An unexpected occurrence of Amblyseius swirskii Athias-Henriot in La Reunion Island (Acari: Phytoseiidae)

The identity of specimens of phytoseiid mites collected inside greenhouses infested by thrips in La Reunion Island was assessed. As the sampled species belongs to the genus Amblyseius, presently containing 415 species which are notoriously difficult to discriminate, morphological and molecular traits were evaluated. Both morphological and DNA comparisons showed that the collected specimens belong to the species Amblyseius swirskii, a successful biological control agent introduced in the market more than ten years ago in Europe and Northern America. This finding was unexpected, as this species was never reported in this part of the world; an accidental introduction is thus suspected.


INTRODUCTION
Biological control is the most environmentally safe and most economical mode of pest management for growers (Cock et al. 2010). Augmentative biological control has been proved to be an efficient alternative to chemical control in vegetable production in greenhouses (van Lenteren and Bueno 2003). The biocontrol industry has made great advances in the last decades with the discovery of more than 230 species of natural enemies available for augmentative biological control worldwide (van Lenteren 2012). Among them, phytoseiid mites are efficient predators of small insects and mites (McMurtry et al. 2013). They are considered important biocontrol agents with many species sold and used all around the world (Wright 2004). The predatory mite Amblyseius swirskii Athias-Henriot, 1962 (Acari: Phytoseiidae) is one of the most efficient; it is currently released in more than 50 countries of the world. It originates from the East Mediterranean coast and has been described in 1962 from almond (Prunus amygdalus [Miller] D.A. Webb) in Bet Dagan, Israel by Athias-Henriot (1962). This species was then reported along the coast of Israel, Middle Eastern countries, Southern Europe, Sub-Saharan Africa and the America (Demite et al. 2016).
-Amblyseius enab El-Badry 1967, described and reported from Egypt on Mangifera indica L. (Anacardiaceae) (Demite et al. 2016 Thus, this species is able to develop not only in the Mediterranean basin but also in subtropical and tropical areas (Zannou and Hanna 2011). Since this species is not entering diapause, it can be used throughout much of the season where daytime temperatures regularly exceed 22°C (Calvo et al. 2015). Amblyseius swirskii is commonly used to control whiteflies and thrips in greenhouse vegetables (especially cucumber, pepper and eggplant) and some ornamental crops, in Europe and North America (Calvo et al. 2015). The biology of this species and its importance for biocontrol were recently reviewed by Calvo et al. (2015) and Buitenhuis et al. (2015).
In 2015, a species looking like A. swirskii was discovered after a thrips outbreak on peppers and roses at the Réunion Island, at several thousand kilometres from its supposed native area. Amblyseius swirskii was furthermore not reported in previous mite biodiversity surveys in La Réunion Island (Quilici et al. 1997(Quilici et al. , 2000Kreiter et al. 2002;Kreiter et al. unp. data). Production of vegetables and ornamentals in greenhouses in this Island correspond to great economic inputs. Furthermore because of recurrent problems with pesticide use (efficiency, environmental and health risks), the development of biological control is very important to improve.
According to the new French regulations on importation of macro-organisms (Anonymous, 2012), A. swirskii is currently authorized for sale in the French Metropolitan Area but La Réunion is an over-seas territory; therefore it is impossible to sell and use this species in this latter area, as it is not indigenous. An importation permit could be requested, but it is expensive and chances to obtain are low. The objective of this paper is thus to assess the identity of the species occurring in the landscape of La Réunion Island in order to determine if the interesting biological control agent A. swirskii is occurring in this territory. Furthermore, as the genus Amblyseius contains approximately 415 species, as no over-all identification key of species of this genus exists, as many species are morphologically very close (with suspicion of synonymies or cryptic species), both morphological and molecular approaches have been carried to ascertain the diagnosis.

Collection of mites and mountings
The specimens were collected on pepper plants Capsicum annuum L. and on roses Rosa sp. inside greenhouses in La Réunion Island in two locations: Bassin-Martin (in city of Saint-Pierre territory), Station Armeflhor (lat. 55°31'9" S, long. 21°20'0" E, altitude 450 m) in greenhouse 1 on roses, 13 V 2015 (coll. Olivier Fontaine); Montvert-les-Bas, EARL Le Montvert (lat. 55°32'19" S, long. 21°19'42" E, altitude 582 m), in greenhouse with peppers, 15 V 2015 (coll. Mrs Solène Callarec and Olivier Fontaine); same location, in another greenhouse with peppers, 15 V 2015 (coll. Ingrid Avril) (Table 1). Phytoseiid mites were stored in 95 % ethanol. Part of this material was used for morphological identification; permanent slides were prepared using Hoyer's medium and kept on a hot plate (50°C) for two weeks. The other part of the material was used for molecular analyses (see below).

Morphological analyses
The concept of generic classification of Chant & McMurtry (2007) is used in this paper. The terminologies for chaetotaxy are those proposed by Lindquist & Evans (1965) as adapted by Rowell et al. (1978) for dorsal idiosomal setae and by Chant  Athias-Henriot (1975). Measurements were performed using a phase and interference contrast microscope (Leica DLMB, Leica Microsystèmes SAS, Rueil-Malmaison, France) (400x magnification). All measurements are given in micrometers (µm). Measurements of specimens collected in La Réunion were compared to measurements of the original description and to measurements of several re-descriptions: specimens from Israel (Porath and Swirski 1965), from several countries in Africa (Zannou et al. 2007), Cape Verde (Ueckermann 1992), Egypt (Abo-Shnaf and Moraes 2014), and from Spain (Ferragut et al. 2010). All voucher specimens are deposited in the mite collection of Montpellier SupAgro Acarology Collection, UMR CBGP, Montpellier, France.

Molecular analysis
As individuals identified morphologically were very close to A. swirskii with only some very minor morphological differences, as many cryptic species exist among many genera of Phytoseiidae and as it is important to ascertain the identity of the species in the perspective of potential further studies and uses, molecular analyses were conducted to assess the identity of mites collected in La Réunion. Specimens of La Réunion Island were compared to specimens of the commercial strain of A. swirskii reared and sold by Koppert BV (Veilingweg 14, 2651 BE Berkel en Rodenrijs, The Netherlands) (Table 1). DNA was individually extracted from females, using a Qiagen DNeasy tissue kit (Qiagen, Hilden, Germany), according to the DNA extraction protocol described in Kanouh et al. (2010). After DNA extraction, females were retrieved from the Qiagen column as described by Tixier et al. (2010). A mitochondrial DNA markers (12S rRNA) was used to allow efficient molecular species diagnosis (see e.g. Okassa et al., 2009Okassa et al., , 2011Tixier et al. 2011Tixier et al. , 2012Tixier et al. , 2014. Primers for the amplification of the DNA fragments were as follows: 12S rRNA, 5'-3' TACTATGTTACGACTTAT and 3'-5' AAACTAG-GATTAGATACCC (Jeyaprakash and Hoy 2002).

Morphological analyses
For the females, except for some slightly longer (s4) or shorter (Sge I) setae and some slightly shorter dimensions of the sternal and genital shields in the La Réunion population, the measurements were very close to all those reported in the original description and further re-descriptions (Table 2). Clearly the differences observed fall into the intraspecific variation as defined in Tixier (2012). Males of the La Réunion population have slightly longer S4, Sge II, Sge III and Sti III and shorter Sge I and JV5 (Table 2). Morphological comparisons seem to show that specimens found in La Réunion belong to the species A. swirskii.

Molecular analyses
A fragment of 418 bp was amplified. The mean genetic distance (1.7 %) between specimens collected in La Réunion Island and specimens of A. swirskii from Koppert was low. Genetic distances range between 0.3 and 3.6 % (Table 3). Such values are similar to distances observed between specimens of the Koppert population. Furthermore, such distances clearly correspond to intraspecific distances reported for other species of Phytoseiidae (e.g. Okassa et al. 2009Okassa et al. , 2011Tixier et al. 2011Tixier et al. , 2012Tixier et al. , 2014. The molecular results thus confirm that the specimens collected in La Réunion Island belong to A. swirskii.

Discussion and conclusion
Molecular and morphological results both allow us to conclude that specimens collected in La Réunion Island belong to the species A. swirskii. The origin of the population collected in La Réunion Island remains unknown. The population may originate from previous releases of a commercial strain of A. swirskii coming from Europe. Amblyseius swirskii has however been reported from another island far away from a continent: Cape Verde, in 1992 (Ueckermann 1992), far before it was sold and used in large numbers in many countries. It can also have come "naturally" or by human commercial activities from Eastern Africa directly or via Madagascar and/ or Mauritius.
Several species of thrips and whiteflies occur in vegetable and ornamental crops in greenhouses in La Réunion (Vayssières et al. 2001), along with the broad Polyphagotarsonemus latus (Banks), and the external demands for alternatives to chemical control are increasing. The potential for development of this indigenous population of A. swirskii as a biocontrol agent for La Réunion crops, mainly in greenhouses but possibly also in outdoor crops such as citrus orchards (Juan-Blasco et al. 2012), is therefore very high.
Manager in Armefhlor, and Jacques Fillatre, responsible of horticultural, strawberry and Papam production at Armeflhor for their investments in the collection and identification of the species. We are very grateful to the staff of Koppert BV (Koppert BV, Veilingweg 14, 2651 BE Berkel en Rodenrijs, The Netherlands) and especially to Dr Markus Knapp for providing us with specimens of the commercial strain of Amblyseius swirskii.