Functional responses and intraspecifi c competition in the ladybird Harmonia axyridis (Coleoptera: Coccinellidae) provided with Melanaphis sacchari (Homoptera: Aphididae) as prey

Functional responses at each developmental stage of predators and intraspecifi c competition associated with direct interactions among them provide insights into developing biological control strategies for pests. The functional responses of Harmonia axyridis (Pallas) at each developmental stage of Melanaphis sacchari (Zehntner) and intraspecifi c competition among predators were evaluated under laboratory conditions. The results showed that all stages of H. axyridis displayed a type II functional response to M. sacchari. Based on Holling’s disc equation, the instantaneous searching rates were highest (a) and handling time was shortest (Th) of fourth instar larvae (a = 0.8818; Th = 3.9 min) and female adults (a = 0.9881; Th = 3.0 min) at larval and adult stages, respectively. The coeffi cients of mutual interference (m) assessed by the intraspecifi c competition equation were higher for fourth instar larvae (m = 0.4764) and f emale adults (m = 0.4183). The present study indicates that fourth instar and female adult were more effective stages of H. axyridis in the context of biological control but suitable predator densities need to be considered before natural enemy release. * Corresponding author; e-mail: zhangrz@ioz.ac.cn INTRODUCTION Melanaphis sacchari (Zehntner) (Hemiptera: Aphididae) is a perennial pest of Sorghum bicolor L. Moench and Saccharum offi cinarum L. which has a wide economic impact (Singh et al., 2004). Melanaphis sacchari originates from Java, Indonesia (Zehntner, 1897). In North America, M. sacchari has been recorded as a sugarcane pest in Florida (Mead, 1978; Denmark, 1988) and Louisiana (Hall, 1987; White et al., 2001). Melanaphis sacchari is initially reported on sorghum when their largely damaging populations invaded in Beaumont, Texas (Villanueva et al., 2014). Melanaphis sacchari is described as a distinct species (Blackman & Eastop, 2006), and their clones are defi ned by geography including Africa, China, Australia, USA and South America rather than by the host plant (Nibouche et al., 2014). Over the past several years, M. sacchari has caused measurable economic effects due to the damaged Eur. J. Entomol. 115: 232–241, 2018 doi: 10.14411/eje.2018.022

plant growth correlated with the sticky "honeydew" covering the plant.Melanaphis sacchari transmits the sugarcane yellow leaf virus causing a 25% reduction in sugarcane yields (Akbar et al., 2010).Control of M. sacchari is diffi cult as pesticides cannot penetrate the greater canopy of sorghum.Pre-and post-harvest restrictions exacerbate the challenge of pesticide use (Armstrong et al., 2016).Melanaphis sacchari cannot destroy sorghum in a short term period, but the large populations growing out of control lead to the chlorosis correlated with damage of plant tissues because of the rapid aphid reproduction (Colares et al., 2015).Melanaphis sacchari can continue injuring the plants at grain-fi lling stage and affecting their seeds both in quality and quantity (Chang & Fang, 1984;van den Berg et al., 2003).Moreover, if the M. sacchari colony has the characteristic of thermal tolerance, it could contribute signifi cantly to its pest status under hot summer conditions

INTRODUCTION
Melanaphis sacchari (Zehntner) (Hemiptera: Aphididae) is a perennial pest of Sorghum bicolor L. Moench and Saccharum offi cinarum L. which has a wide economic impact (Singh et al., 2004).Melanaphis sacchari originates from Java, Indonesia (Zehntner, 1897).In North America, M. sacchari has been recorded as a sugarcane pest in Florida (Mead, 1978;Denmark, 1988) and Louisiana (Hall, 1987;White et al., 2001).Melanaphis sacchari is initially reported on sorghum when their largely damaging populations invaded in Beaumont, Texas (Villanueva et al., 2014).Melanaphis sacchari is described as a distinct species (Blackman & Eastop, 2006), and their clones are defi ned by geography including Africa, China, Australia, USA and South America rather than by the host plant (Nibouche et al., 2014).Over the past several years, M. sacchari has caused measurable economic effects due to the damaged Studying the functional response and intraspecifi c competition will provide insights into the prey-predator and predator-predator interactions between H. axyridis and M. sacchari, which could result in the development of a better strategy for the biological control of M. sacchari using H. axyridis.Thus, the objectives of this study were to determine the functional response of H. axyridis to M. sacchari and intraspecifi c competition among H. axyridis under laboratory conditions.

Insect
Colonies of H. axyridis and M. sacchari were maintained in the laboratory and a greenhouse at the experimental farm of the Florida Agricultural and Mechanical University, FL, USA.Melanaphis sacchari were reared and reproduced on the leaves of sorghum plants at 16L : 8D, 25°C and 40-60% RH.Approximately 20 larvae or adult H. axyridis were reared per plastic container (16 × 22 × 8 cm high) at 16L : 8D, 25°C and 40-60% RH.Harmonia axyridis were re ared on t he sorghum aphid M. sacchari from fi eld populations.Prior to the studies, H. axyridis were transferred into Petri dishes (9 cm diameter) containing sorghum leaves infested with M. sacchari.Neonates were enclosed soon after hatching to avoid sibling cannibalism; female and male adults were also isolated.Melanaphis sacchari were supplied in 12 h intervals to guarantee an abundant H. axyridis population.For the experiment, individuals of H. axyridis were selected for study within 12 h of molting or eclosing.They were starved for 24 h, and then placed on wet paper discs in Petri dishes including less than 12h-old adults of M. sacchari.The dishes were preserved in conditions at 16L : 8D, 25°C and 40-60% RH. Adult M. sacchari were not replaced during the tests and nymphs from adult M. sacchari were removed every 4 h by smooth brushes.

Fun ctional response
To examine the functional response of H. axyridis at each developmental stage on M. sacchari, prey densities tested were 2, 4, 8, 16, 32, 64, 128 and 256 aph id adults for four instars of larvae (fi rst, second, third, fourth instar) and adults in both sexes (male and female).The number of predation events were examined after 24 h by recording the number of aphids consumed using binocular microscopes.Each treatment was replicated 10 times simultaneously.Con trol treatments without predators were performed to take into account the natural mortality of M. sacchari adults and the amount of their newborn nymphs in order to correct M. sacchari consumption as a function of natural mortality.These treatments were each replicated 10 times.

Intraspecifi c competition
To evaluate the effects of intraspecifi c competition on the foraging behavior of H. axyridis, four instars of larvae (fi rst, second, third, fourth instar) and adults in both sexes (male and female) were studied.The prey densities examined were 100, 200, 300, 400 and 500 aphid adults for 1, 2, 3, 4 and 5 predaceous coccinellids at various developmental stages in a Petri dish, respectively.The prey/predator ratio was kept at 100 for each number of coccinellids placed together in a Petri dish, with competition among predators for space increasing with the increasing number of predators placed together.The coeffi cients of mutual interference were calculated by counting the amount of aphids surviving after 24 h using binocular microscopes.Each treatment was replicated 5 times simultaneously.(Pendleton et al., 2009).Melanaphis sacchari is also good at utilizing wild grasses in the fi eld (Singh et al., 2004).
There are 47 species of natural enemies that have been recorded as biological control agents of M. sacchari (Singh et al., 2004).Many studies now indicate that predators often locate their prey by using plant volatiles (Takabayashi & Dicke, 1996;Arimura et al., 2005;Turlings & Ton, 2006).The predators search for aphids through honeydew derived from aphids (Hatano et al., 2008;Verheggen et al., 2008) and volatiles released by damaged plants (James et al., 2005;Sasso et al., 2009).Multicolored coccinellid Harmonia axyridis Pallas has the potential to be a biocontrol agent of aphids throughout Asia, America and Europe (Koch, 2003), and this predacious coccinellid is the effective predator of aphids in natural environments (Mogi, 1969;Choi & Kim, 1985;McClure, 1987;Hong, 1996).The primary factor to determine whether H. axyridis can be a suitable candidate for biological control of M. sacchari is their foraging capacity.Various aphidiphagous responses of H. axyridis have been performed in previous studies (Lou, 1987;Hu et al., 1989;He et al., 1994;Seo & Youn, 2000), but its functional response to M. sacchari is still unstudied.The functional response of a predator to the dynamic of prey densities depends on their instantaneous searching rate and handling time (Hassell et al., 1976).These parameters will provide a valuable reference for augmentative releases of H. axyridis as a biological control agent of M. sacchari in natural habitats.Releases of H axyridis eggs, larvae and adults have been studied (Tedders & Schaefer, 1994;Trouve et al., 1997;Ferran et al., 1998;Kitagami & Ohkubo, 1998;Kuroda & Miura, 2003).Growth stage of natural enemies needs to be considered as their searching rates and handling time vary with the developmental stages (Varley et al., 1973;Dixon, 2000).Complex predator-prey systems are attributed to the interactions not only between predator and prey, but also between predator individuals, especially in circumstances where competition is intense (Papanikolaou et al., 2016).
Predatory coccinellids serve as biological control agents of pests in many crops (Ferran et al., 1996).Interactions among these predators, and competition for resources (prey and space), infl uence their impact on biological control.Our preliminary observations indicated that foraging effi ciency, and hence the effectiveness of biological control, is reduced when too few or too many of these predators are present.Interactive competition is a density-dependent process including indirect and direct interactions between predators (Begon et al., 1996).Intraspecifi c competition occurs via direct interactions between predator individuals of same species (Hassell, 1978).Several models have been performed to quantify mutual interference using phenomenological (Hassell & Varley, 1969) or mechanistic (Beddington, 1975;DeAngelis et al., 1975;Crowley & Martin, 1989) approaches, which indicates that foraging behavior is not only prey-dependent but also a predator-dependent process.Thus, consideration of intraspecifi c competition is necessary during predation under both laboratory and fi eld conditions.

Functional response
The functional responses were studied through two-stage analysis (Juliano, 2001).In the fi rst step, cubic logistic regression analysis proportion of prey consumption as a function of initial density was performed to determine the shape (type II or type III) of functional response: where N a is the number of prey consumed and N 0 is the initial prey density.P 0 , P 1 , P 2 and P 3 are the intercept, linear, quad ratic and cubic coeffi cients, respectively.Negative or positive linear coefficients (P 1 ) from the regression indicate a type II or type III curve, respectively (Juliano, 2001).If a cubic equation non-signifi cantly yields coeffi cients, it is desirable to reduce the model by eliminating the quadratic and cubic coeffi cients from equation 1 and to retest the other parameters (Juliano, 2001).Because the logistic regression analysis indicated that our data fi t type II in each case, further analyses were restricted to the type II functional response.The Holling's disc (Eq.2) (Holling, 1959) was used to model the relationship between the number of prey consumed (N a ) and initial prey density (N 0 ): where N a and N 0 are described in equation 1, T is the total time which in this case is 24 h, a is the instantaneous searching rate and T h is the handling time.A nonlinear regression procedure (NLR) based on the Levenberg-Marquardt method was performed to estimate the parameters a and T h .The starting values of a and T h required by the NLR procedure were found via the linear regression of 1/N a against 1/N 0 .The resultant y-intercept is the initial estimate of T h and the reciprocal of the regression coeffi cient is an estimate of a (Livdahl & Stiven, 1983;Watson et al., 2000).

Intraspecifi c competition
The experiment was performed to calculate the coeffi cients of mutual interference among predators during predation events.Nonlinear regression analysis was performed to estimate parameters of an intraspecifi c competition model by fi tting equation 3 (Hassell & Varley, 1969): where E is the mean consump tion, P is the predator density, m is the coeffi cient of mutual interference and Q is the theoretical maximum consumption rate (%).The values of Q and m were found by power-exponential regressing E and P. Descriptive statistics were given as the mean values and standard errors of the mean.Differences between natural mortality rate and 0 were examined using one sample t-test; P values < 0.05 were considered signifi cant.Statistics were performed with SPSS 20.0 software (IBM, Armonk, NY).Regression analyses were performed using SigmaPl ot 12.0 software (Systat Software Inc., San Jose).

Functional response
Regardless of prey density, natural mortality rates of M. sacchari were not signifi cantly different from 0 (t-test, P > 0.05) as few newborn nymphs were produced during experiments.Thus, the mortality rates of M. sacc hari and amounts of their newborn nymphs were negligible during tests.Parameter estimates from the logistic model (Eq. 1) of the proportion of M. sacc hari consumed by H. axyridis over a 24 h period versus prey density are exhibited in Table 1.Estimates of the linear parameter P 1 were signifi cantly negative for all developmental stages (Table 1).Therefore, the logistic model analysis of all developmental stages performed a type II response to M. sacchari.
The functional response data for prey consumption by H. axyridis over a 24 h period fi tted the Holling 's disc model (Eq.2) well (Table 2), confi rming a type II response for all developm ental stages.The amounts of M. sacchari consumed increased signifi cantly as their densities increased, and foraging capabilities of H. axyridis increased progressively with increasing growth stages (Fig. 1).The coeffi cients of instantaneous searching rate (a) and handling time (T h ) indicated numerica lly this relationship, which had asymptotic 95% confi den ce intervals except 0 at vari- ous developmental stages.The searching rates of female adults (0.9881) were highest among varied developmental stages, followed by those of male adults, fourth, third, second, and fi rst instar larvae.The time of female adults handling a prey (3.0 min) was shortest, followed by that of fourth instar larva, adult male, third, second and fi rst instar larvae (Table 2).

Intraspecifi c competition
When the ratio of prey/predators was kept to 100, total M. sacchari consumption in a Petri dish gradually in-creased as the introduced numbers of predators and prey increased.However, the mean consumption of ladybugs at various developmental stages decreased with increasing predator and prey density due to intraspe cifi c competition associated with space limitation (Fig. 2).Overall, the mean consumption of ladybugs at fi ve prey-predator densities were 53.4 (prey/predator = 100/1), 41.4 (200/2), 37.5 (300/3), 33.3 (400/4) and 25.3 (500/5).Intraspecifi c competition curves fi tted the data at all developmental stages of H. axyridis with equation 3.At all developmental stages, the mean consumption at various predator densities fi tted the intraspecifi c competition equation well (Table 3).Theoretical maximum consumption rates (Q ) and coeffi cients of mutual interference (m) of all developmental stages had asymptotic 95% confi dence intervals that did not include 0. The order of theoretical maximum consumption rates (Q) were highest at the adult female stage, followed by fourth instar, adult male, third, second and fi rst instar stages.The order of coeffi cients of mutual interference (m) were highest at the fourth instar stage, followed by adult female, adult male, and third, second and fi rst instar stages (Table 3).
We found that low foraging success rates were detected in fi rst instars of H. axyridi s due to their smaller sizes and slower movements (Lee & Kang, 2004).The larvae in second and third instars had relatively higher M. sacchari consumption compared to the fi rst instar larvae.Fourth instar larvae accounted for 58.2% of the total prey consumption by larvae, suggesting that fourth instar larvae are the main contributors of aphid consumption by larvae of coccinellids (Hodek & Hon ek, 1996).Prey consumptio n by male and female adults of H. axyridis were 74.3% and 111.3% of that by the fourth instar larvae over a 24 h period, respectively, implying the adult and fourth instar stages were equally effective for biological control.Moreover, fourth instars larvae and female adults of H. axyridis were more voracious than other developmental stages, likely because of higher nutritional requirements for development or for reproduction (Omkar & Srivastava, 2003).

Intraspecifi c competition
Our study indicated that, although the ratio of prey to predators was kept constant at 100, the foraging effi ciency progressively decreased when predator density increased in the Petri dish.Thus, the mean consumption of ladybugs was negatively impacted at a high predator density because of an increased chance of intraspecifi c competition such as interference or cannibalism (as discussed below).Our fi ndings were consistent with previous studies that intraspecific competition increased with the growing population den-sity of the predatory ladybird Chilocorus spp.(Hattingh & Samways, 1990).
The intraspecifi c competition equation for H. axyridis yielded the parameters Q (theoretical maximum consumption rate) and m (coeffi cient of mutual interference), which was consistent with the past studies that the intraspecifi c competition model (Eq.3) was performed to estimate the mutual interference of H. axyridis during their predation on Aphis citricol (Fang et al., 2013) and Cyamophila willieti (Shen et al., 2009).We found that H. axyridis at fourth instar and female stage had higher potential maximum consumption rates and coeffi cients of mutual interference compared to other developmental stages.Coccinellids are digestive-limited predators, so the limitation of theoretical maximum con sumption rate was shown because of predator satiation ( Papanikolaou et al., 2014).The coccinellids became inactive after consuming large numbers of aphids at maximum prey consumption, likely minimizing mutual interference, whereas notable time was likely to be spent on mutual interference during their foraging when the ladybugs had high satiation levels (van Gils & Piersma, 2004;Papanikolaou et al., 2016).Therefore, mature coccinellid individuals had higher coeffi cients of mutual interference due to higher potential maximum consumption rates.
Cannibalism was occasionally observed when multiple predators shared a Petri dish, as conspecifi c competitors attacked each other, resulting in injury and sometimes death.Cannibalism contributed to the sharp decline in per capita consumption as predatory density increased, which was another reason why ladybugs at these stages had more severe intraspecifi c competition.Cannibalism had a negative effect on the overall foraging effi ciency, even though it contributed greater resources such as space and food to superior individuals (Block & Stoks, 2004;Richardson et al., 2010;Bayoumyi & Michaud, 2015).Another negative effect of cannibalism was increasing disease transmission (Saito & Bjørnson, 2006) and decreasing inclusive fi tness (Hamilton, 1964).

Remaining questions and future perspectives
Foraging ca pacities including instantaneous searching rates and handling time are critical for estimating the potential of predators to serve as a biocontrol agent (Lucas et al., 1997).Prey-predator dynamics can be evaluated by a mathematical model (Hassell, 1978).Thus, the dynamics could be established through the functional response curves, and the foraging capacity of H. axyridi s depends on M. sacchari density in natural habitats.However, fi eld studies are needed to validate th e dynamics, because quantitative models built in laboratory studies appear to have limited value in assessing the foraging abilities under fi eld conditions (Gitonga et al., 2002;Lee & Kang, 2004).A series of studies on functional responses of Podisus maculiventris verify that there is a distinction between laboratory and fi eld studies (O'Neil, 1988a(O'Neil, , b, 1990(O'Neil, , 1997;;Wiedenmann & O'Neil, 1991a, b, 1992), likely because of the difference in searching rates of predators between laboratory an d fi eld conditions (Murdoch, 1983).Spatial complexity, critical in the natural environment, cannot be recreated under simple laboratory conditions (Kareiva, 1990).Laboratory studies provide parametric analysis of predator-dependent intraspecifi c competition models, but they are performed only on a non-spatial scale.Thus, intraspecifi c competition in a spatial sense is critical to future studies as it is closer to natural conditions (Sun et al., 2008(Sun et al., , 2014(Sun et al., , 2015)).Intraspecifi c competition may disrupt the foraging capacities quantifi ed by functional response.As such, understanding not only prey-predator but also predator-predator interactions is vital for a reliable predator-based control of aphids.Functional response and intraspecifi c competition models describe the foraging behaviors of predators accurately and also indicate the existence of intraspecifi c competition impacting their foraging effi ciency.Thus, a comprehensive analysis of functional response and intraspecifi c competition will allow further improvement of our understanding of prey-predator-predator interactions in relation to aphid biological control.Fourth instar larvae and adults of H. axyridis can serve as exce llent biological co ntrol agents of aphids in an integrated pest management programme.Field release of third instar larvae has appeared to be economical as mass rearing to fourth instar and adult stages can be avoided (Seko & Miura, 2008).Additionally, a suitable predator density should also be considered to decrease intraspecifi c competition when H. axyridis are released in the fi eld, as too few or too many predators released is likely to result in reduced effectiveness of the biological control agent.Moreover, it could be interesting for further studies to regard the intraguild predation in order to obtain more information on the foraging behavior of coccinellids as these interactions have been fi tted by several models (de Villemereuil & López-Sepulcre, 2011;Sentis et al., 2013).Natural stochasticity needs to be considered in future fi eld experiments on intraspecifi c competition among coccinellids (Papanikolaou et al., 2016).Plant characteristics also need to be considered due to their impacts on the feeding effi ciency of predators (Hodek & Honek, 1996).Future fi eld investigations associated with prey-predator dynamics are critical for effective predator release, which has the potential to reduce pesticide uses and pres erve predator populations (Xue et al., 2009).

Fig. 1 .
Fig. 1.Functional response of each developmental stage of H. axyridis.Solid lines show the functional response curves of H. axyridis attacking M. sacchari obtained by fi tting Holling's disc equation (Eq.2).Circles indicate the number of M. sacchari consumed at each prey density.

Fig. 2 .
Fig. 2. Intraspecifi c competition among individuals of H. axyridis when attacking M. sacchari.Each data point represents the mean number of individuals of M. sacchari consumed by an individual predator.The curve was fi tted using the intraspecifi c competition equation (Eq.3).

Table 1 .
Maximum likelihood estimates (± SE) of the parameters of the logistic model of the proportion of prey consumed versus initial prey density.
R 2 is the coeffi cient of determination estimated by fi tting Holling's disc equation, P is the probability that Holling's disc equation will yield signifi cant parameters, a is the instantaneous searching rate and T h is the handling time.

Table 3 .
Parameter estimates of the intraspecifi c competition equation (Eq. 3) of prey co nsumption rates of H. axyridis at various predator densities.R 2 is the coeffi cient of determination estimated by fi tting the intraspecifi c competition equation and P is the probability that the intraspecifi c competition equation will yield signifi cant parameters.