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Research Article
Systematics and biology of Cotesia typhae sp. n. (Hymenoptera, Braconidae, Microgastrinae), a potential biological control agent against the noctuid Mediterranean corn borer, Sesamia nonagrioides
expand article infoLaure Kaiser, Jose Fernandez-Triana§, Claire Capdevielle-Dulac, Célina Chantre, Matthieu Bodet, Ferial Kaoula, Romain Benoist, Paul-André Calatayud|, Stéphane Dupas, Elisabeth A. Herniou, Rémi Jeannette, Julius Obonyo|, Jean-François Silvain, Bruno Le Ru|
‡ Université Paris-Saclay, Paris, France
§ Canadian National Collection of Insects, Ottawa, Canada
| African Insect Science for Food and Health, Nairobi, Kenya
¶ Université François-Rabelais de Tours, Tours, France
Open Access

Abstract

Many parasitoid species are subjected to strong selective pressures from their host, and their adaptive response may result in the formation of genetically differentiated populations, called host races. When environmental factors and reproduction traits prevent gene flow, host races become distinct species. Such a process has recently been documented within the Cotesia flavipes species complex, all of which are larval parasitoids of moth species whose larvae are stem borers of Poales. A previous study on the African species C. sesamiae, incorporating molecular, ecological and biological data on various samples, showed that a particular population could be considered as a distinct species, because it was specialized at both host (Sesamia nonagrioides) and plant (Typha domingensis) levels, and reproductively isolated from other C. sesamiae. Due to its potential for the biological control of S. nonagrioides, a serious corn pest in Mediterranean countries and even in Iran, we describe here Cotesia typhae Fernandez-Triana sp. n. The new species is characterized on the basis of morphological, molecular, ecological and geographical data, which proved to be useful for future collection and rapid identification of the species within the species complex. Fecundity traits and parasitism success on African and European S. nonagrioides populations, estimated by laboratory studies, are also included.

Keywords

Cotesia, Sesamia, biological control, species complex, Africa, Mediterranean

Introduction

Although the concept of species is questioned in situations characterized by a continuum of genetic differentiation and reproductive isolation between populations (The Marie Curie Speciation Network 2012), well described and identified species are still useful tools in many situations. For instance, in biological control the use of such species, with a defined host range and showing no gene flow with closely related species, limits the risk of confusion and guarantees the stability of its host range. The purpose of this paper is to describe a new species of parasitoid wasp, first considered as a host race of Cotesia sesamiae Cameron (Hymenoptera, Braconidae) (Branca et al. 2011; Kaiser et al. 2015). It is a potential candidate for the biological control of the Mediterranean maize stem borer, Sesamia nonagrioides (Lefèbvre, 1827) (Lepidoptera, Noctuidae).

Cotesia is one of the most diverse genera of the subfamily Microgastrinae (Hymenoptera, Braconidae), with almost 300 species already described (Yu et al. 2016), and probably over 1,000 species worldwide (e.g., Mason 1981). Cotesia was originally considered as a genus by Cameron in the 19th century, and definitively split from the genus Apanteles by Mason (1981) in his generic reclassification of the Microgastrinae, wherein many Apanteles species were transferred to Cotesia. Microgastrine wasps are koinobiont endoparasitoids of lepidopteran larvae, and species attacking large larval hosts are often gregarious (Whitfield 1997, Quicke 2015). Females emit sex-pheromones that attract males and can mate upon emergence (Xu 2014). They locate their host at a distance and initiate oviposition upon recognition of chemical cues (Turlings and Fritzsche 1999, Jembere et al. 2003, Obonyo et al. 2010). The microgastrine wasps use a domesticated virus (called a bracovirus, Polydnaviridae) to inhibit the immune response of host larva. Bracoviruses are produced in the wasps’ ovaries by genes integrated in the wasp genome and injected in the host body together with the eggs (Asgari 2006, Gitau et al. 2007, Herniou et al. 2013). Within the host, the viral particles infect the host cells, which produce the viral proteins, which in turn inactivate the host immune cellular response and regulate the host metabolism to the benefit of wasp larvae (Herniou et al. 2013). Fully developed larvae egress from the host body and spin their cocoons to undergo metamorphosis. Host resistance processes can manifest at all these steps of the life cycle, among which encapsulation of the parasitoid eggs has been often reported and well described (Beckage 1998). Evolution of virulence mechanisms by the parasitoid may have driven the radiation of species within the genus Cotesia (Herniou et al. 2013).

The Cotesia flavipes species-group is a monophyletic complex made up of (until now) four allopatric sister species, all gregarious endoparasitoids of a few families of lepidopteran stem borers (Crambidae, Pyralidae, and Noctuidae) in monocot Poales (Poaceae, Typhaceae and Cyperaceae). The species-group comprises Cotesia chilonis (Munakata, 1912) from eastern Asia, including China, Japan and Indonesia; Cotesia flavipes (Cameron, 1891), from the Indian sub-continent, but also released and established in east Africa and the New World for the purpose of biological control; Cotesia nonagriae (Olliff, 1893), an Australian endemic recently removed from synonymy with C. flavipes (Muirhead et al. 2008, 2012), and Cotesia sesamiae (Cameron, 1906), from sub-Saharan and Southern Africa (Kimani-Njogu and Overholt 1997). Members of this species complex are economically important worldwide as biocontrol agents of cereals and sugarcane stem borer pests (Kfir et al. 2002, Lou et al. 2014, Mindigoyi et al. 2016, Polaszek and Walker 1991, Simões et al. 2012), and their presence in their native settings help regulate populations of important pests (Kfir et al. 2002, Liu et al. 2000).

Additional, cryptic species have been suspected within this complex and several papers have explored this possibility, especially in regard to C. flavipes (e.g., Muirhead et al. 2012) and C. sesamiae. In the latter species, studies made from samples collected in maize fields on a few pest species showed that local adaptation to host resources and environmental factors were major drivers of intra-species genetic diversity (Dupas et al. 2008, Gitau et al. 2007, 2010). Subsequently, Branca et al. (2011) analysed a large sample of C. sesamiae covering most of the species’ distribution area and a large range of host and plant species. They provided evidence that variations of host range were associated with sequence variation of a virulence gene, CrV1, which could be used as a marker of host races. Analysis of microsatellite markers revealed gene flow between the host races, except for one population specialized on the noctuid Sesamia nonagrioides (Branca et al. in prep.). One method to get an insight into the evolutionary stability of host-parasitoid associations is to characterize phylogenetic relationships between so-called host races. Kaiser et al. (2015) performed a phylogenetic analyses of the C. sesamiae samples based on mitochondrial, viral and non-viral nuclear markers, and demonstrated that the samples from the S. nonagrioides host race formed a highly supported monophyletic lineage showing all the hallmarks of a cryptic species. The authors confirmed the species status of this lineage by showing that it was reproductively isolated from the other lineages of C. sesamiae and from C. flavipes. Furthermore they showed that it was the only lineage being virulent against S. nonagrioides, and specifically so. Combined evidence for ecological specialization, selection for divergent host adaptation and for reproductive isolation, allowed them to conclude that this lineage was formed by ecological (adaptive) speciation. In addition, some morphological differences were readily identifiable.

Based on a wealth of information – morphological, molecular, biological, and ecological – we describe this new species of Cotesia from Africa, the fifth member of the flavipes complex, and present the first data showing that it is a successful parasitoid of European populations of S. nonagrioides, a major maize pest in West Africa and in Mediterranean countries.

Materials and methods

Morphological description

We studied 175 specimens from six different countries, representing ten populations from four out of the five known species within the flavipes complex (Table 1). We could not examine specimens of Cotesia nonagriae, but this Australian species has recently been redescribed and illustrated (Muirhead et al. 2008, 2012).

Specimens studied for this paper. F- female specimen, M- male specimen.

Species Country of origin Collecting year # of Specimens Host caterpillar/host plant
C. flavipes Trinidad 1972 & 1980 4 F, 3 M Diatraea lineolata /unknown
C. flavipes Colombia 1978 2 F, 7 M Unknown/unknown
C. flavipes Barbados 1977 2 F Unknown/sugar cane
C. flavipes India 1954 3 F Unknown/unknown
C. flavipes Kenya 2010 25 F, 5 M Chilo partellus/maize
C. sesamiae Kenya (Mombasa) 2010 25 F, 5 M Sesamia calamistis /maize
C. sesamiae Kenya (Kitale) 2012 25 F, 5 M Busseola fusca/maize
C. chilo Japan 2008 2 F, 2 M Unknown/rice
C. typhae sp. n. Kenya (Makindu) 2013 25 F, 5 M Sesamia nonagrioides/
Typha domingensis
C. typhae sp. n. Kenya (Kobodo) 2013 25 F, 5 M Sesamia nonagrioides/
Cyperus dives

We evaluated a number of morphological characters proposed in previous studies (Kimani-Njogu and Overholt 1997, Muirhead et al. 2008), and others characters are explored for the first time in this species complex. Morphological terms and measurements of structures are mostly those used by Mason (1981), Huber and Sharkey (1993), Whitfield (1997), Karlsson and Ronquist (2012), and Fernandez-Triana et al. (2014). All characters used in this paper are illustrated in Figs 18.

In the species description, body ratios and measurement values are presented for the holotype first, followed by the range within the species in parentheses.

Photos were taken with a Keyence VHX-1000 Digital Microscope, using a lens with a range of 10–130 ×. Multiple images were taken of a structure through the focal plane and then combined to produce a single in-focus image using the software associated with the Keyence System. Plates were prepared using Microsoft PowerPoint 2010.

Institution acronyms used:

CBGP Centre de Biologie pour la Gestion des Populations, Montpellier, France.

CNC Canadian National Collection of Insects, Ottawa, Canada.

Molecular characterization

In order to check the molecular-specific characterization of Cotesia typhae, we used the COI (cytochrome oxydase I) sequences from Kaiser et al. (2015) (listed in Appendix 1) to calculate the divergence between pairs of Cotesia species and populations. The divergence corresponds to the number of nucleotide differences divided by the total number of nucleotides. Since there are several samples for each species and population, the minimum and maximum divergence is given for all pairs.

Distribution, ecology and abundance

Knowing that the new Cotesia species was found exclusively on S. nonagrioides on two plant families, Typhaceae and Cyperaceae (Kaiser et al. 2015), its distribution, ecology and abundance are characterized here from a collection of S. nonagrioides larvae on these two plant families, collected in 13 countries in sub-Saharan Africa between 2004 and 2013. Sesamia nonagrioides larvae were sampled from wild plants on banks of streams or rivers and in swamps, the favorite habitat of this species, which is rarely recorded from maize in East Africa. Plants were carefully inspected for stem borer infestations. Symptoms of infestation included scarified leaves, dry leaves and shoots (dead hearts), frass or holes bored. Infested plants were cut and dissected in the field; larvae collected were reared on an artificial diet (Onyango and Ochieng-Odero 1994) until pupation or emergence of parasitoid larvae. Sesamia nonagrioides were identified at the adult stage by dissection of the genitalia. After emergence, adult Cotesia were stored in absolute ethanol and identified by genotyping CrV1 sequence.

Life history traits and parasitoid success in European host populations

Insect material

The C. typhae laboratory-reared strains were collected initially from Kenya localities (Kobodo: 0.68°S; 34.41°E or Luanda: 0.48°S; 34.30°E, depending on the availability of the strains). They were reared on a Kenyan S. nonagrioides strain (collected initially from Makindu: 2.28°S; 37.82°E), according to the method described by Overholt et al. (1994). Parasitoid success was tested on this Kenyan strain and on two S. nonagrioides European strains collected respectively in France (Longage, 43.37°N; 1.19°E) and Italy (Monterotondo scalo, 42.06°N; 12.60°E). The Kenyan and French strains were reared as described above. The Italian larvae were sent from the University of Perugia.

Longevity experiments

Clusters of cocoons were each placed in a 0.5L disposable plastic box with a 1.5 cm diameter opening clogged with a foam cork. One of the three following food sources was placed in the box to test their effects on longevity: honey droplets and a tap water-imbibed cotton ball; a cotton ball imbibed with a 2% saccharose solution or a 20% solution. These small cages were placed at 21°C, with internal relative humidity around 75%. Dead insects were counted every day for the 2% sugar solution and at least every two days for the two other food sources, from 24h following emergence.

Realized fecundity

One-day-old wasps were taken from the cages as above and allowed to oviposit in one host larva per day, for four days. Parasitized larvae were kept individually in Petri dishes (2 cm high) with approximately 10cm3 piece of diet, until emergence of the parasitoid larvae or pupation. The diet was replaced by a piece of toilet paper 12 days after parasitism to facilitate cocoon formation.

Parasitoid success

Four weeks after hatching, i.e. when reaching the 5th- 6th stadium, larvae were exposed each to one wasp, then kept fed with the diet, in the conditions described above, until emergence of the parasitoid larvae, or pupation. Recorded traits are specified in Table 8. Individual cocoon weight was calculated by dividing the weight of the cocoon cluster by the number of emerged adults and dead nymphs.

Data analyses

Kaplan Meyer tables from XLSTAT were used to estimate daily mortality and median longevity. The procedure included three tests of equality of the survival curves (Wilcoxon, Log-rank and Tarone-War) that gave identical P-values, so only Wilcoxon’s result is given in this study. Comparisons of traits of parasitoid success on the three host strains were performed with the R package. As some of the traits did not follow a normal distribution (Shapiro statistic) or did not fulfill homoscedasticity (Bartlett statistic), the Kruskal-Wallis statistic was used to compare the quantitative traits recorded for the three host strains, followed by the Dunn post-hoc multiple comparison test. Chi-square was used to compare the issue of parasitism. Sample sizes are given in Table 8. The percentage of females in the cluster was not included in the analyses when pupal mortality was equal to or exceeded 30%. This occurred for 11 clusters obtained from the French host strain and two clusters obtained from the Kenyan strain.

Results

Morphological study

Cotesia typhae Fernandez-Triana, sp. n.

Figs 1, 2

Holotype

Female (CBGP).

Type locality

Kenya, Makindu, 2.28°S, 37.82°E.

Holotype label details

Kenya, Makindu, xi.2010, ex Sesamia nonagrioides on Typha domingensis Pers. Voucher code: CNC634434. Other code on label: F78.

Paratypes

CBGP, Montferrier s/Lez, France; CNC, Canada; International Centre of Insect Physiology and Ecology, Nairobi, Kenya; Natural History Museum London, UK; Smithsonian National Museum of Natural History, Washington DC, USA. 24 female, 5 male specimens, same locality as holotype; 25 female, 5 male specimens from Kenya, Kobodo, 0.41°S, 34.25°E. iii.2013, ex Sesamia nonagrioides on Cyperus dives Delile.

Previous records

This species has been referred to as the C. sesamiae population, harbouring Cs Snona haplotype on CrV1 locus (Branca et al. 2011), as the C. sesamiae lineage 2 analysed by Kaiser et al. (2015) and as the sample CsBV G4675 sequenced for 3 viral genes in Jancek et al. (2013).

Figure 1. 

Cotesia typhae, holotype, female specimen from Makindu, Kenya. A Habitus, lateral view B Head, frontal view (arrow shows face projection between antennal base) C Wings D Head and mesosoma (partially), lateral view E Propodeum and metasoma, dorsal view F Mesosoma and metasoma, lateral view G Head, mesosoma and tergites 1-2, dorsal view (arrow shows anteromesoscutum punctures).

Figure 2. 

Cotesia typhae, paratype, female specimen from Kobodo, Kenya. A Habitus, lateral view B Head, frontal view (arrow shows face projection between antennal base) C Wings D Metasoma, dorsal view E Head and mesosoma, dorsal view (arrow shows anteromesoscutum punctures) F Metasoma, lateral view.

Diagnosis

The new Cotesia is relatively distinct from other members of the flavipes complex (Table 2). The most distinctive diagnostic characters are the median projection present between the base of the antennae, the punctures on the anteromesoscutum, the length and shape of the paramere, and the relative length of the antennal flagellomere. The median projection between the base of the antennae is depressed (compared to the rest of the face), usually paler than the rest of the face, and has a strongly excavated median longitudinal sulcus (Figs 1B, 2B); all other species within the flavipes complex have a less depressed median projection on the face, usually the same color (or at most slightly lighter) as the rest of the face, and the median sulcus is not defined (nonagriae) or is less strongly excavated (Figs 5B, 6B, 7B, 8B). The anteromesoscutum punctures (Figs 1G, 2E) are the largest, densest, and most widely distributed (present near the posterior margin of the anteromesoscutum) among all species within the flavipes complex (compare against Figs 5G, 6F, 7G, 8H). The paramere length (Figs 3A, B, 4C, F, G) is intermediate compared to the other species (longer than in chilonis/sesamiae and shorter than in flavipes/nonagriae; compare Figs 3D, F, H, 4D, E), and its shape seems to be distinctive, with a somewhat widened part near the apex (Fig. 4F, G). The antennal flagellomeres (Figs 1A, D, 2A, B) are the longest among the entire flavipes complex (compare versus Figs 5A, B, D, 6A, B, 7A, B, 8A, D). The color of metasoma laterally and ventrally (laterotergites, sternites and hypopygium) is light yellow-orange (Figs 1A, F, 2A, F). This character is useful in recognizing typhae, at least in Africa, as all other Cotesia species within this complex generally have a much darker metasoma latero-ventrally (e.g. Figs 5A, F, 6A, E, 7A, F, 8G); however, some populations of C. flavipes we have examined have a light-colored metasoma, so this character is not absolutely diagnostic.

Figure 3. 

Male metasoma in ventral and lateral view. A, B Cotesia typhae, paratype specimen from Kenya C, D Cotesia sesamiae, specimen from Kenya E, F Cotesia flavipes, specimen from Kenya G, H Cotesia chilonis, specimen from Japan.

Figure 4. 

External male genitalia in ventral and lateral view; arrows show length of paramere and sternite 8. A, D Cotesia flavipes specimen from Kenya B, E Cotesia sesamiae, specimen from Kenya C, F, G Cotesia typhae, paratype specimen from Kenya.

Figure 5. 

Cotesia sesamiae, female specimen from Kitale, Kenya. A Habitus, lateral view B Head, frontal view C Wings D Head and mesosoma, lateral view E Scutellar disc, propodeum and metasoma, dorsal view F Mesosoma and metasoma, lateral view G Head, mesosoma and tergites 1-4, dorsal view.

Figure 6. 

Cotesia sesamiae, female specimen from Mombasa, Kenya. A Habitus, lateral view B Head, frontal view C Wings D Mesosoma and metasoma (partially), dorsal view E Mesosoma and metasoma, lateral view F Anteromesoscutum, scutellar disc and propodeum, dorsal view.

Figure 7. 

Cotesia flavipes, female specimen from Mombasa, Kenya. A Habitus, lateral view B Head, frontal view C Wings D Head and mesosoma, lateral view E Mesosoma and metasoma, dorsal view F Mesosoma and metasoma, lateral view G Head, mesosoma and tergites 1-2, dorsal view.

Figure 8. 

Cotesia chilonis, female specimen from Takatsuki, Japan. A Habitus, lateral view B Head, frontal view C Wings D Antennae, front and middle legs, lateral view E Head, lateral view F Propodeum, tergites 1-2, dorsal view G Mesosoma and metasoma, lateral view H Mesosoma and metasoma, dorsal view.

Diagnostic characters within the Cotesia flavipes complex. Data on host caterpillar species from Branca et al. (2011), Muirhead et al. (2012), Sallam (2006), and Kaiser et al. (2015).

Cotesia chilonis Cotesia flavipes Cotesia nonagriae Cotesia sesamiae Cotesia typhae
Scutoscutellar sulcus Straight (Fig. 8H) Curved (Fig. 7E) Curved Curved (Fig. 5F) Curved (Figs 1G, 2E)
Antero-mesoscutum (AMS) punctures Large punctures (diameter larger than distance between punctures) in most of AMS, including most of the posterior half (Fig. 8H) Relatively small punctures on anterior half of AMS, posterior half almost entirely smooth (Fig. 7E, G) Relatively small punctures on anterior half of AMS, posterior half almost entirely smooth Relatively small punctures on anterior half of AMS, posterior half almost entirely smooth (Figs 5G, 6F) Large punctures (diameter larger than distance between punctures) in most of AMS, including most of the posterior half (Fig. 2G, E)
Face projection between antennal base Acute, triangular projection with clearly impressed median longitudinal sulcus (Fig. 8B) Acute projection (sometimes projection less acute, margin almost straight) with clearly impressed median longitudinal sulcus (Fig. 7B) More or less straight margin, with no clearly impressed, median longitudinal sulcus Acute projection (sometimes projection less acute, margin almost straight) with clearly impressed median longitudinal sulcus (Figs 5B, 6B) Acute, triangular projection with clearly impressed median longitudinal sulcus (Fig. 1B, B)
Paramere length (observed externally, without removing genitalia from specimen) Short, around 1.0 × as long as median length of sternite 8 (partially visible in Fig. 3G, H) Large, clearly more than 1.5 × (usually up to 2.0x) as long as median length of sternite 8 (Fig. 4A, D) Large, clearly more than 1.5 × (usually up to 2.0x) as long as median length of sternite 8 Short, around 1.0 × as long as median length of sternite 8 (Fig. 4b, E) Relatively large, around 1.5 × as long as median length of sternite 8 (Fig. 4C)
Paramere shape Rather uniformly narrowing from base to rounded apex Rather uniformly narrowing from base to rounded apex (Fig. 4D) Rather uniformly narrowing from base to rounded apex Rather uniformly narrowing from base to rounded apex (Fig. 4E) With a broad, widened area near apex (Fig. 4F, G)
Antennal flagellomeres Relatively short
(3+ about as long as wide)
Relatively short
(2+ about as long as wide)
Relatively short
(2+ about as long as wide)
Relatively short
(3+ about as long as wide)
Relatively long (1–4 much longer than wide)
Natural known hosts Chilo supressalis, C. partellus
(Crambidae)
More than 7 species (Crambidae & Noctuidae) Bathytricha truncata (Noctuidae) More than 34 species (mostly Noctuidae & Crambidae) Sesamia nonagrioides (Noctuidae)

Description

Head and mesosoma mostly dark brown to black (except for scape, pedicel, wing base and tegula yellow; antennal flagellomeres brown; mandibles and labrums orange-yellow, and face projection between antennal base usually light brown); legs mostly yellow (except for metafemur with brown dorsal tip on posterior 0.1, and metatarsus light brown to brown); metasoma mostly yellow-brown to yellow-orange (except for mediotergites 1 and 2 dark brown to black, and mediotergites 3+ usually with brown spot centrally, near anterior margin). Wings with veins mostly brown, pterostigma brown with pale spot on anterior 0.3.

Head wider than high; face with acute, triangular projection between antennal base, the projection with clearly impressed median longitudinal sulcus; head dorsally smooth; gena laterally and dorsally as wide or wider than eye width; anteromesoscutum with relatively deep, coarse and large punctures (puncture diameter larger than distance between punctures), puncture density similar on most of the anteromesoscutum, including posterior half; scutoscutellar sulcus strongly curved, with 10-12 impressions; scutellar disc mostly smooth, with shallow and sparse punctures; propodeum mostly sculptured with an irregular pattern of strong carinae; mediotergites 1-2 mostly covered by strong longitudinal striae, mediotergites 3+ mostly smooth; hypopygium relatively small, apical tip in lateral view shorter than apical tip of tergites; paramere with broad, widened area near apex; paramere relatively large, around 1.50 × as long as median length of sternite 8.

Body ratios. Length of flagellomere 2/length of flagellomere 14: 1.71 × (1.50–1.86). Metafemur length/width: 3.06 × (2.92–3.25). Length of inner spur of metatibia/length of first segment of metatarsus: 0.48 × (0.46–0.52). Length of inner spur of metatibia/length of outer spur of metatibia: 1.07 × (1.07–1.18). Pterostigma length/width: 2.81 × (2.61–2.88). Length of fore wing vein r/length of fore wing vein 2RS: 0.82 × (0.82–1.00). Mediotergite 1 length/mediotergite width at posterior margin: 1.07 × (0.93–1.20). Length of mediotergite 2/length of mediotergite 3: 0.89 × (0.83–1.00).

Body measurements (all in mm). Body length: 2.40 (2.20–2.50). Fore wing length: 2.10 (2.10–2.20). Length of antennal flagellomere (F), F1: 0.15 (0.14–0.17), F2: 0.12 (0.12–0.13), F3: 0.11 (0.10–0.11), F14: 0.07 (0.06–0.08), F15: 0.07 (0.06–0.08), F16: 0.10 (0.09–0.11). Metafemur length: 0.55 (0.51–0.56). Metafemur width: 0.18 (0.16–0.19). Metatibia length: 0.71 (0.66–0.74). First segment of metatarsus length: 0.31 (0.28–0.31). Length of inner spur of metatibia: 0.15 (0.13–0.16). Length of outer spur of metatibia: 0.14 (0.11–0.14). Ovipositor sheaths length: 0.18 (0.15–0.18). Pterostigma length: 0.45 (0.145–0.49). Pterostigma width: 0.16 (0.16–0.18). Length of fore wing vein r: 0.09 (0.09–0.11). Length of fore wing 2RS: 0.11 (0.10–0.12). Length of mediotergite 1: 0.30 (0.27–0.31). Width at posterior margin of mediotergite 1: 0.28 (0.25–0.32). Length of mediotergite 2: 0.16 (0.14–0.20). Length of mediotergite 3: 0.18 (0.15–0.20).

Etymology

Named after the main host plant on which the wasp parasitizes its host caterpillar, Kaiser et al. (2015).

Notes

Cotesia typhae occurs sympatrically with C. sesamiae and C. flavipes (the latter introduced into Africa). Among these three species, typhae is the largest (body and fore wing lengths usually 0.2–0.3 mm longer than the two others), it also has a more sculptured anteromesoscutum and a longer antenna (especially flagellomeres 1–4 which are significantly longer).

Molecular characterization

Between species, pairwise divergence of COI sequences ranged from 2.6% to 4.2%, and distances observed between C. typhae and the other C. sesamiae species fell in this range Table 3). Within species, divergence was close to zero for C. typhae, C. chilonis and C. flavipes, and ranged from zero to 2.8% in C. sesamiae. The higher within-species values in C. sesamiae are explained by the divergence between the Kitale and Mombassa populations, reflecting their affiliation with different lineages, as shown by Kaiser et al. (2015) (Table 4).

Minimum and maximum divergence of COI sequences between all pairs of species.

C. typhae C. sesamiae C. flavipes C. chilonis
C. typhae 0–0.002
C. sesamiae 0.026–0.035 0–0.028
C. flavipes 0.033–0.035 0.031–0.042 0
C. chilonis 0.035 0.030–0.037 0.037 0

Minimum and maximum divergence of COI sequences between C. typhae and two populations of C. sesamiae.

C. typhae C. sesamiae Kitale C. sesamiae Mombasa
C. typhae 0–0.002
C. sesamiae Kitale 0.03–0.035 0–0.014
C. sesamiae Mombasa 0.026–0.03 0.019–0.028 0–0.003

Distribution, ecology and abundance

Among the ten sampled countries and 65 sampled localities hosting S. nonagrioides on Typhaceae and Cyperaceae, larvae parasitized by C. typhae were found in the three most sampled countries (highest numbers of localities and collected larvae), Ethiopia, Kenya and Tanzania (Table 5), in a total of 12 localities (Table 6). This showed that the probability of discovering C. typhae depended on the sampling effort, so this species may well be present in other sub-Saharan Africa areas inhabited by S. nonagrioides (Kergoat et al. 2015).

Presence of Cotesia typhae in the sampled countries. Results of collections of S. nonagrioides in sub-Saharan Africa from 2004 to 2013. For each country the Table shows the number of localities containing Typhaceae and Cyperaceae plants, the total number of S. nonagrioides larvae collected there during the period, and whether some were parasitized by C. typhae.

Country Number of sampled localities with Typhaceae & Cyperaceae Number of S. nonagrioides larvae presence of Cotesia typhae
Benin 1 26 no
Botswana 1 2 no
Cameroun 1 1 no
Ethiopia 5 167 YES
Kenya 26 1253 YES
R. Congo 2 38 no
R.D.C. 2 26 no
Rwanda 1 7 no
Tanzania 18 463 YES
Tanzania, Pemba 1 1 no
Tanzania, Zanzibar 3 25 no
Uganda 4 26 no

We then estimated the percentage of parasitized S. nonagrioides in the localities where the parasitoid was present. It varied from less than five to more than 70 % (Table 6), with a mean value of 20.3 % (standard error 4.0 %, n=18). All values, except the highest, ranged between 3.4 and 33.3% of parasitized larvae. Among the 660 parasitized larvae, 5 were parasitized by Cotesia other than C. typhae (4 C. sesamiae and 1 C. flavipes). Repeated findings of C. typhae in different years in the same locality, as seen in two Kenyan localities (Mbita Lwanda, 4 collections over 9 years; Makindu, 3 collections over 4 years), showed that locality and plant-host combination were good criteria for finding this new species.

Percentage of parasitism of S. nonagrioides larvae in the localities where C. typhae was found.

Country Locality Latitude / Longitude EDate Plant species Nbr S. n. larvae % parasitism
ETHIOPIA Awasa 7.05°N, 38.47°E Nov.-04 T. domingensis 64 6.3%
ETHIOPIA Chamoleto 5.93°N, 37.53°E Nov.-04 T. domingensis 16 18.8%
ETHIOPIA Omolante 6.16°N, 37.67°E Nov.-04 T. domingensis 27 22.2%
KENYA Kabuto 0.35°S, 34.96°E May-12 C. dives 6 33.3%
KENYA Kobodo 0.86°S, 34.57°E March-13 C. dives 42 7.1%
KENYA Makindu 2.28°S, 37.82°E Nov.-10 T. domingensis 65 10.8%
KENYA Makindu 2.28°S, 37.82°E Feb.-11 T. domingensis 64 4.7%
KENYA Masimba 2.15°S, 37.58°E Dec.-06 T. domingensis 10 30.0%
KENYA Masimba 2.15°S, 37.58°E Apr.-08 T. domingensis 13 15.4%
KENYA Mbita Lwanda 0.89°S, 34.67°E Feb.-05 T. domingensis 68 27.9%
KENYA Mbita Lwanda 0.89°S, 34.67°E Oct.-08 T. domingensis 147 10.2%
KENYA Mbita Lwanda 0.89°S, 34.67°E June-07 T. domingensis 18 72.2%
KENYA Mbita Lwanda 0.89°S, 34.67°E March-13 T. domingensis 59 8.5%
KENYA Rabuor 0.43°S, 34.91°E March-13 C. dives 10 20.0%
KENYA Rabuor 0.43°S, 34.91°E March-13 T. domingensis 6 33.3%
KENYA Sori 0.97°S, 34.28°E March-13 T. domingensis 13 7.7%
TANZANIA Arusha 3.37°S, 36.87°E July-04 T. domingensis 29 3.4%
TANZANIA Ruvu 6.70°S, 38.71°E March-07 C. exaltatus 3 33.3%

Life history traits and parasitoid success on different host strains

Adult longevity

The median longevity was close to three days when adults were fed honey, but equal to two days or less when they were fed with 20% or 2% saccharose solution respectively (Fig. 9). The survival curves were significantly different (W2df = 129.78; P < 10-4). They showed that about 90% adults were dead six days after emergence when fed honey or 20% saccharose, and three days after emergence when fed 2% saccharose (Fig. 9).

Figure 9. 

Survival curves of C. typhae adults fed honey (number of wasps: n=497), or 20% (n=742) or 2% (n=534) saccharose solutions in collective cages at 21°. Median lifespan is the time value observed at 50% survival.

Realized fecundity

Females were given the opportunity to parasitize a maximum of four larvae, but they actually parasitized a mean number of only 2.3 larvae (Table 7), either because they died before the end of the experiment (almost half of them were dead on the third day, Fig. 10), or because they refused to oviposit, as observed for a few females on day 3, and for most of the surviving ones on day 4 (Fig. 10). About 2/3 of the stung larvae allowed successful parasitoid development (Table 7). Finally, females produced about 100 offspring during their lifetime, from two host larvae.

Realized fecundity of C. typhae on Kenyan S. nonagrioides.

adult lifetime (days) stung larvae (nbr) successfully parasitized larvae (nbr) Offspring (total nbr)
Mean (N=40) 2.83 2.3 1.63 102.93
Standard error 1.17 0.11 0.11 6.2

Development of C. typhae in Kenyan and European hosts. Bold characters indicate significant differences between host strains.

S. nonagrioides populations: Kenya France Italy Statistical analyses
N: Nbr parasitized host larvae 58 58 47
Host larval weight at time of parasitism (mg) 295 ± 11 272 ± 12 283 ± 12 KW2df = 1.28 P = 0.331
% successful parasitism % host pupae % host larva mortality 69.0 (b) 12.1 19.0 67.2 (b) 13.8 19.0 89.4 (a) 2.1 8.5 χ22df = 7.95 P = 0.019
N: Nbr of cocoon clusters analyzed below 38 32 33
Cotesia larval development 14.2 ± 0.4 b 14.4 ± 0.2 b 12.9 ± 7.1 a KW2df = 18.29 P = 10-4
Cotesia pupal development (days) 8.2 ± 0.3 b 6.8 ± 0.2 a 7.1 ± 0.1 a KW2df = 19.60 P < 10-4
Cocoon number 60.3 ± 4.6 b 75.0 ± 5.5 a 64.6 ± 4.5 ab KW2df = 7.67 P = 0.022
Individual cocoon weight (mg) 1.3 ± 0.04 1,3 ± 0.05 1,2 ± 0.02 KW2df = 4.20 P = 0.122
% Cotesia pupal mortality 10.4 ± 2.7 a 25.8 ± 4.6 b 3.0 ± 0.7 a KW2df = 16.54 P < 10-3
% females in the cluster 43.9 ± 5.4 c 35.0 ± 8.2 b 72 ± 3.8 a KW2df = 17.98 P < 10-3
Estimated Reproductive Rate (expected viable adults/mother) 37 37 56

In the next experiment, the possibility for C. typhae to develop in European populations was estimated by the incidence of the first oviposition, which ensured more than half of the wasp’s reproductive success.

Figure 10. 

Issue of successive presentations of host larvae to C. typhae (one host per day for four days or less for wasps that died).

Parasitoid success in European host populations

Susceptibility of European S. nonagrioides strains to the parasitoid was equal or even higher than that of the Kenyan strain, with for instance almost 90% of successfully parasitized Italian larvae. Several other traits differed between the host strains, with a trend for better performances in the Italian strain, which ranked “a” for the five progeny traits showing significant differences: faster larval and pupal development, resulting in a development time of 20 days; high offspring number per cluster, showing the lowest pupal mortality and highest ratio of females. Highest immature developmental time (22 days) was observed in the Kenyan host strain, and highest pupal mortality and lowest female ratio was observed in the French strain. From these traits, it is possible to estimate a reproductive rate, i.e. the expected number of viable adults per mother, by multiplying the proportion of successful parasitism (probability of host larvae successfully parasitized) by the mean number of produced cocoons and by the proportion of viable adults (1-proportion of pupal mortality). This approach indicated that a female C. typhae would produce 56 viable offspring from a host larva of the Italian population, and only 37 from the host larvae of the French or Kenyan populations. As discussed hereafter, most differences could be explained by the effect of rearing conditions on host larvae quality.

Discussion

The morphological analysis conducted in this study, as well as the divergence of the CO1 sequences, confirmed the species status of the C. sesamiae lineage specialized on the noctuid S. nonagrioides. The CO1 divergence fell within the range of values observed between species of the flavipes complex. Morphological traits differentiated in this lineage included those used to distinguish species of the flavipes complex. This constitutes evidence for the existence of a fifth species in the flavipes complex. We named this new species C. typhae, based on the main host plant where it is found on its host. Whereas the first four species are allopatric in their endemic range, C. typhae is sympatric with C. sesamiae and may have differentiated from this species through divergent selection for adaptation on S. nonagrioides in Typhaceae and Cyperaceae, a permanent resource, and divergent selection for reproductive isolation (possibly facilitated by Wolbachia) (Kaiser et al. 2015).

It is likely that more species may be found in this complex. For instance, a relatively large CO1 divergence was also observed between C. sesamiae populations from Kitale (inland Kenya) and Mombassa (coastal Kenya), which are two host races with limited gene flow due to Wolbachia infection (Mochiah et al. 2002). Additional studies on the phylogenetic and biological relationships among those lineages, in particular the strength of bidirectional cytoplasmic incompatibilities related to Wolbachia strains, may reveal, in the future, the presence of an additional species.

Male genitalia were one of the differentiated morphological traits. This explains mating abnormalities observed by Kaiser et al. (2015) when crossing males of C. typhae with females of C. sesamiae, i.e. difficulties of males to disengage from females. It is one component of pre-zygotic barriers. In most animal species with internal fertilization, male external genitalia are the most rapidly evolving organs and are usually the first organs to diverge morphologically following speciation (Eberhard 2010; Yassin and Orgogozo 2013). Because of their rapid evolution and species-specificity, their illustration is a common feature in taxonomic literature to discriminate closely related species, particularly in insects (Yassin and Orgogozo 2013), including species of the flavipes complex (Kimani-Njogu and Overholt 1997).

The larger size of C. typhae relatively to the other species of the flavipes complex could result from an adaptation to host size, S. nonagrioides being a rather large noctuid relative to other Poales stem borer hosts for the flavipes complex. The size of a solitary parasitoid has been often reported as a plastic trait varying with host size; in gregarious parasitoids, the clutch size can be plastic and varies with host size (Godfray 1994). An evolutionary relationship between the size of gregarious parasitoids and the host size can exist if there is a genetic constraint on the clutch size, which is very likely, at least due to limits in the number of mature oocytes. The differentiation of other morphological traits may result from selective sweep or genetic correlation with other adaptive traits exposed to differential selection.

The morphological identification of species of the flavipes complex relies on a combination of slight differences, and their observation requires specific expertise, so a molecular diagnoses using CO1 or the virulence gene CrV1 (Dupas et al. 2006; Branca et al. 2011) remains the easiest identification method.

The geographic distribution and ecology of C. typhae have been reported by Kaiser et al. (2015). Here we provide evidence that the probability of collecting S. nonagrioides parasitized by C. typhae depended on the number of collected larvae. In several visited countries, this number was not sufficient to assess the presence of the wasp, so it may well be present over the sub-Saharan distribution of its noctuid host. The percentage of parasitized larvae was highly variable between localities, and even between periods of sampling in the same locality. Abundance of C. typhae within a locality may vary depending on the rainy season. Indeed Mailafiya et al. (2010) found that C. sesamiae was more abundant during the rainy season, and here (Table 6), the highest values of C. typhae abundance were observed in the middle of the rainy season (December), whereas lower values corresponded to the beginning of the season (Makindu-Masimba area, rainy season from November to January, Mailafiya et al. 2010). Other localities in western Kenya with rainy seasons from March to August and October to December had the highest parasitism observed in June. Regarding mean parasitism rates, about 20% of S. nonagrioides larvae were successfully parasitized by C. typhae. The same mean value or range of parasitism rates were observed in stem borers parasitized by C. sesamiae, and by C. flavipes, in maize and sorghum in Kenya (Mailafiya et al. 2010), and by C. chilonis in rice in China (Lou et al. 2014), but lower values were also observed (Jiang et al. 2006). This mean value is much lower than that observed in laboratory conditions. One limiting factor of parasitism success in natural conditions may be the behavior of host larvae, which hide inside the stem galleries with entrances that are naturally plugged with residues from boring, and move about mostly during the night, whereas the wasps are diurnal. Larvae also defend themselves by biting to death the wasps attempting to oviposit, killing 30-40% of them (Potting et al. 1997). Dispersion of these small parasitoids may also limit their efficiency, and would explain why mass releases of C. flavipes performed in sugarcane fields in Brazil successfully raised the parasitism rate to a range of 40-60% (Botelho and Macedo 2002; Dinardo-Miranda et al. 2014). Our data on C. typhae also show that parasitism rates as high as 70% can occur, although this rates was observed only once, and the next closest value was half lower. Even higher seasonal peaks were also reported in the case of the noctuid stem borer Busseola fusca parasitized by C. sesamiae on sorghum in South Africa (Kfir and Bell 1993), and of the crambid rice stem borer C. suppressalis in China (Lou et al. 2014).

Presence of C. typhae in different years in the same place showed that locality and plant-host combination was a good criterion for finding this new species. Very rare occurrence of parasitism of S. nonagrioides by C. sesamiae and C. flavipes, observed in less than 1% of the larvae, means that species identity has to be checked systematically.

The longevity of C. typhae and reproduction dynamics resemble those observed for the other species of the flavipes complex, which are typical short lived pro-ovogenic parasitoid wasps (Quicke 1997), i.e. females emerge with mostly mature oocytes and oviposit shortly after being mated, until egg-depletion. A literature review allows comparisons with three other species of the complex. C. typhae adult longevity was close to that of C. sesamiae, i.e. a mean longevity of about three days when fed honey at 25° and 60% RH (Sallam et al. 2002), but shorter than the longevity recorded in similar conditions for C. flavipes (about 5-6 days, Potting et al. 1997) and for C. nonagriae (about 12 days, Muirhead et al. 2008). Longevity in outdoor conditions may be longer due to cooler temperatures at night and the opportunity to rest in favorable micro-niches provided by plants. In the flavipes complex the dynamic of female reproduction follows their longevity, since most offspring are produced from the first two ovipositions in C. typhae, as in C. sesamiae (Sallam et al. 2002), and along 4-5 ovipositions in C. flavipes (Potting et al. 2007), with the exception of C. nonagriae which produces most offspring during the first two ovipositions, although it can live for several days. This behavior may have been selected in response to the defense behavior of stem borer noctuid larvae, which threatens female survival at each oviposition. With regard to realized fecundity, data available for the species complex were mostly the number of offspring produced from the first oviposition, which can be estimated at 60 offspring in C. typhae. This value is intermediate between higher value observed for C. nonagriae (about 90 offspring from one oviposition, Muirhead et al. 2008), and lower value observed for C. sesamiae and C. flavipes (from 25 to 45, depending on both parasitoid strain and host species, Jiang et al. 2004, Mochiah et al. 2001, NgiSong et al. 1998, Sallam et al. 2002). Altogether, these data indicate an evolution of the reproduction strategy within the flavipes complex. Considering longevity and oviposition dynamic, C. typhae appeared to be closer to C. sesamiae than to C. flavipes and C. nonagriae, which is in accordance with the estimated phylogenetic proximity (Muirhead et al. 2012; Kaiser et al. 2015).

The parasitism success of C. typhae in European host populations, assessed in the present work, was initially questioned because European S. nonagrioides are genetically well differentiated from African populations (Moyal et al. 2011), and they may have evolved immune responses adapted to European parasitoids and pathogens. However, the variation of reproductive success of C. typhae in the different host populations did not depend on the continental origin of the host, because C. typhae performed globally better in the Italian population than in the French and the Kenyan ones. A genetic differentiation of the Italian host population is unlikely because a recent study based on the analysis of micro-satellite markers showed an absence of genetic structure of S. nonagrioides collected in Europe, and in the Near and Middle East (Kader et al., unpublished data). We are more inclined to suspect that the laboratory rearing conditions of the noctuids had an effect on C. typhae parasitism success. Indeed, Italian larvae tested in the present work had been reared in a different laboratory than larvae from the French and the Kenyan populations. The Italian laboratory uses a different diet (Giacometti 1995), and larval food is known to influence immune response of Lepidoptera larvae (Smilanich et al. 2009; Vogelweith et al. 2015). Comparison of diets on susceptibility of the three host populations to C. typhae will allow this hypothesis to be tested.

In the areas where C. typhae have been found, in eastern sub-Saharan Africa, S. nonagrioides is rarely seen on maize, sorghum or sugarcane, whereas this is the case in more western parts of Africa and in Europe and the Near and Middle East. However C. typhae would probably parasitize S. nonagrioides at least on maize, if introduced for biological control, because in laboratory conditions host larvae are readily accepted when fed on maize stem and fecal pellets and eaten stem tissues are highly attractive, triggering intense behavioral examination of the host with antennal tapping.

In conclusion, this study adds a fifth species to the Cotesia flavipes complex. Despite the number of individual studies that illustrate the diversity of ecological adaptations in this complex, a comprehensive analysis of the flavipes species group is still needed. It will require the joint study of all populations across the geographical and ecological range of the Cotesia flavipes complex. The use of an integrative taxonomic approach (combining morphological, molecular, biological and geographical data) will be of paramount importance in recognizing and characterizing this economically important complex of parasitoid wasps. The new C. typhae species is an interesting potential biological control agent of the Mediterranean corn borer S. nonagrioides, because of its strict host-specificity to that species, at least in its native area, precluding potential negative impact on non-target host species populations.

Acknowledgements

We are very grateful to Antoine Branca, Kate Muirhead and Florence Mougel for fruitful discussions on the project; and to the undergraduate students who contributed to the experiments, namely Louise Trouillaud and Sarah Achibet; to Gianandrea Salerno who sent us the Italian S. nonagrioides larvae; to Boaz Musyoka for field collection; to Odile Giraudier, Gerphas Ogola and Sylvie Nortier for insect rearing at Gif and the icipe; to Lionel Saunois, Amandine Dubois and Virginie Héraudet for maize production, to Alice Arnauld de Sartre for editing references; Malcom Eden for linguistic correction. The work of JFT in Canada was supported by project 1558 ‘Arthropods systematics’. This project was also supported by the ANR Bioadapt (ABC Papogen project), and by the other authors’ operating grants from IRD, CNRS, and icipe. It was performed under the juridical frame of a Material Transfer Agreement signed between IRD, icipe and CNRS (CNRS 072057/IRD 302227/00) and the authorization to import Cotesia in France delivered by the DRIAAF of Ile de France.

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Appendix

Genebank accession numbers of CO1 sequences

Species Genbank accession nbr Sample name
Cotesia chilonis KJ882549 P6679
KJ882550 P6680
KJ882551 P6681
Cotesia flavipes KJ882544 P0433
KJ882545 P0434
KJ882546 P0435
KJ882547 P2541
KJ882548 P4706
Cotesia sesamiae Kitale KJ882497 G4540
KJ882501 G4594
KJ882512 G4636
KJ882527 G4701
KJ882528 G4703
KJ882529 G4708
KJ882530 G4907
KJ882532 G4915
KJ882537 G5778
KJ882543 CsK
Cotesia sesamiae Mombasa KJ882495 G4511
KJ882496 G4512
KJ882500 G4572
KJ882513 G4652
KJ882533 G5699
KJ882538 G7338
KJ882541 Mhk
Cotesia typhae KJ882502 G4608
KJ882503 G4609
KJ882507 G4614
KJ882508 G4615
KJ882510 G4618
KJ882511 G4619
KJ882514 G4655
KJ882515 G4656
Cotesia typhae KJ882516 G4664
KJ882518 G4666
KJ882519 G4667
KJ882521 G4675
KJ882522 G4676
KJ882523 G4677
KJ882531 G4909
KJ882534 G5726
KJ882535 G5773
KJ882539 Mbita
KJ882540 MbL
KJ882542 Mkd