Multitrophic Interactions Between Coccinellids and Their Parasitoids in Natural and Managed Plant Systems: Host Plant and Aphid Prey Species Matter


 Background: To better inform conservation biological control of aphids multitrophic interactions between aphids, coccinellids and their parasitoids in natural and managed plant systems were examined over 4 years. Methods: Coccinellid larvae found naturally feeding on aphids on two non-crop plants growing in agricultural landscapes in Pakistan; Aphis nerii (Boyer de Fonscolombe) feeding on Calotropis procera L. and Nerium oleander L.; and four crop host; Lipaphis erysimi (Kalt) feeding on Eurica sativa Mill. and Brassica campestris L., Uroleucon compositae (Theobold) feeding on Carthamus tinctorius L., and Diuraphis noxia (Mordvilko) feeding on Hordeum vulgare L.; were collected throughout the aphid season and reared on their respective aphid-plant combination in the laboratory to identify coccinellid species, their parasitoids and performance (survival and pupal weight).Results: Coccinellid pupal weight on different host plant-aphid combinations varied from 3.4±1.8 to 11.2±0.9 mg (mean ± SE), as did mortality of coccinellid larvae due to diseases and unknown causes (5±2.2 to 31±3.8%). Parasitism of coccinellid larvae by Homalotylus flaminus (Dalman) (Hymenoptera: Encyrtidae) ranged from ca 10±2 to 30±3% in B. campestris and E. sativa, respectively. Parasitism of coccinellid pupae by Oomyzus scaposus (Thomson) (Hymenoptera: Eulophidae) ranged between 17±2.7 (C. procera) to 41±2.9% (B. compestris). One Dinocampus coccinellae Schrank (Hymenoptera: Braconidae) was reared. Conclusions: Coccinellids were host plant and aphid selective. Some coccinellids were never or rarely recorded on a particular aphid-plant combination, suggesting that their prey selection more specific than often assumed. Management decision and conservation biological control cannot be based on superficial categories such as “aphids” and “coccinellids” but needs to be plant and species specific.

other group of predatory organisms and used in pest management programs for over a century [13]. They are often perceived as generalist predators [14,15] and have been used in integrated pest management (IPM) and in augmentative biological control programs against various aphids since the 1950s [16,17].
Conservation approaches, such as intercropping [18] and other modi cations of agriculture practices, such as supplemental food sprays to attract and retain predators and parasitoids in crops [19,20], provide ways to encourage coccinellids to enter managed ecosystems and increase their effect on aphid populations [21,22]. The widespread planting of transgenic crop varieties has reduced insecticide use against lepidopterous pests [23]. Incidental control of non-lepidopterous pests has consequently declined and the importance of Coccinellidae, and landscape elements that may act as refuges for them [24], in IPM for aphid pests of crops has increased [20].
Coccinellids are also attacked by a variety of natural enemies [25], although few of these are thought to signi cantly alter their population dynamics [26]. Coccinellids are attacked in all life stages with nearly 100 species of parasitoids recorded in the Hymenoptera and Diptera [25]. Few parasitoids of entomophagous coccinellids are specialists, some are widely distributed and attack within a subfamily or tribe. One of the best understood parasitoids is Dinocampus coccinellae Schrank (Hymenoptera: Braconidae), whose host preference, life history, ecology, and behavior have been thoroughly investigated [27,28]. Adult coccinellids were preferred hosts, especially older females [29,30], but immature parasitoids have been found in host larvae and pupae [31]. As a solitary endoparasitoid of adults, D. coccinellae prefers larger species of Coccinellinae [25]. Coccinellids vary in their suitability as hosts for D. coccinellae [32], but the mechanisms that in uence realized rates of parasitism of various species are unknown. Comparing primary and secondary parasitism rates suggests that at least some coccinellids are able to overcome parasitoid larvae [33]. Parasitization of coccinellids can lead to a considerable decrease in their populations prior to hibernation, possibly reducing the future effect of these predators against spring outbreaks of aphids [34]. Most reports of coccinellid parasitoids are simple host records with no data on the impact of these natural enemies on population dynamics of their hosts [35].
Here we examine the trophic interactions between various species of prey aphids, their ladybeetle predators and the parasitoids of the lady beetles in managed and natural plant systems. The objectives of this study were to determine the diversity of ladybeetle species in managed and natural plant systems, evaluate coccinellid development and survival on the various aphid-host plant combinations and to record parasitoids of the immature coccinellids in larval and pupal stages. This work will help lay the basis for better management of agricultural systems to improve management of aphid pests.

Insect collection and rearing
Larvae of coccinellid species feeding upon aphids on various plants were collected from the eld and reared in the laboratory on their respective plant-aphid combination. We sampled from two types of plant systems, a disturbed agroecosystem, and an undisturbed or natural plant system in various years (Table 1). Experiments and observations on collected material were conducted under laboratory conditions at the Department of Entomology, Sindh Agriculture University Tandojam, Pakistan. Each week ladybeetle larvae were collected from each host plant-aphid combination growing on the University Campus grounds and nearby experimental elds. The beetle larvae collected from different plant-aphid combinations were divided at random into four groups (designated as replicates) and reared on their respective host plants and aphids in plastic jars (30 × 20 cm) until adult beetle emergence or emergence of parasitoids from larvae or pupae. Collection and rearing started with the appearance of aphids and their coccinellid predators in the eld. Sampling and collection of larvae continued until crops matured and were harvested, or the disappearance of aphids andt their coccinellid predators from plants due to high temperatures at the onset of summer. The six host plants and their associated aphid species (Table 1) included four plants grown as cultivated crops and two non-crop plants: calotropis is a widespread weed growing on uncultivated lands and channel embankments and oleander is garden and roadside ornamental. Menochilus sexmaculatus F. and Brumus suturalis F.
The coccinellid larvae were provided with fresh food daily until they pupated. Final instar fully-grown larvae and pupae were kept individually in labelled plastic vials (70 × 30 mm) and the check twice daily to determine when parasitoids emerged or adult ladybeetles eclosed. After emergence, the parasitoids were preserved in 70% alcohol, labeled and sent to CABI Bioscience, Central and West Asia, Rawalpindi, Pakistan for identi cation. Weights of coccinellid pupae and adult beetles were recorded on a balance ((Ohaus®, Galexy 160) one day after pupation and eclsion respectively. Coccinellids could only be identi ed to species with certainty after adult eclosion, so records of parasitoids from immatures could only be associated with host plant and aphid species.

Results
Coccinellid species associated with different host-aphid plant combination   on rocket. The relationship between pupal weight and number of parasitoids per coccinellid pupa was signi cant in most cases and larger pupae typically supported more parasitoids than smaller pupae (Table 4). In 2010, a single Dinocampus coccinellae Schrank (Hymenoptera: Braconidae) was reared from a coccinellid collected on sa ower.

Discussion
Greater numbers of larval and pupal parasitoids emerged from coccinellid larvae and pupae collected from crop plant hosts than non-crop plant hosts. The higher number of larval and pupal parasitoids emerging from a single larva and pupa, and higher pupal weight supporting more parasitoids from crop plants compared with non-crop plants, may have a negative impact on development of coccinellid populations in crop ecosystems and the biological control of insect pests. Coccinellids are important natural enemies of phytophagous insects in natural and managed ecosystems [42] and play a signi cant role in biological control of pest insects in agroecosystem [12,22]. There was a signi cant relationship between the number of parasitoids emerging per pupa and pupal weight of hosts. Higher resource availability for a developing parasitoid is assumed in larger hosts [35,36]. Better parasitoid survival, larger body size, increased longevity and higher fecundity rates are expected with greater resource availability than for parasitoids emerging from low quality hosts [37]. The increased parasitization of predatory coccinellids may have negative impact on IPM of phytophagous insects in agriculture settings.
Host plant in uences the nutritional quality of aphid prey for predatory coccinellids [38,42]. The positive (nutritive) and negative (toxic) trophic effects are manifested in the biological performance of predators.
In the present study signi cant variation was found in pupal and adult weight of coccinellids feeding on different host plant-aphid prey combinations. [39] reported that the adult weight of Adalia bipunctata L. depended on the host plant of the prey and adults of A. bipunctata fed with aphids from S. alba. and B. napus were larger than those fed with M. persicae reared on V. fabia. [43] found signi cant host plantaphid effects for adult mass at emergence. The suitability of each aphid species was improved by rearing on its host plant of origin and diminished by rearing on alternative host plants. [44], observed that prey species signi cantly affected adult weights of Hippodamia convergens Guérin-Méneville, surviving adults weighed less when provided with Brassica-specialist aphids, L. erysimi or Brevicoryne brassicae (L.), than when fed the generalist M. persicae. The specialist aphids sequestered high levels of indole glucosinolates from their host plants that were toxic to aphid predators.
Several species of hymenopteran parasitoids parasitize different life stages of coccinellid beetles in various regions [25]. The most important among them are D. cocinellae, several species of Homalotylus and O. scaposus [45]. In the present study, three parasitoid species were recorded on coccinellid beetles, two gregarious species (H. aminius on larvae (10-36% parasitism rate) and O. scaposus (17-42% parasitism rate) and a solitary species (D. coccinellae) which was very rare (a single individual recorded from a coccinellid on sa ower). Dinocampus coccinellae is typically found in cooler regions [46,47] and is not prevalent in the comparatively hot climatic conditions of present study area.
Herbivorous insects are conventionally grouped into categories based on their degree of dietary specialization. Specialist herbivores have physiological adaptations to cope with the plant defenses, which allow greater tolerance of secondary plant substances produced by plants to defend against herbivory than most generalists [48]. There are natural enemies which are adapted to consuming toxic prey. Feeding by herbivores induces volatile organic compound (semiochemicals) emissions from host plants which help natural enemies locate and recognize their host or prey [49]. [50] investigated the electrophysiological and behavioral responses of the ladybird parasitoid Dincampus coccinellae to the alkaloid precoccinelline of C. septempunctata, which is thought to provide a toxic defence for the ladybird.
Brassica plants are important vegetable and oil seed crops of agroecosystem and produce glucosinolates as defense against herbivory. The phytophagous insects and their predators have evolved mechanisms to cope with these compounds. The brassica specialist, B. brassicae sequesters glucosinolates as a defense against predators. Glucosinolates have been shown to affect performance of coccinellid predators in a concentration (dose)-dependent manner [51] and in this study adult weight of C. septempunctata and C. undecimpunctata was signi cantly lower when these predators fed on L. erysimi collected from rocket rather than mustard.
The toxicity of milkweeds comes from bitter-tasting steroids glycosides (cardenolides) that have toxic effects on most animals. Cardenolides bind to the Na, K-ATPase pump making them a vertebrate toxin that functions in a dose-dependent manner and their production is known to be induced following damage by several species of herbivores [52,53]. Aphis nerii feed in the cardenolide-rich internal phloem of host bicollateral vascular bundles [54], and these chemicals are present in A. nerii honeydew and body tissue [55,56]. This aphid is found wherever milkweeds and oleander occur and it is known to sequester cardenolides, and use them as a chemical defense against a range of non-adapted natural enemies [57,60]. Cardenolides are toxic to many coccinellids, including C. septempunctata [12] and Adonia variegata [58]. Aphis nerii is not toxic to M. sexmaculata [45]. In our study adult weight of M. sexmaculata feeding of A. nerii was signi cantly higher from oleander and calotropis as host plants compared with other aphid-host plant combination. [61] studied host preference of M. sexmaculata on ve aphid species and found it developed best on A. nerii; see also [62]. Some generalist natural enemies of cardenolidecontaining insects are deterred by the compounds [63]. In the present study, coccinellid larvae consuming A. nerii as a prey supported the lowest number of H. aminus adults per larva compared with other prey species feeding on non-toxic host plants. Pupal weight of coccinellids was signi cantly less for coccinellids reared from calotropis and oleander compared with other host plants. Similarly, the number of adult parasitoids emerging from single coccinellid pupa was lowest when feeding on A. nerii on calotropis and oleander. Similarly, for parasitoids emerging from coccinellids pupae feeding on A. nerii compared with other host plants. This suggests that cardenolides negatively affected the fourth trophic level. C. septempunctata and C. undecimpunctata were not collected from calotropis and oleander plants indicating that these coccinellids are not adapted to feed on A. nerii and this aphid is toxic to them [64].

Conclusion
Coccinellids are important natural enemies of aphids, scale insects and other soft bodied insects infesting natural and managed plant systems. Coccinellid predators frequently move between crop and non-crop habitats that act as reservoirs, and provide biological control of aphid pests in agriculture habitats. Growing owering plants around border rows of crops will encourage and attract coccinellid predators to colonize and to multiply and control aphid pests in crop habitats. The suppression of aphid populations by coccinellid predators provides environmental and economic bene ts and may reduce yield loss without the negative environmental impacts of toxic pesticide use, but as we have found not all coccinellids feed equally as well on all aphids -host plant combinations. Designing better agricultural landscapes needs to take this into consideration.  Interactions between host plants, aphid species and coccinellids under natural and managed plant systems. The arrows are proportional to how often different coccinellids are reared from each aphid-plant system.