Sub-lethal effects of a copper sulfate fungicide on development and reproduction in three coccinellid species.

Copper-based fungicides reliably control various foliar diseases in citrus production, although they are suspected to exacerbate mite problems through various mechanisms. Studies have shown negative effects of various copper formulations on entomopathogenic fungi, nematodes, and parasitoids, but few have sought to measure its impact on the biology of predatory insects. We exposed the larvae of three species of ladybeetle (Coleoptera: Coccinellidae) to field rates of copper sulfate in combination with petroleum oil, a formulation commonly applied in Florida citrus. First instar larvae of Curinus coeruleus Mulsant, Harmonia axyridis Pallas, and Olla v-nigrum Mulsant received a 24 h exposure to residues on Petri dishes, and another 24 h exposure in the third instar. Treated larvae of all three species survived to adulthood at the same rate as control larvae, but larvae of O. v-nigrum experienced a significant increase in developmental time. Female adults of C. coeruleus and H. axyridis receiving copper sulfate exposures as larvae did not differ from control adults in pre-reproductive period, fecundity or fertility over ten days of reproduction. Treated O. v-nigrum females had significantly longer pre-reproductive periods than control females and laid significantly fewer eggs, although egg fertility was equivalent. We conclude that copper-sulfate fungicides are unlikely to disrupt biological control processes in citrus groves that are mediated by these coccinellid beetles.

Introduction

Citrus production has long relied on copper-based fungicides for disease control (Winston ) and copper is still the most widely used fungicide in Florida citrus (McCoy ). Cost is an important factor in the selection of control measures, and copper fungicides remain cheap and effective relative to many alternatives. Modern strobilurin fungicides such as azoxystrobin and fenbuconazole are also effective against many of the diseases controlled by copper-based fungicides, but concerns about resistance development limit their recommended application rate to once per year in a given grove (2003 Florida Citrus Pest Management Guide, 2003). The need to rotate these compounds with others of different modes of action frequently leads to the inclusion of copper in disease management programs.

Environmental concerns about the side effects of copper-based fungicides have addressed their potential for accumulation in soils and effects on soil biota such as earthworms (Paoletti ) and nematodes (Jaworska and Gorczyca, 2002). Some studies have demonstrated adverse impacts on beneficial mite species (Buschkovskaya, 1974; Childers ), although other studies have found no negative effects (Reis ). In citrus, copper fungicide applications have been associated with outbreaks of the citrus red mites, Panonychus citri (Tetranychidae) (Kim ), and Phyllocoptruta oleivora (Eriophyidae) (Winston ). This effect has been attributed to chemical interactions between copper and miticides such as zineb that reduce their effectiveness (Griffiths and Fisher, 1949; Johnson, 1960). However, Childers (1994) presented evidence to indicate stimulatory effects of copper on P. oleivora populations independent of interactions with miticides. The primary biological control of eriophyid mites in citrus is the entomopathogenic fungus Hirsutella thompsonii and various studies have attributed outbreaks of citrus rust mite and other eriophyids to suppression of H. thompsonii with copper fungicides (van Brussel, 1975; Ureta, 1980; McCoy and Lye, 1995). Nevertheless, studies on mite control in viticulture have found copper-based fungicides to be more IPM-compatible than alternatives such as carbamates in sparing predatory phytoseiid mites (Morando ; Rumbos ).

The potential impacts of copper on insect biology and ecology have been less well studied. Ropek and Para (2002) showed that copper fungicides inhibit the growth and infectivity of Verticillium lecanii, an entomopathic fungus that can be important for aphid control in citrus (Rondon ; Michaud, 1999). Glowacka studied heavy metal loads in 14 species of psyllids (Homoptera: Psyllidae) from polluted regions in Finland and found that wax and honeydew secretions were major routes of copper elimination that largely prevented significant bioaccumulation in these insects. They speculated that certain psyllid species might be important in the transfer of heavy metals to predators such as ants.

Jalali and Singh (1995) noted some mortality of Aphytis sp. (Hymenoptera: Aphelinidae) following a 24 h exposure to 0.2% copper oxychloride, and reduced ability to parasitize San Jose scale, Quadraspidiotus perniciosus (Homoptera: Diaspididae). Mani found that exposure to copper oxychloride reduced the longevity and fecundity of the citrus mealybug parasitoid Leptomastix dactylopii (Hymenoptera: Encyrtidae). Similarly, Havelka and Bartova (1991) concluded that copper fungicides were not compatible with IPM programs for greenhouses because of their adverse effects on the aphid predator Aphidoletes aphidimyza (Diptera: Cecidomyiidae). However, Teran concluded that a copper sulfate fungicide was safe for two Aphytis parasitoids (Hymenoptera: Aphelinidae) important for scale control in citrus.

Coccinellid beetles comprise a complex of species important as predators of various homopteran pests of citrus including scales, whiteflies, aphids and psyllids. Lo et al. (1992) reported reduced scale predation by a coccinellid, Orcus chalybeus, when foraging on surfaces treated with a copper sulfate fungicide. Previously, Michaud (2001a) found that a combination of copper sulfate fungicide and petroleum oil caused significant mortality to the coccinellid Cycloneda sanguinea, whether larvae were exposed to residues or direct topical sprays. However, another coccinellid, Harmonia axyridis Pallas, was not similarly affected. The results indicated that copper-oil formulations may have direct negative effects on some, but not all, coccinellid species. However, possible chronic, sub-lethal effects of copper on the developmental and reproductive biology of these insects were not sought. The current study was undertaken to determine if exposure to copper sulfate could have chronic, sublethal effects on the development and reproduction of three coccinellid species important in biological control in Florida citrus, H. axyridis, Curinus coeruleus Mulsant, and Olla v-nigrum Mulsant. H. axyridis is an invasive species in Florida (Michaud, 2002a) that is nevertheless a very effective predator of aphids and other citrus pests (Michaud 1999). Although C. coeruleus has a limited distribution in Florida citrus, it is an effective predator of scales and psyllids (Michaud 2002b), and O. v-nigrum is a key predator of Asian citrus psyllid (Michaud, 2001b).

Materials and Methods

Insect Colonies

We established colonies of H. axyridis and O. v-nigrum from adults that were field-collected in Polk County, Florida in May, 2002. A colony of C. coeruleus was established using adults field-collected in St. Lucie County, Florida in August, 2001. Adult beetles were maintained in 1 liter, wide-mouth mason jars filled with shredded wax paper and covered with muslin fabric. Beetles were fed a combination of bee pollen and frozen eggs of the flour moth Ephestia kuehniella (Lepidoptera: Pyralidae). Distilled water was continuously available on a cotton wick. For oviposition, females were removed from the jars and isolated in plastic Petri dishes (5.5 cm diameter. × 1.0 cm). Insects were each provided with approx. 5–20 mg. of frozen Ephestia eggs every 48 h and water encapsulated in polymer beads (Entomos, LLC, 4445 SW 35th Terrace, Suite 310, Gainesville, Florida 32608) every 3 days. Eggs of H. axyridis and O. v-nigrum were usually laid directly on the surface of the plastic Petri dish and were harvested daily by moving the female to a clean dish. In nature, eggs of C. coeruleus are laid singly in cryptic locations, so females of this species were provided with small, trimmed squares of black synthetic carpet fiber (5.0 – 7.0 mm square) into which they readily oviposited. Eggs of all three species were held in a Plexiglass incubator until eclosion 3–4 days later (H. axyridis and O. v-nigrum) or 7–8 days later (C. coeruleus). Newly eclosed larvae were reared in Petri dishes (as above) on a diet of frozen Ephestia eggs with water made available in polymer beads.

Copper Sulfate Treatments

Larvae of each species were treated twice, once in the first instar (24 ± 6 h old), and again in the third instar (24–48 h following the second larval molt). A copper-sulfate – oil emulsion was formulated to correspond to the field rate of these materials normally applied in Florida citrus for control of greasy spot, Mycosphaerella citri (Florida Citrus Pest Management Guide, 2003). An emulsion of 0.12 % copper sulfate (40% metallic copper, Elf Atochem Corporation) and 1.0 % (by volume) petroleum oil (Sunspray® 9E, Sunoco Inc.) was prepared in de-ionized, distilled water and applied directly to the bottom of plastic Petri dishes (5.5 cm diameter × 1 cm) using a Potter Precision Spray Tower (Burkard Manufacturing Co. Ltd., www.burkard.co.uk). The copper sulfate-oil emulsion was well agitated prior to withdrawing each aliquot. Treatment dishes (n = 60 for each species) were each sprayed with 1.0 ml of the test material and control dishes (n = 60 for each species) were each sprayed with 1.0 ml of de-ionized, distilled water. Dishes were air-dried for at least 30 min and then a small measure of Ephestia eggs (2.0 – 3.0 mg) was added to each. A single larvae was transferred to each dish for a period of 24 h and then removed to a clean dish and provisioned with fresh food and water for the duration of larval development.

Larval Survival and Development

The number of larvae alive in each treatment was tallied daily until all larvae either pupated or died. Larval developmental time was calculated in days from the date of hatching to the date the larva formed a pre-pupa. Survival was determined as the percentage of larvae that emerged successfully as adults. Adults were transferred to mason jars (prepared as above for stock colonies) as they emerged, grouped separately as ‘treatment’ and ‘control’ insects for each species. Survival was compared between treatment and control groups for each species using a Chi-squared Goodness of Fit test (P ≤ 0.05) and developmental time, by one-way ANOVA (SPSS, 1998).

Adult Reproduction

Following the addition of the last emerging adult to a jar, adults were left in the jar for another seven days to ensure all females were mated. On the eighth day, all insects were removed from the jar and isolated in individual Petri dishes with food and water (as above). Only females were retained for assessment of reproductive performance. In the case of C. coeruleus it was possible to reliably sex individuals based on external coloration. Since neither H. axyridis nor O. v-nigrum could be sexed reliably on the basis of external features, individuals of these species were confined in pairs until sex could be determined by direct observation of copula, or by oviposition. Sex ratio was tested for deviation from 50:50 using a Chi Square, Goodness of Fit test (P ≤ 0.05).

As females began to oviposit they were numbered sequentially and their eggs collected daily (as above) for a total of ten days of reproduction. Eggs were labeled, placed in the incubator, and the number hatching recorded. The number of eggs laid was compared between treatment and control females by one-way ANOVA. The percentage of eggs hatching was compared between treatments by one-way ANOVA. As we were unable to determine the exact emergence date for individual females following their removal from jars, the female pre-reproductive period was estimated as the period from date of removal from the jar to the date of first oviposition, plus the median number of days spent in the jar. These data were also compared by one-way ANOVA. Values of rm were calculated for each species as described by Price (1997). As these values are all based on only ten days of reproduction from each female, they underestimate rmax, but are useful for comparative purposes.

Results

The percentages of larvae of each species surviving the copper sulfate and oil treatments, along with their respective mean developmental times and standard errors, are given in Table 1. One C. coeruleus larvae in the treatment group was accidentally killed in the course of rearing, reducing sample size to 59. Survival was not significantly different from 100% for any species in any treatment, nor were differences between treatment and control groups significant (Chi Square, P > 0.05 in all cases). The copper treatment extended the developmental time of O. v-nigrum larvae by 8.5% but had no measurable effect on the other two species (Table 1).

Table 1.

Percent survival (± SEM) of control and treatment larvae of three coccinellid species surviving two 24-h exposures to residues of copper sulfate and petroleum oil and their mean developmental times (± SEM).

The sex ratios of emerging adults were not significantly different from 0.5 in any treatment, nor were there significant differences between control and treatment groups (Chi square, P > 0.05 in all cases). Pooled sex ratios for each species were (C. coeruleus: 0.38; H. axyridis: 0.42; O. v- nigrum: 0.49). A total of 2, 1 and 1 adult females died during the 21-d period in control groups of C. coeruleus, H. axyridis and O. v-nigrum, respectively, and 3, 2, and 1 in treatment groups, respectively. None of these females yielded ten days of oviposition data so all were excluded from reproductive analyses. The mean pre-reproductive periods for control and treatment females of all three species and their standard errors are given in Table 2. The mean fecundity of adult females of all three species for ten days of reproduction are depicted in Fig. 1. Female O. v-nigrum receiving copper treatments as larvae laid fewer eggs than did their control counterparts (F = 5.598, 1,53 df, P = 0.023) and had their pre-reproductive period extended by 6.6%. The mean percentage of eggs hatching was not significantly different between control or treatment females for any species (Table 3). The lower percentage of O. v-nigrum eggs hatching relative to the other two species was partly due to some egg cannibalism by early hatching larvae of this species that could not be prevented.

Table 2.

Mean pre-reproductive periods (± SEM) in days for adult females of three coccinellid species surviving two 24-h exposures to residues of copper sulfate and petroleum oil as larvae.

Figure 1.

Mean numbers of eggs laid (± SEM) by adult females of three coccinellid species over 10 days of reproduction. Treatment females received two 24-h exposures to copper sulfate/petroleum oil residues as larvae. Asterisk indicates a significant difference between treatment and control groups (ANOVA, P < 0.05).

Table 3.

Mean percentage of eggs hatching (± SEM) for adult females of three coccinellid species surviving two 24-h exposures to residues of copper sulfate and petroleum oil as larvae.

Rm values were calculated for control insects only in C. coeruleus (rm = 0.054) and H. axyridis (0.113) as these insects did not experience any measurable effect of treatment on any life history trait. The value for O. v-nigrum control insects was rm = 0.120, as opposed to rm = 0.111 for treatment insects, as reduction of 8.1%.

Discussion

While we did not directly measure the amounts of copper taken up by beetles in this study, the two 24-h treatments likely represented heavy exposures of copper sulfate in comparison to anything larvae of these insects would naturally encounter in process of foraging on treated leaves in a citrus grove. While residues may persist on foliage for many days following treatment, the deposits take the form of isolated spots, clearly visible by their blue coloration. Consequently, insects only make sporadic contact with these deposits while foraging, as opposed to the continuous contact they had with coated surfaces in these experiments.

Three different life history parameters of O. v-nigrum suffered impact from larval exposures to the copper sulfate/petroleum oil formulation, whereas both C. coeruleus and H. axyridis were unaffected by the same treatments. This difference among species in apparent sensitivity to copper may arise from variation in rates of physiological uptake, elimination efficiency, or even behavioral avoidance in the case of C. coeruleus. The copper-oil combination seemed to exhibit some repellency to larvae of C. coeruleus, although not to those of H. axyridis or O. v-nigrum. Larvae of C. coeruleus have lower activity levels than those of the latter species (Michaud and Grant 2003) and appeared to minimize their contact with the treated surface by remaining on top of their food for extended periods. This may have reduced their uptake of the material relative to the other two species that were more active on treated surfaces. Michaud and Grant (2003) also found that third-instar O. v-nigrum larvae had higher activity levels than third-instar H. axyridis larvae, as measured by rates of larval exodus from filter paper circles. Higher activity levels may have increased exposure of O. v-nigrum larvae to copper sulfate during treatments. However, the provision of food on the treated surface of Petri dishes during exposure periods likely resulted in some oral ingestion of copper by all species in addition to dermal contact.

Some evidence suggests relatively inefficient uptake of copper in some insects, and effective elimination by others, factors that may explain some of the variation in the toxicity of copper across insect species. Stone found relatively constant levels of copper in bodies of the ground beetle Pterosthicus oblongopunctatus (Carabidae) collected from five sites along a pollution gradient in Poland, while body burdens of zinc, lead, and cadmium all varied greatly along the gradient. Edwards and Hodgson (1973) found copper oxychloride to have no immediate toxic effects on the mite predator Stethorus nigripes (Coccinellidae). Mani and Thorntakarya (1988) found both Bordeaux mixture and copper oxychloride to be safe for the coccinellid Scymnus coccivora. Mani found that copper oxychloride exposure had no negative effects on the longevity or reproduction of the mealybug predator Cryptolaemus montrouzieri (Coccinellidae). Niranjan observed that exposure to sub-lethal doses of copper sulfate caused marked changes in the protein concentration of the hemolymph, fat bodies, and gonads of the beetle Hydrophilous olivaceous ((Hydrophilidae). In one of the more detailed studies of effects of copper on beetle biology, Bayley reared larvae of Pterostichus cupreus (Carabidae) on copper-contaminated food and soil and measured both acute and chronic toxicity including elevated larval mortality and depressed adult locomotor functions. Izhevskii (1976) fed mineral salts to larvae of Leptinotarsa decemlineata (Chrysomelidae) on potato leaves and found that copper and sulfate salts depressed enzymatic activity in the beetles and delayed larval development.

The small increases in developmental time (0.7 days) and pre-reproductive period (0.8 days) observed in O. v-nigrum following the copper sulfate treatment were followed by a 13% reduction in female fecundity. Although these impacts on life history resulted in an 8.1 % reduction in the intrinsic rate of population increase, it seems unlikely this would translate into measurable reductions of field populations of this insect. We conclude that copper sulfate fungicides are relatively safe for field populations of these coccinellid species and are unlikely to disrupt the biological control processes in citrus groves that rely on them.