(Diptera: Culicidae) Larval Development

ARTICLE

Laboratory and Field Evaluation of Metallic Copper on Aedes albopictus (Diptera: Culicidae) Larval Development ROBERTO ROMI,1 MARCO DI LUCA,1 WALTER RAINERI,2 MARIA PESCE,2 ANTONIO REY,2 SILVANA GIOVANNANGELI,3 FABIO ZANASI,3 AND ANTONINO BELLA4

J. Med. Entomol. 37(2): 281Ð285 (2000)

ABSTRACT Laboratory bioassays and Þeld trials were carried out to study the effect of metallic copper on the development of Aedes albopictus (Skuse). Multiwire electric cable was used as a source of metallic copper. Three different doses were used in laboratory tests (5, 10, and 20 g/liter) and two in Þeld tests (20 and 40 g/liter). In the laboratory, 10 g/liter induced high mortality and a lack of development in Ae. albopictus larvae and doses of 20 g/liter completely inhibited development. Larval mortality was higher in earlier instars than in third through fourth instars and pupae. No effects were reported on egg hatching. Copper ion concentration in water increased up to 574 ppb for 5 g/liter dose, 710 ppb for 10 g/liter dose, and 1,210 ppb for 20 g/liter dose, within week 6. The increasing concentration of copper in water was correlated positively with the decreasing production of adults. Copper ions concentration ⬍500 ppb did not or only slightly affected larval development and mortality of Ae. albopictus in laboratory tests. Copper concentrations between 500 and 1,000 ppb delayed larval development and caused high mortality. Copper concentrations ⬎1,000 ppb inhibited larval development completely killing all the larvae. This last result has been achieved by the use of a 20 g/liter dose of metallic copper in water. Copper also affected adult weight. In Þeld trials, 20 g/liter reduced the number of larvae in treated pots by 90%, and 40 g/liter completely prevented oviposition. Moreover, the persistence of the toxic action of metallic copper in the Þeld lasted for several months. KEY WORDS Aedes albopictus, larval development, mosquito control, metallic copper

IN 1991, THE Þrst clearly established population of Aedes albopictus (Skuse) in Italy was discovered at a tire depository in Padua, Veneto region (Dalla Pozza and Majori 1992). By the end of 1998 the Asian tiger mosquito had been found in 107 municipalities belonging to 22 provinces of the country (Romi et al. 1999). Ae. albopictus found favorable conditions for its development mainly in the urban areas of Northern regions of Italy, where the rapid spread of the species involved a wide variety of artiÞcial water holding containers and the street drains of the rainwater drainage system (Romi 1995). Control activities in Italy consisted mainly in a campaign of source reduction and in antilarval treatments. Focal adulticide treatments were carried out only in cases of heavy infestation in speciÞc areas such as tire depositories and cemeteries (Romi et al. 1999). Control of Ae. albopictus is very difÞcult, particularly in cemeteries, where microhabitats (all types of ßower-holding containers) are readily available. It is almost impossible to reduce the sources of infestation because of religious customs. 1 Laboratorio di Parassitologia, Istituto Superiore di Sanita`, Roma, Italy. 2 Museo Civico di Storia Naturale “Giacomo Doria,” Genova, Italy. 3 Laboratorio di Alimenti, Istituto Superiore di Sanita`, Roma, Italy. 4 Laboratorio di Epidemiologia e Biostatistica, Istituto Superiore di Sanita`, Roma, Italy.

In some areas, such as the city of Genoa, cemeteries became the main infested foci of this species in the urban environment (Raineri et al. 1991, 1993). In the summer of 1996, during a survey carried out at the Staglieno Cemetery in Genoa, Ae. albopictus immature stages were found in ⬎70% of the containers inspected (unpublished data). Although OÕMeara et al. (1992a) reported reduced numbers of larvae in bronze vases in Florida cemeteries, we found high densities of mosquito larvae in all types of ßower-holding containers, including bronze and copper vases (which are commonly coated with an antioxidant paint that avoids the release of copper ions). The toxicity of copper to aquatic organisms has been extensively studied in nature and in laboratory. Some acute and chronic effects on aquatic insects have been pointed out (Clements et al. 1988, Hare 1992). Research carried out in Florida reported the toxic effect of bronze as a limiting factor in the development of Ae. albopictus and Ae. aegypti L. larvae (OÕMeara et al. 1992a, 1992b). More recently, laboratory tests documented the lethal effect of metallic copper on Ae. albopictus and Ae. aegypti larvae (Della Torre et al. 1993, Rayms-Keller et al. 1998, Bellini et al. 1999). From 1997 to 1998 we carried out preliminary laboratory bioassays and Þeld trials to evaluate the effect of metallic copper on Ae. albopictus development. These results are reported in the current article, to

0022-2585/00/0281Ð0285$02.00/0 䉷 2000 Entomological Society of America

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provide more details on the toxicity of metallic copper to mosquito larvae and to deÞne a possible role of this element for the control of Ae. albopictus larvae in small artiÞcial containers. Materials and Methods Laboratory Bioassays. Three different assays have been performed to determine if metallic copper may affect larval development, egg hatching, and oviposition. Tests were carried out from March to November 1997 in a climatic chamber at 27 ⫾ 1⬚C, 70% RH, and a photoperiod of 16:8 (L:D) h. The Ae. albopictus laboratory strain used in these tests was maintained in the insectary of the Istituto Superiore di Sanita` since 1992, originating from the city of Padua. All tests have been replicated two times in different occasions, involving totals of 4,800 larvae, 1,600 adults, and 3,200 eggs. Larval Development Assay. Batches of 100 Þrst instars (8 h old) were placed in plastic trays (30 by 15 by 10 cm) containing 1 liter of dechlorinated tap water and covered with a “tulle” sheet to prevent adult dispersion. Larvae were exposed to three doses of metallic copper (5, 10, and 20 g/liter). Dosages were obtained by placing a weighed piece of multiwire electric cable (ø 6 mm, 110 wires) in trays containing water. Tests were carried out in duplicate with two trays as control. This experience was repeated three times in succession, adding new batches of Þrst instars in the same trays after the complete emergence of the previous group to verify the persistence of the toxic action. Daily, larvae were fed with cat biscuits (Kit & Kat, Dolma S.p.a, Pavia, Italy), evaporated water replaced, larval stages checked, dead larvae removed, and adults were collected by an electric aspirator and weighed. The assay was replicated twice. Every week, 20 ml of water were sampled from each tray for chemical analysis. Ionic copper concentration in the weekly water samples was measured by atomic absorption spectrometry, using a JY 38S ICP sequential spectrometer (Jobin Yvon, Division of Instruments S.A., Lonjumeau, France) at a wavelength of 324.75 nm, following the technique described by Cameron (1970). Egg Hatching Assay. Batches of 200 eggs (5 d old) were placed in trays containing 1 liter of tap water and exposed to the three doses of metallic copper as described above. After 48 h the number of hatched larvae was counted. Tests were carried out in duplicate with two trays as control. The whole assay was replicated twice. Oviposition Assay. Small glass containers (ø 5 cm) Þlled with 20 ml of dechlorinated tap water and treated with the three different doses of metallic copper were put in cages (25 by 25 by 35 cm) with 100 gravid females. Filter paper strips were placed on the inner surface of the containers to ncourage ovoposition. After 48 h, Þlter paper strips were collected and eggs were counted under a dissecting microscope. Tests were carried out in duplicate with two cages as control. The whole assay was replicated twice. Field Trials. Field trials were carried out in the Staglieno Cemetery in Genoa during the summer 1997

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and 1998. A neglected area was selected, apart from the main body of the cemetery, that was not involved in control operations. Black plastic ßower pots (500 ml capacity) were used as ovitraps. A strip of masonite (2 by 12 cm) was suspended vertically in the middle of the pots to provide a suitable surface for oviposition. The pots were distributed in pairs (treated and not treated) at random in shaded sites of the selected area. Pots were Þlled with 300 ml of dechlorinated tap water. Every week the strip was changed, pots were reÞlled, and checked for eggs and preimaginal stages. The number of eggs was counted by observing the strips under a dissecting microscope. Batches of pots were treated with two doses of multi-wire electric cable, 20 and 40 g/liter, while other batches were used as control. In 1997, from mid-July to mid-October, 30 pairs of ßower pots (treated with 20 g/liter) were placed and followed for 12 wk. At the end of the trial the pots were left in place until the next season. In 1998, from June to the Þrst week of November, 50 more pairs of pots (25 with 20 g/liter ⫹ 25 with 40 g/liter) were placed in the same area and observed for 16 wk. In addition, the 30 old pairs were reused, after cleaning, to verify the persistence of the action after 1 yr. Statistical Analysis. The odds ratio and the related conÞdence interval were calculated to evaluate the inßuence of the different metallic copper concentrations on larval survival. Linear regression was used to evaluate relationships existing between copper concentration and adult emergence rate. The analysis of variance (ANOVA) was used to evaluate the difference in weight of Ae. albopictus adults treated with different copper concentrations. Multiple comparisons were carried out between each copper concentration and control by Student test for unpaired samples. All statistical tests were considered signiÞcant at the ␣ ⫽ 0.05 probability level, and multiple comparisons were corrected by BonferroniÕs formula (Edwards 1985). Statistical analysis were processed by BMDP (1988) and Epi-Info (1995) software. Results Laboratory Assays. The results of the larval development assay are reported in Fig. 1. The development of the Þrst batch of larvae from Þrst instar to adult emergence was delayed by metallic copper at 10 and 20 g/liter. Compared with an average of 11.5 ⫾ 0.5 d in the control and 12 ⫾ 0.7 d at 5 g/liter, larvae at 10 and 20 g/liter required 26 ⫾ 0.7 and 28 ⫾ 1.4 d, respectively, to complete the cycle. The mean adult emergence rate was 96.2% in the control, 94% at 5 g/liter, 88% at 10 g/liter, and 58% at 20 g/liter. Larval mortality occurred mainly at Þrst and second instar. The development of the second batch was slightly delayed at 5 g/liter, an average of 17 ⫾ 1.6 d versus 12 ⫾ 0.0 in the control, and strongly delayed at 10 g/liter (34.5 ⫾ 2.2 d) and 20 g/liter (41 ⫾ 2.2 d). The mean adult emergence rate was 94.4% in the control, 91% at 5 g/liter, 37% at 10 g/liter, and 2.8% at 20 g/liter. At this last dose, adults died soon after emergence.

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Fig. 1. Mean number of days occurred for the development of three batches of Þrst-instar Ae. albopictus to adults in presence of three different doses of metallic copper. Second and third batches of Þrst-instar were added in the test trays after the complete emergence of the previous batches. Values above the columns are the mean rate of adult emergence (%). In brackets ⫾SD.

Larval mortality occurred mainly at earlier instars and within the Þrst 3 d. The development of the third batch was signiÞcantly delayed at 5 g/liter, an average of 26 ⫾ 1.6 d versus 11.5 ⫾ 0.5 of the control, strongly delayed at 10 g/liter (39 ⫾ 1.9 d), whereas all larvae died at 20 g/liter. At 10 g/liter, larval mortality occurred mainly during the Þrst 3 d (⬇80%). At 20 g/liter, all the larvae died in 24 h. The mean adult emergence rate was 95% in the control, 68% at 5 g/liter, and 3.2% at 10 g/liter. At this last dose, adults died soon after emergence. The concentration of copper ions in the test trays increased progressively during the Þrst 6 wk, then it slightly ßuctuated and remained quite stable up to the 16th wk when tests were concluded. A mean copper ion concentration in water, during the Þrst 6 wk, is reported in Table 1. The increase was up to 574 ppb for 5 g/liter dose, 710 ppb for 10 g/liter dose, and 1,210 ppb for 20 g/liter dose. Copper concentration in the controls was stable, with a mean value of 7.5 ⫾ 0.3 ppb. Table 1. Concentration (mean ⴞ SD) of copper ion in water after exposition to 3 doses of metallic copper within a period of 6 weeks Time, wk 1st 2nd 3rd 4th 5th 6th

Copper concn. at 5 g/liter



147 ⫾ 32.9 344 ⫾ 25.6 410 ⫾ 37.1 487 ⫾ 45 522 ⫾ 38.9 574 ⫾ 26.6

214 ⫾ 11.8 431 ⫾ 28.6 520 ⫾ 14.7 580 ⫾ 26.8 685 ⫾ 8.5 710 ⫾ 18.7

323 ⫾ 33.5 645 ⫾ 12.2 891 ⫾ 21.6 1,014 ⫾ 32.2 1,182 ⫾ 23.7 1,210 ⫾ 38.6

Values are expressed in ppb.

At the dose of 5 g/liter, duration of the larval development and mortality were quite similar to those of the control for the Þrst batch of larvae. The second batch of larvae was added during the second week of exposition (mean copper concentration ⬍500 ppb) and adult emergence was achieved at fourth week, with slight effects on the duration of the cycle and mortality. The third batch of larvae was added at the Þfth week (mean copper concentration of ⬎500 ppb) and it was affected both in the duration of the larval development and mortality (32%). At the dose of 10 g/liter, larval development and mortality of the Þrst batch of larvae were both affected by copper ion concentration that reached an average of ⬎500 ppb within the third week. The second batch of larvae was added at the fourth week (mean copper concentration of ⬎500 ppb) with heavy effects on both development and mortality (63%). The third batch of larvae was added over the sixth week (mean copper concentraton of ⬎700) with very strong effects on both larval development and mortality (96.8%). At the dose of 20 g/liter, mean copper ion concentration reached 500 ppb within the second week, causing ⬎40% of mortality in the Þrst batch of larvae. The second batch of larvae was added at the fourth week (mean copper concentration ⬎1,000 ppb) with very strong effects on both larval development and mortality (97.2%). The third batch of larvae, added over the sixth week (a stable mean copper concentration ⬎1,200 ppb), did not survive the Þrst day. The odds ratio calculated between treated and not treated batches of larvae showed that exposure to metallic copper reduced the Ae. albopictus larval abil-

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Table 3. Mean ⴞ SD number of Ae. albopictus eggs hatched in water samples containing 3 doses of metallic copper and mean number of eggs laid by Ae. albopictus females on containers treated with the same doses

Fig. 2. Linear relation between mean copper concentration in the test water and adult Ae. albopictus emergence rate (R2 ⫽ 0.82, P ⬍ 0.001).

ity to survive the different concentrations. The odds ratio calculated at 5 g/liter was not signiÞcant (odds ratio ⫽ 0.62, conÞdence interval ⫽ 0.34 Ð1.10) when compared with control, whereas it was highly significant at 10 g/liter (odds ratio ⫽ 0.02, conÞdence interval ⫽ 0.0 Ð 0.15) and 20 g/liter (odds ratio ⫽ 0.0, conÞdence interval ⫽ 0.0 Ð 0.06). The effect of increasing copper concentrations and production of Ae. albopictus adults is shown in Fig. 2. The linear relation between mean copper concentration in the water and survival was very signiÞcant (R2 ⫽ 0.82, P ⬍ 0.001). All the Ae. albopictus adults that survived to emergence where weighed and a mean weight per batch was calculated. Results are shown in Table 2. The ANOVA showed that weights of adult Ae. albopictus that survived at different metallic copper concentrations were signiÞcantly different (F ⫽ 206.2; df ⫽ 3, 76; P ⬍ 0.001). Moreover, a signiÞcant difference between each concentration and the control resulted from multiple comparisons (P ⬍ 0.001). The results of the egg hatch and oviposition assays are shown in Table 3. The presence of metallic copper in the water at different doses did not affect eggs hatching and oviposition. No signiÞcant difference was detected among groups if compared with control in both assays. Field Trials. The results of Þeld trials are reported in Table 4. In 1997, out of 30 pots treated with 20 g/liter, 10 (33%) were found positive for eggs at the second or third week of exposure. After that, they Table 2. Mean ⴞ SD weight (in mg) of adults Ae. albopictus emerged after reared in trays containing 3 different doses of metallic copper Dose of copper

Mean adult wt 1⬚ batch



5 g/liter 10 g/liter 20 g/liter Control

1.87 ⫾ 0.08 1.52 ⫾ 0.10* 1.14 ⫾ 0.13* 2.01 ⫾ 0.15

1.65 ⫾ 0.08 1.13 ⫾ 0.13* No adult 1.89 ⫾ 0.12

1.08 ⫾ 0.18* No adult No adult 1.98 ⫾ 0.08

The second and third batches of Þrst instar were put in the trays after the complete emergence of the previous batch, and copper concentration in the water was increasing. *, Within the columns, the values followed by an asterisk are signiÞcantly different with control when StudentÕs t-test is used.

Dose

Mean no. of eggs hatched

Mean no. of eggs laid

Control 5 g/liter 10 g/liter 20 g/liter

76.3 ⫾ 4.1 75.0 ⫾ 4.2 70.3 ⫾ 6.8 73.0 ⫾ 0.8

615.3 ⫾ 162.6 568.3 ⫾ 108.9 607.7 ⫾ 167.3 634.3 ⫾ 221.0

were found negative at the following checks: 90% (27/30) of the control pots became positive for eggs or larvae between the second and the sixth week of exposure and all of them remained positive for the duration of the trial. In 1998, none of the 25 ßowerpots treated with 40 g/liter was found positive for the duration of the trial. Out of 25 pots treated with 20 g/liter, two were found positive for eggs at the ninth week of exposure and remained positive for eggs for the following 7 wk. Eightty two percent (41/50) of the control pots became positive for eggs or larvae between the second and the seventh week of exposure and all they remained positive for the duration of the trial. In 1998, the 30 pairs of ßowerpots previously used in 1997 were reused for a further test. Out of the 30 treated, 28 (94.3%) became positive between the 9th and 14th weeks and remained positive until the trial was over. Among controls, 100% of the pots became positive between second and ninth week. The difference between treated and untreated pots was very signiÞcant both in 1997 and 1998. The analysis of the odds ratio showed that the presence of 20 g/liter of metallic copper reduced the presence of eggs in treated pots versus control pots by ⬇94% in 1997 (odds ratio ⫽ 0.06, conÞdence interval ⫽ 0.01Ð 0.26) and 98% in 1998 (odds ratio ⫽ 0.02, conÞdence interval ⫽ 0.0 Ð 0.15). In addition, it should be stressed that in positive treated pots only eggs were found both in 1997 and 1998. In control pots, Ae. albopictus larvae at various stages have been found associated with eggs in ⬎70% of the cases (unpublished data). After a year of use, metallic copper ceased to release ions into the water. There was no signiÞcant difference between treated and untreated 1997 pots when reused in 1998. Table 4. Field trials with plastic ovitraps treated with 2 doses of metallic copper

Year 1997 1998 Treated the previous year

Dose of metallic copper, g/liter

No. of ovitraps treated/ control

Treated

Control

20 20 40 20 (1997a)

30/30 25/25 25/25 30/30

10/30 2/25 0/25 28/30

27/30 20/25 21/25 30/30

No. positive/total

Trials were carried out in 1997 for 12 wk (JulyÐOctober) and in 1998 for 16 wk (JuneÐNovember). a The 30 ovitraps used in 1997 were re-used in 1998.

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ROMI ET AL.: EFFECT OF METALLIC COPPER ON Ae. albopictus Discussion

The results of our laboratory assays showed that metallic copper may induce high mortality and a lack of development in Ae. albopictus larvae as previously observed by Della Torre et al. (1993) and Bellini et al. (1999). At 10 and 20 g/liter, larval development is strongly delayed and high larval mortality is induced, mainly at the Þrst and second instars. Our assays showed that copper ion concentrations ⬍500 ppb did not (or only slightly) affect larval development and mortality of Ae. albopictus in laboratory tests. Copper concentrations between 500 and 1,000 ppb delayed larval development and caused high mortality. Copper concentrations ⬎1,000 ppb completely inhibited larval development killing all the larvae. This last result has been achieved by the use of a 20 g/liter dose of metallic copper in water. According to our statistical analysis, concentrations of 10 and 20 g/liter of metallic copper reduce the ability of Ae. albopictus larvae to survive by 98 and 100%, respectively. Our results do not agree with those obtained in laboratory tests by Della Torre et al. (1993) in which doses of metallic copper of 2 g/liter completely inhibited Ae. Albopictus larval development and killed all the larvae in the Þrst instar. Using a 10 g/liter dose of metallic copper, our experimental model gave similar results to those obtained by Bellini et al. (1999) with a dose of 8 g/liter. However, we reached a total inhibition of larval development with the use of a 20 g/liter dose. At the doses we experienced, copper ion concentration seemed to not affect Ae. albopictus eggs hatch. First and second instar were the most sensitive to the toxic action of copper, and the larvae that survived up to the third or fourth instar developed into adults. These results agree with those obtained by Rayms-Keller et al. (1998) on Aedes aegypti (L.) larvae in which egg hatch rate was signiÞcantly affected by copper over 3,200 ppb and the concentration resulting in 50% third-instar mortality (LC50) was estimated in 33,000 ppb. Finally, adult Ae. albopictus weight was affected by copper concentration, as previously reported by Bellini et al. (1999). This fact probably inßuences their ability to survive. In the Þeld, both ovitraps treated with 20 and 40 g/liter of metallic copper worked well. The dose of 40 g/liter seemed to work better, with no egg positivity in the 1998 trial versus 8% in the 20 g/liter pots of the same year and 33.3% in those of 1997. In the Þeld, metallic copper seemed to also inßuence Ae. albopictus females on their choice of the oviposition site, contrary to what happened in laboratory tests. Probably the high concentration of copper ions in the pots treated with 40 g/liter may induce Ae. albopictus to avoid these habitats. Further trials should be carried out to assess the minimum copper ion concentration required for killing larvae without avoid oviposition. In conclusion, our experience conÞrmed the toxic activity of metallic copper on Ae. albopictus. Pieces of multiwire electric cables introduced into small containers of water, at least 20 g/liter, may prevent the development of Ae. albopictus larvae into adults. Our

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Þeld trials showed that this is an effective control method at 40 g/liter in a natural environment such as a cemetery. Moreover, the persistence of the toxic action lasts several months. We think that the use of this alternative, economic, and safe method may be effective for the control of Ae. albopictus larvae in small afÞxed containers. As a part of a more complex control program, it could help to reduce the density of the species in selected environments. References Cited Bellini, R., M. Carrieri, M. Bacchi, P. Fonti, and G. Celli. 1998. Possible utilization of metallic copper to inhibit Aedes albopictus (Skuse) larval development. JAMCA 14: 451Ð 456. Cameron, A. G. 1970. Determination of copper in foods by atomic absorption spectrophotometry. J. Sci. Food Agric. 21: 535Ð536. Clements, W. H., D. S. Cherry, and J. Cairns Jr. 1988. Structural alterations in aquatic insects communities exposed to copper in laboratory streams. Environ. Toxicol. Chem. 7: 715Ð722. Dalla Pozza, G., and G. Majori. 1992. First record of Aedes albopictus establishment in Italy. JAMCA 8: 318 Ð320. Dean, A. G., J. A. Dean, D. Coulombier, K. A. Brendel, D. C. Smith, A. H. Burton, R. C. Dicker, K. Sullivan, R. F. Fagan, and T. G. Arnier. 1995. EPI INFO, version 6: a word processing, database and statistics program for epidemiology on microcomputers. Centers for Diseases Control and Prevention, Atlanta, GA. Della Torre, A., V. Raineri, and G. Cancrini. 1993. Effetto del rame metallico sullo sviluppo larvale di Aedes albopictus: primi dati di laboratorio. Parassitologia 35: 51Ð53. Dixon, W. J. 1988. BMDP, Biomedical Computer Programs, University of California Press, Los Angeles. Edwards, A. L. 1985. Experimental design in psychological research. Harper & Row, New York. Hare, L. 1992. Aquatic insect and trace metals: Bioavailability, bioaccumulation, and toxicity. Crit. Rev. Toxicol. 22: 327Ð369. O’Meara, G. F., L. F. Evans, Jr., and A. D. Gettman. 1992a. Reduced mosquito production in cemetery vases with copper lines. JAMCA 8: 419 Ð 420. O’Meara, G. F., A. D. Gettman, L. F. Evans. Jr., and F. D. Scheel. 1992b. Invasion of cemeteries in Florida by Aedes albopictus. JAMCA 8: 1Ð10. Raineri, V., F. Rongioletti, and A. Rebora. 1993. Osservazioni sulla presenza di Aedes albopictus in Liguria. Parassitologia 35: 31Ð32. Raineri, V., G. Trovato, A. Sabatini, and M. Coluzzi. 1991. Ulteriori dati sulla diffusione a Genova di Aedes albopictus. Parassitologia 33: 183Ð185. Rayms-Keller, A., K. E. Olson, M. McGraw, C. Oray, J. O Carlson, and B. J. Beaty. 1998. Effect of heavy metals on Aedes aegypti (Diptera:Culicidae) larvae. Ecotoxicol. Environ. Saf. 39: 41Ð 47. Romi, R. 1995. History and updating of the spread of Aedes albopictus in Italy. Parassitologia 37: 99 Ð103. Romi, R., M. Di Luca, and G. Majori. 1999. Current status of Aedes albopictus and Ae. atropalpus in Italy. JAMCA 15: 425Ð 427. Received for publication 26 July 1999; accepted 2 December 1999.