Mitochondrial DNA phylogeography of the Cotesia flavipes complex of parasitic wasps (Hymenoptera: Braconidae)

The Cotesia flavipes species complex of parasitic wasps are economically important worldwide for the biological control of lepidopteran stem borers. The complex currently comprises three species: Cotesia flavipes Cameron, C. sesamiae (Cameron) and C. chilonis (Matsumura) (Hymenoptera: Braconidae), which appear morphologically similar. Despite their economic importance, little is known about the genetic diversity and phylogeography of these parasitoids. Differences in the biology of geographic populations have generally been interpreted as genetic divergence among strains, but direct genetic evidence is lacking. In Australia, several stem borer pests in neighbouring countries have been identified as significant threats to the sugar industry. However, the status of C. flavipes in Australia is unknown. To examine the genetic variation among worldwide populations of the C. flavipes complex and investigate the status of the Australian C. flavipes-like species, partial sequence data were generated for mitochondrial gene regions, 16S rRNA and COI. Parsimony, minimum evolution and Bayesian analyses based on 21 geographic populations of the complex and four outgroups supported the monophyly of the complex and the existence of genetically divergent populations of C. flavipes and C. sesamiae. The geographically isolated Australian haplotypes formed a distinct lineage within the complex and were ~3.0% divergent from the other species. The results indicated that historical biogeographic barriers and recent biological control introductions play an important role in structuring lineages within these species. This study provides a phylogeographical context for examining adaptive evolution and host range within biologically divergent strains of the C. flavipes complex.     

Sensilla on antennae,ovipositor and tarsi of the larval parasitoids,Cotesia sesamiae (Cameron 1906) and Cotesia flavipes Cameron 1891 (Hymenoptera: Braconidae): a comparative scanning electron microscopy study

Two braconid parasitoids of cereal stemborers in eastern Africa, Cotesia sesamiae and Cotesia flavipes, have been shown to display a similar hierarchy of behavioural events during host recognition and acceptance. In order to understand the mechanisms underlying host recognition and acceptance, the morphology of antennal sensilla on the last antennomeres, on the ovipositor, and on the fifth tarsomere and pretarsus of the prothoracic legs tarsi were studied using scanning electron microscopy followed by selective silver nitrate staining. It appeared that female C. sesamiae and C. flavipes shared the same types and distribution of sensory receptors, which enable them to detect volatiles and contact chemical stimuli from their hosts. In both parasitoids, four types of sensilla were identified on the three terminal antennomeres: (i) non-porous sensilla trichodea likely to be involved in mechanoreception, (ii) uniporous sensilla chaetica with porous tips that have gustatory functions, (iii) multiporous sensilla placodea, which are likely to have olfactory function, and (iv) sensilla coeloconica known to have thermo-hygroreceptive function. The tarsi of both parasitoids possessed a few uniporous sensilla chaetica with porous tips, which may have gustatory functions. The distal end of the ovipositor bore numerous dome-shaped sensilla. However, there were no sensilla coeloconica or styloconica, known to have gustatory function in other parasitoid species, on the ovipositors of the two braconid wasps.     

Calyx fluid proteins of two Cotesia sesamiae (Cameron) (Hymenoptera: Braconidae) biotypes in Kenya: implications to biological control of the stem borer Busseola fusca (Fuller) (Lepidoptera: Noctuidae)

Cotesia sesamiae (Cameron) (Hymenoptera: Braconidae) is an indigenous larval endoparasitoid of Busseola fusca (Fuller) (Lepidoptera: Noctuidae) in sub-Saharan Africa. In Kenya, reports suggest that C. sesamiae occurs as two biotypes. Biotype avirulent to B. fusca gets encapsulated by haemocytes in this host and is unable to complete development. Biotype virulent to B. fusca is able to overcome immune defences. Factors present in the calyx fluid such as the PolyDNAviruses (PDV), venom and calyx fluid proteins have been implicated in the variation of C. sesamiae virulence against B. fusca. In the present study, calyx fluid proteins of the two C. sesamiae biotypes were compared using 2-D gel electrophoresis. More protein spots were observed in the virulent parasitoid calyx fluid, but some proteins were specifically observed in the avirulent parasitoid calyx fluid while others were observed in both. To study changes in proteins due to parasitism of B. fusca larvae by the two strains, SDS-PAGE gel were performed on fat body tissues and the haemolymph at three time points. Differences between the two strains were observed in both the fat body and haemolymph tissues. Parasitism-specific protein bands were detectable in fat body tissues of B. fusca larvae parasitized by the two C. sesamiae strains. These proteins were absent in unparasitized larvae. Implications for using C. sesamiae as a biocontrol agent of B. fusca in Africa are discussed.     

Importance of contact chemical cues in host recognition and acceptance by the braconid larval endoparasitoids Cotesia sesamiae and Cotesia flavipes

The ability of the congeneric braconid parasitoids Cotesia sesamiae (Cameron) and Cotesia flavipes Cameron to discriminate between stemborer larval cues upon contact was studied using their natural hosts, namely the noctuid Busseola fusca (Fuller) and the crambid Chilo partellus (Swinhoe), respectively, and the pyralid non-host Eldana saccharina (Walker). When the natural host larvae were washed in distilled water, parasitoid behavior was similar to that displayed when in contact with E. saccharina, characterized by the absence of ovipositor insertion. When washed host or non-host larvae were bathed with water extracts of their natural host, the parasitoids showed a significant increase in ovipositor insertions. However, the water extracts of host-larvae deposited on cotton wool balls did not induce ovipositor insertion in either C. sesamiae or C. flavipes. Nevertheless, the extracts enabled the parasitoids to discriminate between natural and non-hosts as indicated by the intensive antennating of the former. For both parasitoids, frass was found to be important in short-range host recognition as indicated by differences in the time spent on antennating between frass sources. In addition, the regurgitants of B. fusca and C. partellus induced ovipositor insertion in C. flavipes only. These results indicated that C. sesamiae and C. flavipes used different chemical cues for acceptation and oviposition in a stemborer larva, and that B. fusca and C. partellus shared the same chemical cues to induce oviposition in C. flavipes.     

Geographic variation in male courtship acoustics and genetic divergence of populations of the Cotesia flavipes species complex

Courtship behaviors of insect populations can vary across the range of a species. Populations exhibiting divergent courtship behavior may indicate genetic divergence or cryptic species. Courtship acoustic signals produced by male wing fanning and genetic structure (using amplified fragment length polymorphisms) were examined for seven allopatric populations of the Cotesia flavipes (Hymenoptera: Braconidae) species complex, using four C. sesamiae (Cameron) and three C. flavipes Cameron populations. Members of this species complex parasitize lepidopteran pests in gramineous crops including sugarcane, maize, and rice . Significant variation was detected in courtship acoustic signals and genetic structure among populations of both species. For C. sesamiae, courtship acoustic signals varied more between populations of two biotypes that were collected near an area of sympatry. The two biotypes of C. sesamiae were also genetically divergent. For C. flavipes, significant differences in acoustic signals and genetic structure occurred among allopatric populations; these differences support the recent designation of one population as a new species. Courtship acoustics play a role in reproductive isolation in this species complex, and are likely used in conjunction with chemical signals. Ecological factors such as host range and host plant use may also influence the divergence of both courtship acoustic signals and genetic structure among populations in the C. flavipes complex.     

Release and establishment of Cotesia flavipes Cameron (Hymenoptera: Braconidae) an exotic parasitoid of Chilo partellus (Swinhoe) (Lepidoptera: Crambidae) in East and Southern Africa

Cotesia flavipes Cameron (Hymenoptera: Braconidae) was first imported into Kenya in 1991 from Pakistan for control of Chilo partellus (Swinhoe) (Lepidoptera: Crambidae). First releases were made at the Kenya coast in 1993 and a few recoveries of the parasitoid were made the following year. Additional foreign exploration for C. flavipes was conducted in central India in 1996 and 1998, which resulted in additional importation of the parasitoid for subsequent releases in eastern and southern Africa. Region-wide releases commenced with releases in Mozambique in 1996, Somalia in 1997 and Uganda in 1998. By 2005, many releases had been made in 10 countries in East and Southern Africa with establishment being reported in all of them except Eritrea but including Ethiopia where releases had never been made. This paper describes the progress made in the release and establishment of C. flavipes in East and Southern Africa and quantifies the rate of spread from the initial release sites in Kenya.     

Dispersal of the exotic parasitoid Cotesia flavipes in a new ecosystem

A study on the dispersal of the exotic larval endoparasitoid, Cotesia flavipes Cameron (Hymenoptera: Braconidae), was conducted in a maize field in the northern Kilifi District in the coastal area of Kenya. Because C. flavipes did not previously occur in the release area, it was possible to use a unique indirect method to estimate dispersal by examining the distribution of parasitised hosts. Parasitoids released in the centre of the field moved as far as 64 meters during their life span, and dispersal was dependent on wind direction. The level of parasitism was influenced by the location of hosts in plants. The majority of parasitised stemborers (88.4%) were found inside the plant (stems and tassel stems), where 74.3% of the suitable hosts were found, which indicates that female parasitoids were not searching randomly for hosts. Aggregation of parasitoids in response to plants with different host densities was not detected. Implications of the release of C. flavipes on stemborers population in the agroecosystem of East Africa are discussed.     

Host Suitability of Four Cereal Stem Borers (Lepidoptera: Crambidae, Noctuidae) for Different Geographic Populations of Cotesia sesamiae (Cameron) (Hymenoptera: Braconidae) in Kenya

This study focused on the suitability of four species of cereal stem borers for the development of five geographic populations of Cotesia sesamiae (Cameron). C. sesamiae, an indigenous larval parasitoid of gramineous stem borers, is widespread in Africa. Four stem borers, Chilo partellus (Swinhoe), Chilo orichalcociliellus Strand (Lepidoptera: Crambidae), Busseola fusca Fuller, and Sesamia calamistis Hampson (Lepidoptera: Noctuidae), were offered to C. sesamiae for oviposition. Parasitoid individuals originated from five locations in Kenya. Biological parameters such as developmental time, percentage parasitism, progeny production, mortality of immature parasitoids, and proportion of female progeny were compared across host species. The two populations from western Kenya developed well on B. fusca. However, populations from the coast and the Eastern Province could not successfully parasitize B. fusca. With the exception of B. fusca, the percentage of hosts successfully parasitized by the different C. sesamiae populations was not different. The size of the host appeared to be an important factor influencing the development and reproductive potential of the parasitoid. We conclude that the different parasitoid populations were adapted to location-specific characteristics. Parasitoid–host compatibility must be evaluated before release for better establishment and colonization.     

Host–parasitoid community model and its potential application in biological control of cereal stemborers in Kenya

A two-host–two-parasitoid model was constructed to assess the effects of the introduced larval parasitoid, the braconid Cotesia flavipes, on its primary target host, the invasive crambid Chilo partellus, and on secondary host species, in inter-specific competition with Cotesia sesamiae, the main native parasitoid species of stemborers in Kenya. The model assumed that: (1) there was no host discrimination by either parasitoid species; (2) Cotesia flavipes was the superior competitor that out-competed Cotesia sesamiae when the host was suitable; and (3) Cotesia flavipes could only develop in an unsuitable host if it had been previously parasitized by Cotesia sesamiae. Model parameters were estimated from surveys conducted in Kenya and from laboratory experiments. Different scenarios of host and parasitoid species composition and host suitability occurring in the different ecological zones in Kenya were analyzed. Results indicated that: (1) the coexistence of stemborer host populations are determined by their population growth rates, the degree of aggregation of the parasitoids and their searching efficiency; (2) in the regions where both the invasive and the predominant native host species were suitable to either parasitoid species, stemborer densities would be reduced to and controlled at low densities, and Cotesia flavipes would become the dominant parasitoid species. However, the extinction or predominance of the native stemborer species depends on the ratio of the growth rates of exotic and native stemborers and their relative searching efficiencies; and (3) if the native host species was acceptable but unsuitable to Cotesia flavipes, the parasite would not become established.     

Influence of Adult Size on Mate Choice in the Solitary and Gregarious Parasitoids, Cotesia marginiventris and Cotesia flavipes

Female and male mate choice in relation to adult size were examined for the solitary and gregarious parasitoids, Cotesia marginiventris (Cresson) and Cotesia flavipes Cameron, respectively. In addition, male precopulatory behaviors were observed for evidence of male competition or a large-male advantage in mate acquisition. Male parasitoids are not known to offer female mates direct benefits, but females that mate high quality males may obtain indirect benefits, such as offspring that are more successful in obtaining mates. Female choice experiments for C. marginiventris found that large males approached females first more frequently than small males, and that females mated large males significantly more often than small males. Male choice experiments for C. marginiventris did not demonstrate a male preference for female size. In contrast, female choice experiments with C. flavipes found that females mated equally with large or small males, while male choice experiments showed that males attempted copulation and mated more frequently with smaller females. Male competition was not observed in the gregarious species C. flavipes, but competition in this gregarious parasitoid could be moderated by dispersal.     

Assessment of the impact of natural enemies on stemborer infestations and yield loss in maize using selected insecticides in Mozambique

The effect of natural enemies on stemborer infestations and maize grain yields was estimated using an insecticide exclusion method. Field experiments were conducted at low, mid and high elevation zones, which vary in the stemborer species composition. Dimethoate was applied to exclude natural enemies and Cypermethrin to suppress stemborers, while other plots served as control. At all study sites more stemborer larvae and pupae were collected when natural enemies were excluded. Parasitism as well as maize grain weight in the unprotected plots were significantly higher than in the exclusion plots. Yield losses increased by 28.9 % in unprotected to 43.3 % in exclusion plots. The most abundant parasitoids of Chilo partellus (Swinhoe) (Lepidoptera: Crambidae) were Cotesia sesamiae (Cameron), Cotesia flavipes Cameron (Hymenoptera: Braconidae) and Dentichasmias busseolae Heinrich (Hymenoptera: Ichneumonidae). While for Busseola fusca (Fuller) (Lepidoptera: Noctuidae) they were C. sesamiae, Sturmiopsis parasitica (Curran) (Diptera: Tachinidae) and Porcerochasmias nigromaculatus Heinrich (Hymenoptera: Ichneumonidae). It was concluded that exclusion of natural enemies caused an increase in stemborer populations, thus, the parasitoids play an important role in suppressing stemborer infestations and increase maize yield.     

Host suitability of the New World stalkborer Diatraea considerata for three Old World Cotesia parasitoids

Biological control ofstalkboring Lepidoptera often has beensuccessful when the braconid parasitoids in thegenera Cotesia and Apanteles werethe natural enemies of choice. Constraints inusing these gregarious, koinobiont,endoparasitoids have included host suitability,especially as influenced by the host's immuneresponse. The suitability of a novel host, theNew World stalkborer Diatraeaconsiderata (Lepidoptera:Pyralidae), for parasitization by three OldWorld braconids, Cotesia chilonis, C. flavipes and C. sesamiae (Hymenoptera: Braconidae), wascompared to the suitability of another NewWorld novel host, Diatraea saccharalis. D. considerata was less suitable for allthree parasitoids than was D.saccharalis. The frequent occurrence ofparasitized D. considerata larvae thatdid not yield parasitoids, or pupate within anappropriate time interval, suggestedencapsulation of the parasitoid progeny, whichwas visible through the host cuticle. Giventhe suitability results, these threeparasitoids would not be appropriate candidatesfor use against D. considerata. Theresults also have important implications forthe narrow host range expressed by theseparasitoids.     

The temporal correlation and spatial synchrony in the stemborer and parasitoid system of Coast Kenya with climate effects

The spatial synchrony and the temporal auto-correlationship of the exotic stemborer Chilo partellus (Swinhoe) (Lepidoptera: Crambidae) and the indigenous Sesamia calamistis Hampson (Lepidoptera: Noctuidae) and Chilo orichalcociliellus (Strand) (Lepidoptera: Crambidae), and crosscorrelationship with their indigenous and introduced larval parasitoids Cotesia sesamiae (Cameron) and Cotesia flavipes Cameron (Hymenoptera: Braconidae) was studied using 3-year data collected in coastal Kenya. An autoregressive model was used to study the effect of climatic stochasticity or population density-dependent factors on stemborer populations. It appeared that rainfall did have a direct impact on stemborers in the south coast and an indirect one in the north coast. Spatial nonparametric correlation functions (SNCF) and cross-correlation functions (SNCCF) were applied for spatial synchrony analysis. The regional synchrony of Ch. partellus and S. calamistis decreased and that of Ch. orichalcociliellus increased after the introduction of Co. flavipes. The positive crosscorrelation coefficient between stemborers and parasitoids suggests a synchrony between the pest and its natural enemy.     

Quality control of Cotesia flavipes (Cameron) (Hymenoptera: Braconidae) from different Brazilian bio-factories

The goal of this study was to evaluate the quality of Cotesia flavipes from different bio-factories as biological control agents. We evaluated biological characteristics of the parasitoids throughout their lifespan, and measured the body length and width, abdomen width, thorax width and width and length of the right forewing of female and male parasitoids. Our results showed that the number of males and pupal viability were similar among the bio-factories; the number of emerged females was greater in bio-factories I and II; the egg-pupa period and the pupal period were shorter in bio-factory IV; and a greater longevity was found in bio-factories II and III. Sex ratio (at approximately 60% females) was satisfactory (in terms of suitability for release) across all bio-factories. For morphometric measurements, the body, abdomen and wing widths were similar in males; however, thorax width was greater in the males from bio-factory I; bio-factory III produced females with the highest body length; bio-factory I produced females with the greatest abdomen width; bio-factories III and IV produced females with the greatest wing length. Among the bio-factories studied, bio-factory IV produced the best quality C. flavipes, with respect to the greatest number of parasitoids per pupal mass (a mean of 57% more parasitoids) in a satisfactory sex ratio, and with the shortest developmental time, which facilitates faster rearing in the laboratory. Studies such as this, which assess the quality of a mass-produced C. flavipes, are crucial for the continued use of this parasitoid in controlling Diatraea saccharalis in the field.     

Encapsulation and hemocyte numbers in three lepidopteran stemborers parasitized by Cotesia flavipes-complex endoparasitoids

Determination of the potential and actual host range of a natural enemy is crucial before its importation and release for biological control. We studied some of the factors that are important in determining the physiological host range of insect parasitoids attacking lepidopteran hosts. Our experimental system consisted of novel host-parasitoid associations, with two New World pyralid stalk borers, Diatraea saccharalis and D. grandiosella; one Old World crambid borer, Ostrinia nubilalis as hosts; and three Old World microgastrine braconids, Cotesia chilonis, C. sesamiae, and C. flavipes as parasitoids. Experiments on the chronology of encapsulation of the parasitoid progeny by host hemocytes indicated that lepidopteran stemborers that are taxonomically, behaviorally and ecologically very similar differ in their ability to encapsulate a parasitoid species. D. saccharalis encapsulated C. flavipes sometimes, whereas D. grandiosella consistently encapsulated C. sesamiae and C. flavipes. C. chilonis was not encapsulated by either Diatraea host. If encapsulation occurred it did not start until four days after parasitization and continued during the following days. O. nubilalis was an unsuitable host for all three parasitoid species; parasitoid eggs were killed within 24 hours of parasitization. O. nubilalis had nearly twice as many hemocytes present in the hemolymph compared to the Diatraea species. In many of the host-parasitoid combinations, there was an initial increase of hemocyte number soon after parasitization, which was not due to mechanical damage at oviposition. There was no correlation between total numbers of hemocytes present in the host hemolymph and the observed encapsulation levels. By understanding the encapsulation response we may be able to make better predictions about the host range of a parasitoid species before its release as a biological control agent.     

Species diversity of lepidopteran stem borer parasitoids in cultivated and natural habitats in Kenya

Field surveys were conducted during 2005 to 2007 to assess the species diversity of stem borer parasitoids in cultivated and natural habitats in four agroecological zones in Kenya. In total, 33 parasitoid species were recovered, of which 18 parasitized six stem borer species feeding on cereal crops, while 27 parasitized 21 stem borer species feeding on 19 wild host plant species. The most common parasitoids in cultivated habitats were Cotesia flavipes Cameron, Cotesia sesamiae (Cameron), Pediobius furvus Gahan and the tachinid Siphona sp., whereas in natural habitats, Siphona sp. was the most common. The majority of parasitoids were stenophagous species; only five species –Cotesia sp., Enicospilus ruscus Gauld and Mitchell, Pristomerus nr. bullis, Sturmiopsis parasitica (Curran) and Syzeuctus ruberrimus Benoit – were monophagous. In both cultivated and natural habitats, parasitoid species diversity was highest on the most dominant stem borers Busseola spp. and Chilo spp. On cereal crops, parasitoid diversity was highest on maize and among wild host plants, it was highest on Setaria spp. The ingress‐and‐sting attack method was the most common strategy used by parasitoids in both habitats. In all agroecological zones, parasitoid species diversity was significantly higher in natural than in cultivated habitats. Furthermore, the majority of parasitoid species were common to both cultivated and natural habitats. It was concluded that natural habitats surrounding cereal crops serve as refugia for sustaining the diversity of stem borer parasitoids from adjacent cereal fields.     

Multiple Parasitism by Cotesia sesamiae and Cotesia flavipes (Hymenoptera: Braconidae) on Busseola fusca (Lepidoptera: Noctuidae)

Busseola fusca (Fuller) is one of the most important pest of cereals in sub-Saharan Africa. Cotesia sesamiae (Cameron) is the predominant parasitoid attacking B. fusca larvae in many parts of Africa. An exotic parasitoid, Cotesia flavipes Cameron, was introduced into Kenya in 1993 for the control of Chilo partellus (Swinhoe). Laboratory studies indicated that although C. flavipes would search for, and attack B. fusca , it was not able to complete its development in this host. The aim of the present study was to investigate the outcome of multiple parasitism of B. fusca by the two Cotesia species. The study showed that when both parasitoid species stung a B. fusca larva at the same time, both parasitoids emerged from more than half of the host larvae, C. flavipes alone emerged from 17%, and C. sesamiae alone emerged from 9%. When the larvae were parasitized by C. sesamiae first, and then 2 h later by C. flavipes , and vice versa, most of the progeny were C. flavipes . However, when B. fusca larvae were stung by C. sesamiae three days before oviposition by C. flavipes , significantly more C. sesamiae emerged from the larvae. When C. flavipes oviposited first, no larvae produced C. flavipes only. The interaction of parasitoids and the host immune system, and the implications of these results for the biological control of stem borers in East Africa are discussed.     

A review of sugarcane stem borers and their natural enemies in Asia and Indian Ocean Islands: an Australian perspective

This paper provides a review on lepidopteran stem borer pests of graminaceous crops in Asia and Indian Ocean Islands which have the potential to invade Australia. Information on the geographical distribution, host plants and potential of invading Australia is provided for 36 stem borer species. A literature review of all natural enemies of 18 key pest species is provided. A knowledge of possible biological control options is essential to determine which natural enemies are to be considered for introduction following an incursion. The Braconid, Cotesia flavipes Cameron (Hymenoptera: Braconidae), stands out as a promising candidate for introduction into Australia following a borer incursion. Studies are currently being conducted on a native Cotesia species in Australia, which may be able to parasitize larvae of exotic borers, therefore minimizing the need for other parasitoids introductions.     

Foraging behavior and life history of the stemborer parasitoidCotesia flavipes (hymenoptera: Braconidae)

The gregarious parasitoidCotesia flavipes (Hymenoptera: Braconidae) attacks larvae of pyralid and noctuid stemborers by entering the stemborer tunnel. The short-range foraging behavior of femaleC. flavipes was studied on stemborerinfested plants, in patches with host-related products and in artificial transparent tunnels. In addition, the longevity under specific conditions and the potential and realized fecundity of femaleC. flavipes were determined. Larval frass, caterpillar regurgitate, and holes in the stem are used in host location byC. flavipes. The response to host products byC. flavipes seems not to be host species specific. FemaleC. flavipes respond to frass from four stemborer species and one leaf feeder. No differences are found in the behavior ofC. flavipes on maize plants infested with the suitable host,Chilo partellus (Lepidoptera: Pyralidae), or the unsuitable host,Busseola fusca (Lepidoptera: Noctuidae). Attacking a stemborer larva inside the stem is risky for the parasitoid. The mortality rate of the parasitoids inside the stem is high: 30–40% of the parasitoids are killed by the spitting and biting stemborer larva.C. flavipes is relatively shortlived: without food the parasitoids die within 2 days; with food and under high-humidity conditions they die within 5–6 days.C. flavipes is proovigenic and has about 150 eggs available for oviposition. A relatively large proportion of the available egg load (20–25%) is allocated to each host, so femaleC. flavipes are egg depleted after parasitizing only five or six hosts.     

Evidences of non-additive effects of multiple parasitoids on Diatraea saccharalis Fabr. (Lep., Crambidae) populations in sugarcane fields in Brazil

Biological control is a relatively benign method of pest control. However, considerable debate exists over whether multiple natural enemies often interact to produce additive or non‐additive effects on their prey or host populations. Based on the large data set stored in the São João and Barra sugarcane mills (state of São Paulo, Brazil) regarding the programme of biological control of Diatraea saccharalis using the parasitoids Cotesia flavipes and tachinid flies, in the present study the author investigated whether the parasitoids released into sugarcane fields interfered significantly with the rate of parasitized D. saccharalis hosts. The author also observed whether there was an additive effect of releasing C. flavipes and tachinids on the rate of parasitized hosts, and looked for evidence of possible negative effects of the use of multiple parasitoid species in this biological control programme. Results showed that C. flavipes and the tachinids were concomitantly released in the Barra Mill, but not in the São Jão Mill. Furthermore, in the Barra Mill there was evidence that the parasitoids interacted because the percentage of parasitism did not increase after the release of either C. flavipes or tachinids. In the São João Mill, when both parasitoid species were released out of synchrony, both the percentage of parasitism by C. flavipes as well as that of the tachinids increased. When large numbers of tachinids were released in the Barra Mill, they caused a significant lower percentage of parasitism imposed by C. flavipes. The implications of the results as evidence of non‐additive effects of C. flavipes plus tachinids on D. saccharalis populations are discussed.