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.

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

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 19^th 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.

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.

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.