First report in Colombia and diagnosis of Diaphorencyrtus aligarhensis (Hymenoptera: Encyrtidae), a parasitoid wasp of Diaphorina citri (Hemiptera: Liviidae) Primer

Diaphorencyrtus aligarhensis (Hymenoptera: Encyrtidae) is herein reported for the first time from Colombia based on specimens collected in the municipality of Palmira, department of Valle del Cauca. Adult male and female wasps of this endoparasitoid are diagnosed based on published literature and character states taken from specimens collected in the present study. The adult parasitoids were extracted from parasitized nymphs (mummies) of the Asian citrus psyllid, Diaphorina citri (Hemiptera: Liviidae). Information is provided on the differences in the morphology of parasitized nymphs of D. citri with exit holes made by the two main primary parasitoids, i.e., Tamarixia radiata (Hymenoptera: Eulophidae) and D. aligarhensis . Rates of parasitization on D. citri ranged from 1.5 to 24.2 % for T. radiata and 0.3 to 1.0 % for D. aligarhensis . With the present study, the presence of D. aligarhensis in Colombia becomes the second confirmed report of the species in South America, after Ecuador.


INTRODUCTION
In Colombia, efforts to prevent the spread of Huanglongbing (HLB), one of the most devastating diseases of citrus worldwide, have been carried out through chemical control of the insect vector (D. citri) and the eradication of infected trees (Kondo et al. 2020). On the other hand, in areas free of HLB, since 2013 the Colombian Corporation for Agricultural Research -Agrosavia has been conducting a mass rearing and release program of the wasp Tamarixia radiata (Waterston, 1922) (Hymenoptera: Eulophidae), the main parasitoid of D. citri, for research purposes. The program also is aimed at maintaining populations of D. citri at low levels in house gardens, hedge trees, and shrubs of the family Rutaceae (e.g., Citrus spp., Swinglea glutinosa (Blanco) Merr. and Murraya paniculata (L.) Jack) in urban areas because these populations can act as reservoirs for the HLB causing bacteria (Kondo 2018, Kondo et al. 2020. During a study carried out in a citrus orchard in Agrosavia, Palmira Research Station, in order to determine the rates of parasitization of T. radiata, a second parasitoid which was hitherto not detected in the area, was identified as Diaphorencyrtus aligarhensis (Shafee, Alam and Agarwal, 1975) (Hymenoptera: Encyrtidae). Until now in Colombia, there was a record of Diaphorencyrtus sp. from the department of Caldas (Arias-Ortega et al. 2016), however, this is the first record of D. aligarhensis in Colombia. The present paper provides information on i) rates of parasiti-zation of T. radiata and D. aligarhensis on D. citri under field conditions; ii) differences between these two primary parasitoids of D. citri, based on the field morphology of the adult wasps and of the parasitized nymphs; and iii) diagnoses and photographs of the adult male and female of D. aligarhensis.

MATERIALS AND METHODS
Eight field surveys were carried out between February 2020 and February 2021 in order to monitor the rates of
The surveys conducted in 2020 were aimed at determining the natural rate of parasitization of D. citri in the field prior to mass releases of T. radiata, and the survey in 2021 was carried out to determine the rate of parasitization of D. citri after a field release of adult parasitoids of T. radiata. Surveys between February and October 2020 were non-destructive and the impact of T. radiata was determined by field counting of the number of mummies (with and without exit holes). With the aid of a magnifying glass, mummies can be recognized easily in the field by their dark brown to reddish brown coloration (Fig. 1a). A non-destructive method was chosen to prevent the removal of parasitoids from the field, which could affect the populations of the parasitoid in the next generation and result in a lower rate of parasitization in the following survey.
Surveys carried out in December 2020, and January and February 2021 were destructive; psyllid-infested shoots and/or leaf buds were trimmed off the trees and put in Petri dishes for daily observations in the laboratory. In the laboratory, the number of parasitized nymphs of D. citri was determined based on the presence of eggs, larvae, and pupae of T. radiata, as well as nymphs of D. citri with a parasitoid emergence hole. Nymphs were individually checked with the help of a fine pencil brush and entomological forceps under a stereo microscope. The shoots with parasitized nymphs were placed individually in 16-ounce plastic tubes to observe the emergence of the parasitoids and confirm that T. radiata had parasitized the psyllids. The shoots were checked daily for an average period of seven to ten days, counting the adults of T. radiata that emerged each day. To reduce the dehydration of the citrus shoot, the basal part of the stem where the cut was made was covered with a piece of cotton moistened with distilled water and cotton mesh was fitted in the lid of the plastic tube to allow the passage of air. This destructive method was adapted from various studies (Pluke et al. 2008, Chong et al. 2010, Cortez et al. 2010, Branco and Postali 2012, Chávez et al. 2017 and was used in the last three surveys because other natural enemies, especially coccinellids, were consuming the nymphs of D. citri that were being evaluated, making it difficult to calculate the parasitization rate of the parasitoids. In February 2021, 320 adults of T. radiata were released in the citrus orchard. The parasitoids were obtained from a mass rearing program established at Agrosavia, Palmira Research Center, and were packed in 60 cc plastic bottles with a screw cap (each bottle contained 40 unsexed adults of T. radiata) for a total of eight bottles. One bottle of parasitoids was released at each of eight sites in the lot, following a diagonal x-shaped pattern covering as much area as possible within the orchard. The number of nymphs on each of the selected shoots was counted just prior to the field release of the parasitoids. Observations were carried out daily from day one until either the emergence of the parasitoids, the adult psyllids emerged, or the nymphs disappeared due to the reasons described above. The rate of parasitization was determined by using the following formula: Rate of parasitization (%) = [(number of parasitized nymphs)/(total number of evaluated nymphs)]×100 Most of the emerged parasitoids identified as Diaphorencyrtus sp. were preserved in 75 % ethyl alcohol. A number of the alcohol-preserved specimens were critical-point-dried and mounted following the techniques described by Noyes (1982). Some individuals were point-mounted without previous treatments and others were mounted on slides in Hoyer's mounting medium (distilled water 50 cc, gum Arabic 30 cc, chloral hydrate 200 cc and glycerin 20 cc) or Euparal. Except for the antennae, which are used to determine the distribution pattern of coloration, the specimens for slide-mounting were cleared by heating them for about 10 to 20 minutes in potassium hydroxide (10 % w/v) prior to mounting. The specimens were identified to genus and species level by using the keys of Noyes and Hayat (1984) and Hayat (2006) and the descriptions of Robinson (1960), Shafee et al. (1975) and Hayat (1981). Morphological terminology used for the parasitoids follows that of Gibson (1997).

Parasitized nymphs (mummies): Tamarixia radiata vs Diaphorencyrtus aligarhensis
While studying the parasitization rate of T. radiata in the field using a non-destructive method, the authors first recognized the presence of a second parasitoid by the position of the exit hole of the adult parasitoids. In the field, the position of the exit hole on the mummies of D. citri can be used to distinguish nymphs parasitized by D. aligarhensis from those parasitized by T. radiata, i.e., the emerging adult of T. radiata exits dorsally through the thorax, whereas the adult of D. aligarhensis exists dorsally through the abdomen. There are also other differences induced by both parasitoids such as the shape of the mummies. The parasitized nymphs or mummies of D. citri hosting T. radiata and D. aligarhensis can be differentiated by the following combination of features [features of D. aligarhensis in square brackets]: 1) exit hole of the adult parasitoid usually found on the thorax (Hoy 2005); the plant substrate visible inside the exit hole (Figs. 1a, b) [exit hole usually found on the abdomen; dried thin ventral derm of the parasitized nymph visible inside the exit hole (Figs. 1d, e)]; 2) mummies normally attached to the plant substrate by silk webbing that is visible around the margins of the mummy, mainly around the posterior abdomen (Figs. 1a, b) [mummies attached ventrally to the plant substrate by a sticky substance; without silk webbing around the mummy (Figs. 1d, e)]; 3) body shape similar to a non-parasitized nymph, rather flat, dark brown (Figs. 1a, b), with the ectoparasitoid larva found externally under the psyllid body [body shape different from non-parasitized nymphs, becoming cylindrical (Fig. 1d), the endoparasitoid larva found within the psyllid body]; and 4) parasitized nymph with a meconium on posterior part of body, its abdomen without pigmentation, usually of the same color as rest of body (Figs. 1a, b) [parasitized nymph without a meconium on posterior part of body; abdomen often with a pigmentation on posterior abdominal segments, clearly darker than rest of body (Figs. 1d, e)].

The adults: Tamarixia radiata vs Diaphorencyrtus aligarhensis
Considering there are only two primary parasitoids of D.

Note. Descriptions of nymphs of D. citri parasitized by T.
radiata can also be found in Aubert and Quilici (1984), Chen and Stansly (2014), and Hoy (2005); information on nymphs of D. citri parasitized by D. aligarhensis are found in Rohrig (c2010, c2014) and Qing (1990). Character states of the adults of T. radiata were taken from Kondo et al. (2012) and Qing and Aubert (1990), and those of D. aligarhensis taken from Rohrig (c2014). These reports agree well with our observations. Additionally, some features were taken from the studied material, i.e., the dark coloration around posterior abdominal segments and the dried thin ventral derm of the parasitized nymph visible inside the exit hole in nymphs of D. citri parasitized by D. aligarhensis.

Diagnosis of the adults of Diaphorencyrtus aligarhensis
According to Qing and Aubert (1990), D. aligarhensis can be diagnosed by the following combination of features: female body 1.5-1.8 mm long, head and mesosoma black, basal four metasomal segments yellow, apical metasoma dark brown; antennae yellowish brown, funicle 6-segmented, funiculars wider than long, gradually larger towards clava; clava 3-segmented, subcylindrical, rounded apically; legs yellow, with 3-6 spines on apical parts of mid tibiae and 1 st -3 rd tarsi of middle leg. Male antennae yellowish, funicle 6-segmented, funiculars longer than wide, clava slender, 1-segmented; legs white-yellowish, basal abdomen yellowish brown. The studied material agrees well with the above diagnosis.
Taxonomic notes. Diaphorencyrtus can be recognized by a combination of features of the antennal segmentation and coloration (Figs. 2a-c) in which the clava is slightly darker than the funicle segments and apically rounded, the robust and convex mesosoma, the yellow or orange terga at the base of the metasoma (Figs. 2a, b), and the wing venation (Fig. 2c) with a pronounced postmarginal vein that is shorter than the long stigmal vein. The metapleuron is covered in conspicuous silvery setae (Fig. 2a) (Robinson, 1960) can be distinguished from D. aligarhensis by having only a single yellow tergum at the base of the metasoma (at least two in D. aligarhen-sis), the antennal scape which is dark brown in the basal half (all yellow in D. aligarhensis) and differences in leg coloration (see description in Robinson (1960)).
According to Prinsloo (1985), cleared, slide-mounted female specimens of D. aligarhensis from Reunion Island differ slightly from Indian specimens by having the antennal pedicel about as long as the basal two funicle segments together (a little longer in Indian specimens), with the head and thorax black (brownish-black to dark brown in Indian specimens), although he considered these differences as part of the morphological variation of the species. Arias-Ortega et al. (2016) reported Diaphorencyrtus sp. from Colombia as a possible undescribed species, stating that unlike the features described by Mani (1989) and Rohrig (c2014), the postmarginal vein of the anterior wing is not slightly longer than the marginal vein, the female has setae on the antennae and the antennae of the male are highly pilose, which may indicate that it is a different species. However, we disagree with the character states listed by Arias-Ortega et al. (2016) as evidence for a different species because in the photograph they provide the postmarginal vein is slightly longer than the marginal vein as stated in the original description of Shafee et al. (1975). Concerning the pilosity of the antennae, Mani (1989) does not illustrate nor mention the pilosity for either male or female antennae, and Rohrig (c2014) described the female antennae as smooth and clubbed and the male antennae as slightly longer than those of the female, lacking a club and being covered with short hairs. Although Rohrig (c2014) describes the antennae of D. aligarhensis as being smooth, the photos of the adult female in his factsheet clearly shows small hairs covering the antennal segments. Thus, we consider that the report of Diaphorencyrtus sp. by Arias- Ortega et al. (2016) from the municipality of Manizales, department of Caldas, Colombia refers to D. aligarhensis.

DISCUSSION
In South America, D. aligarhensis has been recorded previously only from Ecuador where the parasitoid was probably accidentally introduced with its psyllid host, D. citri (Portalanza et al. 2017). In a checklist of natural enemies of D. citri of the world compiled by Kondo et al. (2015a), the authors erroneously listed Argentina as part of the distribution of D. aligarhensis citing a data sheet by García-Darderes (2009), however, this publication mere-ly mentioned the species as a biological control agent. The distribution of D. aligarhenisis in South America is now revised to include only Colombia and Ecuador. It is likely that D. aligarhensis is more widespread in Colombia and elsewhere in South America because D. aligarhensis is outcompeted by T. radiata (see below for discussion) and thus not as easy to detect, and intraguild predation by coccinellids and other predators may generally keep their populations very low.
Diaphorencyrtus aligarhensis is native to the Oriental region, known from Afghanistan (CABI c2021), China (Yang et al. 2006), India (Shafee et al. 1975), Philippines and Vietnam (Aubert 1987); and has been introduced deliberately to Reunion Island (Aubert and Quilici 1984), Taiwan (Chien and Chu 1996), South Africa (Prinsloo 1985 However, Hoddle et al. (2014) confirmed that most species reported by Hussain and Nath (1927) are hyperparasitoids or parasitoids of other insect species and that there are only two primary parasitoids of D. citri in the province of Punjab, i.e., T. radiata and D. aligarhensis. Another study also reports these two primary parasitoids in the Asian Pacific region, and 13 secondary or tertiary parasitoids (Qing 1990). Mummies from which hyperparasitoids emerge, generally have exit holes on the lateral side of the psyllid nymphs (Qin 1990), thus they can be differentiated from mummies induced by primary parasitoids in which the exit holes are found in the midline of the mummies (Qing 1990).
Between 2014 and 2017, over 300 000 D. aligarhensis wasps were released in urban areas of Southern California, U.S.A. by the California Department of Food and Agriculture (CDFA) and the University of California, Riverside (Milosavljević et al. 2017). More than 20 million parasitoids (T. radiata and D. aligarhensis combined) were mass-produced and released at >1 500 sites in southern California by the CDFA, however, only T. radiata spread rapidly and established, whereas D. aligarhensis apparently did not become established (Milosavljević et al. 2021).
A study conducted under laboratory conditions indicated that when D. aligarhensis and T. radiata compete, T. radiata will have an advantage, suggesting that it may be difficult for D. aligarhensis to contribute significantly to the biological control of D. citri when both parasitoids are present (Vankosky and Hoddle 2019b). When a nymph of D. citri is parasitized by T. radiata before or within five days following oviposition by D. aligarhensis, the emerging parasitoid will be an offspring of T. radiata (Rohrig et al. 2012). Nevertheless, in Reunion Island, in the absence of hyperparasitoids, both parasitoids have been reported to be extremely efficient in controlling populations of D. citri (Aubert and Quilici 1984).
In a study conducted in Punjab, Pakistan, on nymphs of D. citri on Citrus reticulata L. and Citrus sinensis Osbeck (Rutaceae), average rate of parasitization of 26 % by T. radiata and 17 % for D. aligarhensis have been reported . During its lifetime, an adult female of D. aligarhensis can kill up to 280 nymphs of D. citri through a combination of host feeding and parasitization (Chien 1995). On the other hand, an adult female of T. radiata can kill up to 500 nymphs of D. citri through a combination of host feeding and parasitization during its lifetime (Chien 1995).
During the present study, we observed that the number of nymphs of D. citri usually started to decline from day one in the field due to predation and other undetermined factors. In some cases, all nymphs disappeared from a shoot from one day to the next, making it impossible to evaluate the rate of parasitization. In the Valle del Cauca region, apart from T. radiata, numerous other natural enemies of D. citri have been reported, including ladybird beetles (Coleoptera: Coccinellidae), hover flies (Diptera: Syrphidae), assassin bugs (Hemiptera: Reduviidae), vespid wasps (Hymenoptera: Vespidae), lacewings (Neuroptera: Chrysopidae), ants (Hymenoptera: Formicidae), and a dragonfly (Odonata: Gomphidae), among others (Kondo et al. 2015a(Kondo et al. , b, 2018. A study conducted in California, U.S.A., determined that D. aligarhensis shows a high preference for the Asian citrus psyllid, D. citri, but may parasitize the potato psyllid Bactericera cockerelli (Sulc.,1909), at low levels (< 14 %) aligarhensis (Skelley and Hoy 2004).
Diaphorencyrtus aligarhensis wasps oviposit on second-, third-and fourth-instar nymphs of D. citri and host feed on first-to fourth-instar nymphs (Skelley andHoy 2004, Rohrig et al. 2011). On the other hand, T. radiata prefers to parasitize third-, fourth-and fifth-instar nymphs of D. citri  (Chien 1995, Rohrig et al. 2011. The developmental stages of D. aligarhensis include an embryonic stage (ca. 2 days), four larval instars (ca. 6 days), a prepupal stage (ca. 1 day), and a pupal stage (ca. 7 days) (Rohrig c2014). After about 8 days post oviposition, the parasitized nymphs of D. citri die and harden, becoming a brown mummy (Rohrig c2014). There are no significant differences in the mean number of progeny produced by females of D. aligarhensis when given second-, third-, or fourth-instar hosts (Skelley and Hoy 2004).
The population of D. aligarhensis introduced to Florida from Taiwan are infected by the intracellular endosymbiont Wolbachia (Jeyaprakash andHoy 2000, Meyer andHoy 2007), which probably explains why they are thelytokous, comprised of only females (Chien 1995). The population in Colombia produces both males and females.

ACKNOWLEDGMENTS
Thanks to the Colombian Ministry of Agriculture and Rural Development (MADR) for funding the project "Technologies for the integrated management of Diaphorina citri -HLB pathosystem in citrus cultivation in Colombia", convention tv19. Many thanks to Dr. Mohammad Hayat (Aligarh Muslim University) for comparing images of our material with paratypes of D. aligarhensis in his collection, and for helpful comments on the identity of our material. The authors thank the Corporación Colombiana de Investigación Agropecuaria (Agrosavia) for partially funding this study. Many thanks to Penny Gullan (The Australian National University) for reviewing an earlier version of the manuscript and the anonymous reviewers whose comments greatly improved the manuscript.