrera 1965). Inseveral Eryngium spp. known from Brazil and Ar-gentina, overlapping leaves arranged in whorls cap-ture and retain water in their axils (Fig. 1). Theaquatic fauna occurring in axils of Eryngium pan-danifolium Cham. and Schlecht in temperate Ar-gentina (Vucetich and Rossi 1980) and of Eryngiumfloribundum Cham. in tropical Brazil (Machado1976) have been inventoried. Mosquitoes were rec-ognized in these previous studies, but not identifiedbeyond family level.

rCentro de Estudios Parasitol6gicos y de Vectores,CONICET-Universidad Nacional de La Plata, Calle 2no. 584 (1900) La Plata, Argentina.

2 To whom correspondence should be addressed. Uni-versity of Florida, Florida Medical Entomology Labora-tory,2N 9th Street SE, Vero Beach,FL32962.

Four species of Culex (Culex) have been de-scribed from specimens collected as immaturesfrom the axils of unidentified species of Eryngiumin Brazil and Argentina (Lane and Ramalho 1960;Casal and Garcia 1967a, 1967b; Da Silva Mattos etal. 1978). To date, the immatures of these speciesof Culex are not known from other habitats. Hereinwe report on the bionomics of 3 of these species,Culex heppen Casal and Garcia, Culex renatoiLane and Ramalho, and Culex castroi Casal andGarcia, occurring in 2 species of Eryngium thatgrow sympatrically along the Rio de La Plata, Ar-gentina.

MATERIALS AND METHODS

Field studies were conducted from December1994 through March L997 in the Punta Lara area(34'51'33'5, 57"52'23'N) of Rio de La Plata inBuenos Aires Province. Annual rainfall in this re-gion was 828.6 mm in 1995 and 857.3 mm in 1996.Mean daily temperatures range from 9'C in July to28'C in January. A gallery forest, composed of na-tive trees and the introduced Ligustrum lucidumAit., borders the river in this area and, under naturalcircumstances, is flooded daily at high tide. How-ever, the artificial containment of tides by man-made levees has curtailed daily flooding and al-lowed for the invasion of exotic plant species inthis forest. On the other side of the gallery forestwas an open field.

Eryngium cabrerae Pontiroli (Fig. 1) occurredonly in the field blut E. pandanifulium was commonin both the field and gallery forest. Both specieshave a similar growth form, but the maximumheight of E. pandanifolium (2.5 m) is much greaterthan that of E. cabrerae (0.6 m) (Cabrera 1965).

493

DECEMBER 1999 ERvNcruM spp. AND Currx INeumNns w AnceNrINl 495

Eryngium p/4dtnlfq/tum: fi'�ld

+ Cdex ca&oi-.o . C,/axlbppE�ri

D J F M A M J J A S O N D J F M A M J J A S O N O J F M

o

D J F M A M J J A S O N D J F M A M J J A S O N D J F M

1995 Months 1996 1997

Fig. 3. The total numbers of individuals of 2 Culexspecies identified from monthly samples of 3O Eryngiumpandanifulium in the forest and field sites.

dt : 27), indicating that habitat availability per seis not the major determinant of Culex abrrrdance inthese phytotelmata. For the largest observed abun-dance peak of Cx. hepperi, 55.7Vo of immatures inDecember 1996 and 74Vo in January 1997 were 1stinstars.

We compared the proportionof E. pandanifuliumcontaining immatures of Cx. heppen in forest andfield habitats. The proportion of forest plants posi-tive (Fig. 4A') for Cx. hepperi (2O.2Vo) was not sig-nificantly different from the proportion of fieldplants positive (l5.6Vo) (Gri : 0.16, P > 0.50).Further, the mean density of Cx. hepperi in mos-quito-positive plants in the forest (0.86 + O.O3 SE)did not differ significantly from that of the fieldhabitat (0.8S * 0.04 SE) (t": -O.21, P : 0.84, df-- 299).

To determine whether developmental success ofCx. hepperi was greater in the forest or field, wecompared instar distributions of the immature stag-es collected from these 2 habitats (Fig. 4A). Firstinstars predominated in both sets of samples, andthe stage-specific distributions of Cx, hepperi did,not differ significantly when tested by a Kolmoro-gov-Smirnov 2-sample test (D.* : 0.2O, P > 0.1,n = 5 ) .

To evaluate whether Cx. hepperi and Cr. castroi

o

5 ' *oo 0

aj zsoo

2 n

D J F M A M J J A S O N D J F M A M J J A S O N D J F M

1995 Months

1996 1997

Fig.2. The monthly percentages of 3O Eryngium pan-

dantfolium that contained measureable water (>l ml) in28 months of sampling field and forest sites.

0.05, n = 20). Based on these width measurements,plants were categorized as either small (0.8-2.3cm), medium (2.4-3.7 cm), or large (3.8-5.2 cm).

RESULTS

Eryngium pandanifuliun and its culicidinhabitants

The proportion of plants with standing waterfluctuated seasonally (Fig. 2). Plants were mostlikely to be dry between January and March when,on 2 occasions, no E pandanifoliulrz in the fieldheld measurable water. The average monthly pro-portion of samples with water in the forest (64.5 *6.0 SE) was significantly greater than that for Epandanifulium in the field (48.6 + 5.5 SE) (Wil-coxon signed ranks 7. : 62.5, P : 0.002).

Both Cr. hepperi and Cx. castroi were identifiedfrom E pandanifuliun phytotelmata, with Cx. hep-peri more abundant in both field and forest collec-tions (Fig. 3). Abundance peaks of both species oc-cured in either the May-June or December-January periods, but did not coincide in the fieldand forest. Moreover, the number of larvae collect-ed and the proportion of plants with water were notsignificantly conelated in either the field (r : O.21,P : O.27, dt : 27) or forest (r: O.33, P : 0.08,

496 JoURNAL oF Tr# AMERTCIN Mosquno CoNrnol AssocnrroN Vol . 15 , No.4

Cubx tBp'Ert

@ Forest;n=170/840I Fi€ld; n=13' l /840

Stages

Fig. 4. (A) Numbers of Culex hepperi in each im-mature stage from all collections of Eryngium pandani-

folium. lnset shows proportions of samples positive forCx. hepperi in forest and field. (B) Numbers of individualsin each immature stage of Culex renatoi collected fromEryngium cabrerae.

co-occurred in the same E. pandanifulium plantsmore often than chance would predict, we per-formed a2 x 2 association analysis and calculatedthe C, coefficient of interspecific association, whichvaries from *1 to -1 (Hurlbert 1969). The C* of

0.5 and a highly significant chi-square value (cor-rected for continuity) of 52.5 (P < 0.001) for theassociation analysis indicated that these 2 specieswere highly likely to co-occur in the same plantsamples.

Eryngium cobrerae and its culicid inhabitants

More than 507o of plants of this species held wa-ter on every sampling date except March 1997 (Fig.5). On 5 of 14 occasions, IOOVo of the sampledplants contained measurable water.

The mosquitoes Cx. castroi and Cx. renatoi werecollected on all 14 sampling dates (Fig. 5). On 2occasions after the field had flooded from excessiverainfall, larvae and pupae of the ground-water spe-cies Aedes albifasciatus Macquart were recoveredfrom many E. cabrerae axils. Because the presenceof Ae. albifasciatus in this phytotelm was regardedas accidental, this species was not considered fur-ther in the present study.

On 11 of 14 occasions Cr. renatoi was moreabundant than Cx. castroi in monthly collections(Fig. 5). The monthly numbers of mosquito im-matures were not significantly correlated with theproportion of E. cabrerae with water for either Cr.renatoi (r: 0.16, P : 0.59, df: 13) or Cx. castroi(r : -0.15, P : 0.60, df : 13).

The stage-specific distribution of Cx. renatoi in-dicated a prevalence of lst instars (Fig. 4B). Todetermine whether the distribution of Cx. renatoiimmatures by stage differed from that of field-col-lected Cx. hepperi it E. pandanifolium (Fig. aA),the 2 were compared with a Kolmorogov-Smirnov2-sample test, which indicated no significant dif-ference between the distributions of the 2 speciesin the 2 plants (D.^ : O.2O,P > 0.1, n : 5\.

The co-occurrence of Cx. castroi and Cx. renatoiin the same E. cabrerae plants was positive in the2 X 2 association analysis, which yielded a signif-

Io

n n ]

]oa

6 0 Q

o

4 0 t

uE

m s

r $ oI3. soo

3 250oz_a 200E

E 150E2 r o o

A

1996

A S

Months

F

1997

Fig. 5. The monthly percentages of 30 Eryngium cabrerae that contained measureable water (>1 ml) and the totalnumbers of 2 Culex species in these samples for 14 consecutive months.

Dpcprr4sen 1999 Enyxctuu spp. AND Currx hteunNrs rN AncBNrrnh 497

Erynglum cabrera,e

(n=71311 - clilexca,*o'

( n = 8 / 8 9 ) = Culexrcnatoi

( n = 1 / 5 )

(n = 30/89)

(n = 4/31)

(n = 0/5)

Small

( 0 . 8 - 2 . 3 )

Large

( 2 . 4 - 3 . 7 ) ( 3 . 8 - 5 . 2 )

Size

Fig. 6. The mean numbers of Culex castoi and Culex renatoi immatures in Eryngium cabrerae plants positive forthese mosquito species, segregated into size categories by width measurements of basal leaves.

ci. l E

(D '"

ooo:+ 1 0ooa-goz

o=

icantly positive chi-square value (corrected for con-rinuity) of 3.86 (P < 0.05). However, the C, valuefor interspecific association between these 2 specieswas <O.Ol, indicating a relatively weak association.

The sampling of all E cabrerae plants in the 2X 2-m plots yielded 5 large, 3l medium, and 89small plants with measureable water. The averagedensity of Cx. renatoi was highest in small plants,and no ifimatures were recovered in the largeplants (Fig. 6). Significantly more small plants wereoccupied by Cx. renatoi than were medium-sizedE. cabrerae (G"oi : 4.19, P < 0.05).

Comparative success of Cx. castroi

Because Cx. castroi occurred in both E. pandan-ifulium and E. cabrerae, it was of interest to com-

pare the occupancy of the 2 phytotelmata by thisspecies. The proportion of E cabrerae positive(Fig.7) for Cx. castroi (0.203) was significantlyhigher than the proportion of E pandanifulium thatwere positive for this species (0.073) (G., : 39.8,P < 0.001). Distributions of instars (Fig. 7) did notdiffer between plant species (Kolmorogov-Smirnov2-sample test, D^^: 0.60, P > 0.1, n : 5).

In the 2 X 2-m plot of E. cabrerae, Cx. castroioccurred in plants of all 3 sizes (Fig. 6). A G-testfor independence indicated no significant differenc-es in the proportion of plants of the 3 size classesoccupied by Cx. castroi (G^dnzr: 3.72, P > 0.1).

DISCUSSION

Thirty-seven species of Culicidae are knownfrom the Punta Lara area (Ronderos et al. 1992,Campos 1994), but only 7 species from 5 genera(Culex; Haemagogus, Wyeomyia, Runchomyia,Toxorlrynchites) have been recovered from phyto-telmata. Larvae of the 3 Culex species of the pres-ent study are known only from Eryngium spp. axils.Interestingly, Culex dolosus Lynch Arirbalzaga,which was recovered from Eryngium axlls in MinasGerais State, Brazil (Da Silva Mattos et ^1. 1978),was not observed in Argentinian Eryngium, despitethe abundance of Cx. dolosus in temporary and per-manent ponds along the Rio de la Plata (Maci6 etal. 1996). Culex spinosus Lutz has been reportedfrom the axils of an unidentified, Eryngium in Pa-ran6 State, Brazil (Lozovei and Luz 1976), but thisculicid is not known from phytotelmata in otherparts of its range, which includes Argentina (Darsieand Mitchell 1985).

Thus, 3 levels of host-plant specificity may occuramong the 6 species of Culex known from Eryn-

€ zoo

::s tso

= 100z

Culex 6s'.ol

ffiM E. cab6ne; n = 7AU1- E. pandanifolium: n = 71872

ill

Stages

Fig. 7. The numbers of individual Culex castroi indifferent immature stages collected from the 2 species ofEryngium. Inset indicates proportions of samples of the 2plants that were positive for Cx. castroi.

498 JounNlr- oF rnE AwenrceN Moseurro CoNrnol AssocrerroN VoL . 15 , No .4

gium axils. Culex hepperi arrd Cx. renatoi are themost host-speciflc, occurring only in L species ofEryngium. Culex machadoi, described from an un-identified species of Eryngium from Minas GeraisState, Brazil (Da Silva Mattos et al. 1978), may fallin this category or the next. Culex castroi repre-sents a 2nd level of specificity, insofar as it occursin more than 1 species of Eryngium but is notknown from any other larval habitat. Finally, Cx.dolosus and Cr. spinosus, both of widespread dis-tribution in South America, represent a class ofhabitat generalists that occupy phytotelmata, in-cluding Eryngium, only occasionally.

Habitat specificity of phytotelm-inhabiting mos-quitoes is frequently mediated by plant-producedchemical cues for oviposition (Fish 1983). For ex-ample, fluid from the bracts of Heliconia caribaeastimulates oviposition by the specialist Wyeomyiaulocoma (Theobald) (Lounibos and Machado-Alli-son 1993), and a water-soluble extract from Sar-racenia purpurea induces oviposition by its host-specific inquiline, Wyeomltia smithii Coq. (Istock etal. 1983). The selectivity of gravid Cx. hepperi andCx. renatoi for E. pandanifulium and E. cabrerae,respectively, suggests the use of host-specificchemical cues by these mosquito species to locatetheir host plants. Culex castroi, on the other hand,may use less specific chemical cues, if any.

The occupancy patterns of immatures in plantsof 3 size classes is consistent with the expectationfrom chemical stimulation of oviposition by themost specialized mosquito species. The youngestphytotelm habitats, such as freshly opened, Sarra-cenia leaves or Heliconia bracts, are the most at-tractive to specialist mosquitoes, such as Wy. smi-thii or Wy. ulocoma, lured by host-plant chemicals(Fish and Hall 1978, Machado-Allison et al. 1983).Thus, it is not surprising that Cx. renatoi shouldsignificantly favor oviposition in the smallest.E cd-brerae, which distinguishes it in host choice fromthe less-specific Cx. castrot (Fig. 6).

The distribution of immature stages of all 3 spe-cies of Culex was highly skewed towards younger,especially lst, instars (Figs. 4 and 7), suggestinghigh mortality early in larval life. Causes of thismortality remain to be resolved. Unlike E. floribun-dum in Minas Gerais, Brazll, where inquiline odo-nate larvae prey on mosquitoes (Machado 1976,Corbet 1983), the axils of E. pandanifulium and E.cabrerae did not harbor predators of mosquito lar-vae (Vucetich and Rossi 1980; Campos, personalobservations). On the other hand, a fungus (Lop€zLastra 1997) and a microsporidan (Micieli 1996),both isolated from Cx. renatoi, are capable of con-tributing to mortality of larval mosquitoes.

The absence of a correlation between larvalabundance and habitat availability, as observed forCx. hepperi and Cr. castroi, is common to otheraxil-inhabiting mosquitoes. Heavy rainfall is knownto wash mosquito eggs from axils (e.g., Pajot 1976,Frank and Curtis 1977), which together with the

occasional floods that washed Ae. albifasciatrrr intoaxils may make the rainiest periods less propitiousfor the Calex colonists.

ACKNOWLEDGMENTS

We thank J. E Day and J. H. Frank for criticalreviews of this work. Research was supported byUniversidad Nacional de La Plata, Argentina. Thispaper is Florida Agricultural Experiment StationJournal Series R-06720.

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