Host plant quality factors that influence the growth and development of the Melaleuca quinquenervia biological control agent Oxyops vitiosa

G. S. Wheeler

(wheelerg@saa.ars.usda.gov)

Abstract.  Biological control efforts of Melaleuca quinquenervia in Florida, USA have resulted in the 1997 release of the Australian weevil Oxyops vitiosa.  The larvae of this biological control agent are flush-feeders, only found on the growing tips of their host.  Knowledge concerning this and additional nutritional requirements may assist in the establishment and dispersal of this species.  Studies were conducted where O. vitiosa survival was assessed when neonates were fed M. quinquenervia leaves from branches that had dormant buds or emerging bud leaves. Additionally, the influence of leaf quality from different sites and within sites was determined by feeding neonates emerging bud leaves collected at three sites and from three leaf qualities (poor, intermediate, and high).  Within-site leaf qualities were described in the field by leaf color, and in the laboratory by percent dry mass, and nitrogen.  Larval survival was lowest when fed leaves from branches that had dormant buds.  Associated with this low survival were high leaf toughness and percent dry mass. When larvae were fed emerging bud leaves, most of the variation in larval survival and performance was attributed to differences in within-site plant quality.  Larval survival generally decreased when fed the poor, and in one site, the intermediate quality leaves.  Larvae required less time to develop to adults when fed the high quality leaves.  Development time increased in females but not males when the larvae were fed the poor quality leaves.  Adult biomass of both females and males was generally increased when the larvae were fed the high quality leaves from two of the three sites.  The results indicate that the larvae of O. vitiosa are restricted to feeding on flush foliage with low toughness.  Additionally, variations in foliar percent dry mass and nitrogen will influence larval survival and performance.

Introduction

Biological control efforts against the Australian melaleuca tree Melaleuca quinquenervia (Cav.) Blake (Myrtaceae) have resulted in the 1997 release in south Florida of the leaf-feeding weevil Oxyops vitiosa Pascoe (Coleoptera: Curculionidae) (Center et al., 2000).  Both the adults and larvae of this species have been observed feeding primarily on flush-foliage on tip leaves of the melaleuca tree (Purcell and Balciunas, 1994).  In Australia this insect is most abundant in situations where trees are growing rapidly, often from suckers or landscaped plants (Purcell and Balciunas, 1994).  This suggests that larval survival, growth and development may be influenced by the host nutritional quality and therefore, establishment efforts of this species in Florida for biological control of M. quinquenervia could benefit from knowledge of these nutritional requirements. 

The melaleuca tree is an aggressive weed threatening the biodiversity of the south Florida everglades ecosystem (Turner et al., 1998). Originally from eastern Australia, this tree was introduced to south Florida around the turn of the century by horticulturists (Morton, 1966; Dray, personal communication). Following its introduction, the tree has spread through the natural areas of south Florida that include, and are adjacent to, the Everglades National Park and Big Cypress Preserve.  With the dramatic decreases in water flows of south Florida for flood control, protecting agriculture and urban areas, this species and many other exotic weeds have flourished.  The tree now occupies more than 200,000 ha in the region and because of its ability to resprout from periodic freezes has the potential to invade coastal wetlands of southern Louisiana and eastern Texas (Turner et al., 1998). 

The nutrition of herbivores feeding on tree foliage may be limited by several key factors, among them moisture and nitrogen content (Mattson and Scriber, 1987).  Compared with herbaceous plants, tree foliage typically has lower levels of water, nitrogen, and mineral elements (Mattson and Scriber, 1987).  Additionally, fiber content and leaf toughness may be important factors limiting the survival, growth, and development of tree-feeding folivores (Coley and Barone, 1996).  Consequently, the herbivores that feed on tree foliage typically have lowered performance and conversion efficiencies compared with herb-feeders (Scriber and Feeny, 1979, Mattson, 1980).  The goals of this study were to quantify select plant quality factors and to determine their impact on the survival and performance of the M. quinquenervia biological control agent O. vitiosa.

Methods and Materials

Preliminary test 

Plant quality. A preliminary test was conducted to investigate the degree of dependence of O. vitiosa neonates on flush-foliage.  This was determined by recording O. vitiosa neonate survival when fed M. quinquenervia branches with and without emerging bud leaves.  Branches were collected at Tree Tops Park (TT), Broward Co., FL, USA during spring 1997. The branches without emerging leaves had dormant buds and were characterized as stage 1 and those with leaves that were fully emerged from the bud were characterized as stage 4 (Van et al., personal communication).  The leaves were analyzed for leaf toughness (n = 10) using a modified gram gauge (Wheeler and Center, 1996). Leaf toughness was estimated for 15 consecutive leaves from the apical tip toward the base of the branch. All statistical analyses were conducted on SAS/PC (SAS Institute, 1990). To determine if leaf toughness changed with leaf position on the stem, the data were analyzed with linear regression.  To determine if leaf toughness differed between the branches with dormant buds and those from emerging buds, the regression coefficients were compared with ANCOVA.  Percent dry mass of leaves was determined gravimetrically (n = 16) by combining all the leaves in each branch and comparing the mass of leaves weighed fresh and after drying at 60 °C for 48h.

Larval survival. Neonates (n = 40) were fed leaves on freshly collected M. quinquenervia branches until the adult stage in petri dishes (15 x 2 cm) lined with moistened filter paper and sealed with parafilm to retain moisture. The filter paper was moistened, the frass was removed, and the M. quinquenervia branches were replaced about every 3 d. Larvae were reared at 27 °C, 90% RH, and L14:D10 h photophase. Data were collected on larval survival to the prepupal, pupal, and adult stages.

Second test –Among and within sites test

Plant quality. Subsequent collections included branches that had buds with emerging leaves (stage 4 buds; Van et al., personal communication) collected from three south Florida sites located at Holiday Park (HP), Tree Tops Park (TT), both Broward Co., and Krome Avenue & Highway 27 (Krome), Dade Co. All collections were conducted during the spring of 1997. At each site leaves were classified into one of three categories based upon leaf color, which may be a useful predictor of relative plant quality and overall suitability of select herbivores.  Leaves were classified by the chromatic colors green or yellow using the notation of G or Y (or an intermediate of the two; e.g., GY), respectively, based upon the Munsell color chart (Anonymous, 1977).  Each color may be further subdivided with numbers that refer to the darkness of the hue, where dark is symbolized as 0/ and light is 10/, and the degree of its saturation, where the greater the value, symbolized as /10, the greater its saturation. The leaves collected at sites were classified as poor (5Y 8/8 to 5GY 7/8), intermediate (5GY 5/6 to 7.5GY 4/4), and high (5GY 4/6 to 7.5GY 3/4). 

Percent dry mass of leaves collected at the three sites and three categories within each site were determined gravimetrically by leaf position (n = 16). The percent dry mass was determined as described previously for individual leaves. Additionally, leaves were digested using a modified Kjeldahl method (Hach et al., 1987) and percent nitrogen content was determined (n = 16) by an ammonia-selective ion method (Greenberg et al., 1992).  Standard reference tomato leaves (National Institute of Standards, Gaithersburg, MD, USA) were analyzed as controls and the values were adjusted for percent recovery.  To determine if leaf percent dry mass or nitrogen differed among sites and qualities within sites, a 2-way ANCOVA was performed, where leaf position served as the covariate.  The means were compared with the Ryan’s Q multiple comparison test (P = 0.05). To determine differences in elevations and slopes of percent dry mass or nitrogen over leaf positions on a branch within each site, the regression coefficients were compared with ANCOVA.

Larval performance. Neonates (n = 40) were fed leaves individually in petri dishes as described above until they developed to the adult stage.  Data were collected on several performance parameters including larval survival, biomass gain and development time to the adult stage.  To determine if site, quality, or adult sex significantly influenced these insect performance parameters a three-way ANOVA was performed.

Results

Preliminary test 

Leaf toughness. Leaf toughness was significantly greater for leaves from branches with dormant buds compared with those from branches that had buds with emerging leaves (F _1,218 = 27.78; P < 0.0001; Fig. 1). Toughness of the leaves from dormant buds ranged from 750 to nearly 800 g/mm², whereas the leaves from branches with emerging bud leaves ranged from 200 to < 350 g/mm². The leaf toughness from branches with dormant buds did not change with leaf position (P > 0.09) in contrast to those with emerging bud leaves that increased toward the branch base.  Dry mass of the leaves was significantly greater (F _{1, 45} = 685.02; P < 0.0001) from branches with dormant buds (43. 8 ± 0.7%) compared with leaves from branches with emerging bud leaves (23.2 ± 0.5%).

Larval survival.  Survival of neonates to the prepupal, pupal and adult stages was significantly reduced when fed leaves from branches with dormant buds (F _1,10 = 41.63; P < 0.0001).  Only 7.5 (± 4.8%) of the neonates survived to the prepupal, pupal, and adult stages when fed leaves from branches with dormant buds compared with 47.5 (± 3.7%) for those fed leaves from branches that had emerging bud leaves.

Second test – Among and within sites test

Plant quality. Leaf percent dry mass was influenced by site (F _2,2143 = 26.52; P < 0.0001), quality (F _2,2143 = 459.59; P < 0.0001), the leaf position (covariate) on the branch (F _{1, 2143} = 241.70; P < 0.0001), and the interaction of site and quality (F _4,2143 = 225.14; P < 0.0001).  Among site comparisons indicated that leaf percent dry mass was greatest for poor quality leaves collected at the TT site followed by those collected at the Krome and the HP site (F _2,717 = 191.31; P < 0.0001; Fig. 2).  

Leaves of the intermediate quality had the greatest percent dry mass from the HP and Krome sites (F _2,717 = 60.64; P < 0.0001).  The high quality leaves had the greatest percent dry mass at the Krome site, followed by the HP site, which was greater than that collected at the TT site (F _2,710 = 140.60; P < 0.0001).  Additionally, within site analyses indicated that leaf percent dry mass was greatest for leaves from the poor quality at the TT (F _2,716 = 1369.11; P < 0.0001) and Krome (F _2,716 = 93.63; P < 0.0001) sites and greatest for the high and poor quality leaves at the HP site (F _2,712 = 9.36; P < 0.0001).  

Leaf percent dry mass decreased significantly with distance from the tip for all site x quality combinations except for the high quality branches, which either had positive slopes or slopes that were not significantly different from zero (Table 1; Fig. 3).  Moreover, the slopes of the lines for high quality were greater than those of the intermediate quality, which were generally greater than, or equal to (e.g., Krome), those of poor quality.  Additionally, for each site the elevations of the lines for poor quality leaves were significantly greater (Table 1) than those for intermediate leaves which were greater than, or equal to (e.g., Krome), those for high quality leaves. 

As the percent dry mass of the leaves changed according to site, quality, and leaf position, the nutrients available to herbivores would be diluted differently.  Therefore, herein, nitrogen content is expressed on a fresh mass basis. Leaf percent nitrogen was influenced by site (F _2,119 = 99.40; P < 0.0001), quality (F _2,119 =15.75; P < 0.0001), the leaf position (covariate) on the branch (F _1,119 = 25.49; P < 0.0001) and the interaction of site and quality (F _4,119 = 3.92; P = 0.0050; Fig. 4).  Percent nitrogen of foliage of all three quality levels (high: F _2,40 = 40.24; P < 0.0001; intermediate: F _2,41 = 46.96; P < 0.0001; and poor: F _2,39 = 22.57; P < 0.0001) was highest when collected at the Krome site, followed by that of HP and the TT site (Fig. 4).  Furthermore, within site analyses indicated that the percent nitrogen was greatest for the high quality leaves, followed by that of the intermediate and poor quality leaves at the HP (F _2,45 = 5.78; P = 0.0058) and TT sites (F _2,42 = 27.32; P < 0.0001), whereas, no differences occurred among the leaf qualities from Krome (Fig. 4).  

Foliar percent nitrogen was distributed differently from the branch tip to the base according to the quality of branch collected.  Leaf percent nitrogen decreased significantly with distance from the tip for all site x quality combinations except for the high quality branches, which all increased toward the branch base (Table 2; Fig. 5).  The slopes of the lines for high quality were significantly greater than those of the poor and intermediate qualities at all sites.  Additionally, the elevations of the lines for the intermediate and poor quality branches from HP and Krome were generally greater than those for the high quality leaves (Table 2). 

Insect survival.  Neonate survival to the prepupal stage decreased significantly when fed the poor quality leaves at the TT (F _2,9 = 31.65 = ; P < 0.0001) and HP (F _2,9 = 13.29 = ; P = 0.0021) sites and on the intermediate leaves at the TT site (Fig. 6). However, regardless of within site quality differences, overall first instar survival differed little when fed leaves from different sites (F _{2, 33} = 0.85; P > 0.4). 

Insect growth and development. Development time to the adult stage was significantly affected by both site (F _2,183 = 17.19; P < 0.0001) and quality (F _2,183 = 10.90; P < 0.0001) and the interaction of these two factors (F _4,183 = 6.94; P < 0.0001; Fig. 7).  Among site comparisons indicated that adult development time was significantly shortest when fed the intermediate quality leaves from the HP site (F _2,60 = 27.74; P < 0.0001) compared with the same quality of leaves from the other two sites. Little difference among sites occurred in adult development time for the other quality leaves.  Within site comparisons indicated that development time was generally shorter for larvae fed the high quality leaves from the TT (F _2,65 = 13.42; P < 0.0001) and the Krome (F _2,70 = 10.08; P < 0.0001) sites compared with those fed the other category leaves. Adult development time was also influenced by the interaction of insect sex and plant quality (F _2,183 = 3.93; P = 0.0214). However, only female adult development time (F _2,106 = 7.07; P = 0.0013) was greater when fed the poor quality leaves (35.7 ± 1.1 d) compared with those fed the intermediate (33.1 ± 0.7 d) and high quality leaves (32.1 ± 0.4 d).  Male development time was not influenced by leaf quality (32.6 ± 0.3 d; P > 0.05).

Adult biomass was also significantly affected by site (F _2,185 = 5.72; P = 0.0039), plant quality (F _2,185 = 3.43; P = 0.0346), and their interaction (F _4,185 = 4.38; P = 0.0021; Fig. 7).  Little difference occurred in adult biomass among sites, however it was greatest for larvae fed the intermediate quality leaves from the HP and Krome sites (F _{2, 61} = 5.00; P = 0.0097) compared with the same quality leaves from the TT site.  Within site analyses indicated that adult biomass values were significantly greatest (F _{2, 67} = 8.72; P = 0.0004) when larvae were fed the high quality leaves from the TT site compared with the other quality leaves from the same site. Additionally, larvae fed the intermediate quality leaves from the Krome site had greater adult biomass (F _{2, 70} = 2.83; P = 0.0661) compared with the larvae fed the poor quality leaves from the same site.  Insect sex also influenced adult biomass where the females had significantly greater biomass (41.8 ± 0.4 mg) compared with the males (36.0 ± 0.5 mg; F _1,185 = 71.38; P < 0.0001). However, none of the interactions were significant suggesting that the effects of site and quality influenced the male and female biomass similarly.  

Discussion

These results suggest that both within and among site variations in M. quinquenervia leaf percent dry mass and nitrogen influenced the survival, growth and development of O. vitiosa larvae. Additionally, the most dramatic effect was found in the low larval survival when feeding on leaves from branches with dormant buds.  Associated with this low larval survival were high leaf toughness and percent dry mass. However, when larvae were fed leaves that were emerging from buds, little difference in larval performance was found among sites. Most of the variation in larval survival and performance could be directly related to within-site plant quality differences, namely, percent dry mass and nitrogen of the leaves.  Although larval survival and adult biomass were similar in larvae fed leaves from different sites, development time for larvae fed the intermediate and poor quality leaves from HP was generally shortest.  

Other site factors, not investigated here, may influence O. vitiosa survival such as hydroperiod, natural enemies, and secondary plant chemistry.  This species completes larval development on leaves in the tree canopy then drops or climbs to the ground where it excavates a pupal cell in the soil (Purcell and Balciunas, 1994).  This tree species occupies coastal wetlands in Australia (Turner et al., 1998) and many sites infested with M. quinquenervia in south Florida are flooded for extensive periods (Center et al., 2000).  The prepupae of O. vitiosa are not expected to survive long periods in water and weevil populations have not established at sites in Florida with long hydroperiods (Center et al., 2000).  Additionally, natural enemies may be important regulators of O. vitiosa populations in their native range, however in Florida only a few localities occur where generalist predators have been observed attacking larvae, including the predacious pentatomid Podisus mucronatus (Pratt personal communication).  Other factors that potentially may impact herbivore populations are the numerous foliar terpenoids of M. quinquenervia.  These terpenoids have been well documented in Australia (Brophy et al., 1989) and in other countries (e.g., Ramanoelina et al., 1994), however, little is known about their composition in Florida.  Even less is known about the biological relevance of M. quinquenervia terpenoids to the associated herbivores, including this biological control agent.  Different sites in Australia and Florida may be dominated by distinct M. quinquenervia chemotypes that vary in the concentrations of the principal terpenoids (Ireland, 1999; Dray and Wheeler unpublished data), many of which are well known mediators of mammalian (Lawler et al., 1999) and invertebrate (Gershenzon and Croteau, 1991) herbivore behavior, growth and development.  Examples include a-pinene, a well known factor that influences diverse insect-plant interactions (Gershenzon and Croteau, 1991), 1-8 cineole, an attractant of the banana weevil (Ndiege et al., 1996) and E,S-nerolidol an antifeedant of the gypsy moth (Doskotch et al., 1980). The distribution and biological significance of these, and other M. quinquenervia terpenoids, on O. vitiosa larvae and adult performance and behavior need to be determined. 

The classification of M. quinquenervia plant quality by leaf color (Anonymous, 1977), was proposed as a useful field estimate of relative plant nutrient levels and suitability for flush-feeding herbivores like O. vitiosa.  In several cases the technique accurately characterized the relative levels of percent nitrogen of leaves and O. vitiosa larval survival and performance.  For example at the TT site, percent nitrogen was greatest for high quality leaves, followed by the intermediate and lowest for poor quality leaves.  Larvae fed the high quality leaves from this site had their greatest survival, shortest development time, and greatest adult biomass, followed by the larvae fed the intermediate and poor quality leaves.  Although larval survival and adult biomass did not differ significantly when the larvae were fed the intermediate and poor quality leaves from TT, development time was significantly greater for larvae fed the intermediate compared with those fed the poor quality leaves.  However, the leaf quality classification system applied to leaves collected at the Krome site appeared to conflict with the percent nitrogen determination as no difference was found in the latter values among the different leaf quality categories.  However, larvae fed the high quality leaves from the Krome site completed development more rapidly than larvae fed the other two quality leaves and adult biomass was generally greater for larvae fed the high and intermediate quality leaves.  Finally, for the HP site, only larval survival was greater for larvae fed the high and intermediate compared with those fed the poor quality leaves.  Additional studies are needed to address other among and within site factors, such as the terpenoid levels of these leaves, which may have influenced larval survival, growth and development.

The larvae of O. vitiosa minimize the physical barriers of leaf toughness and nutritional deficiencies of limited water and nitrogen by feeding on the young leaves or flush growth of M. quinquenervia trees.  The results indicate that the apical leaves in branches with growing tips are softest and the leaf toughness increases toward the branch base.  Additionally, the distribution of foliar percent dry mass (moisture) and nitrogen from the branch tip toward its base was influenced by the quality of leaves.  Percent leaf dry mass was generally greatest, and so nutrients were less dilute, in leaves near the branch tip especially in the intermediate and poor quality leaves and this level generally decreased toward the branch base. Percent nitrogen levels increased toward the base in the high quality branches and decreased toward the base in the intermediate and poor quality branches.  Eggs of O. vitiosa are generally laid singly or in small clusters on young leaves in or near the branch tip (Purcell and Balciunas, 1994).  After egg hatch the larvae begin feeding on the same leaves near the branch tip.  This oviposition behavior provides the neonates with the softest leaves on branches that have growing tips.  These leaves from the intermediate and poor quality branches also have the greatest percent dry mass (lowest moisture) and nitrogen content.  Possibly O. vitiosa larvae are not water-limited feeding on these drier leaves, but more importantly, essential nutrients like nitrogen are more concentrated.  Subsequently, larval development is completed on leaves farther from the tip with decreases in both percent dry mass (greater moisture) and nitrogen for the intermediate and poor quality leaves with greater distance from the branch tip. On the high quality branches the opposite occurs, where the leaves farthest from the tip have increased percent dry mass and nitrogen.  Possibly O. vitiosa eggs and larvae are distributed similarly to take advantage of the nutritional differences on high versus intermediate and poor quality branches.  However intriguing, this relationship has yet to be determined.  Most likely, the relatively high leaf toughness and its associated high O. vitiosa larval mortality has selected for oviposition, larval growth and development on the low toughness and often high nitrogen content apical leaves. 

References

References cited