The Pupal Pigmentation Pattern and Pupal Development in the Species of Aphytis Howard (Hymenoptera: Aphelinidae)

Simple Summary Aphytis species (Hymenoptera: Aphelinidae) have been considered as the most important natural enemies in biological control of armoured scale insects (Hemiptera: Diaspididae). However, it is difficult to identify the species of the genus, particularly that of the A. lingnanensis group, based on adult morphological characters. Although the differences in pupal pigmentation of Aphytis species have been documented previously, they have not been much used as taxonomic characters for Aphytis species identification. In this study, we present four characteristic pigmentation patterns of Aphytis pupae, together with photographs, including the categories of: entirely yellow, partly dark brown, entirely or predominantly black, and partly black pupae. No significant intra-specific variation in pupal colour pattern was detected despite relatively high numbers of specimens examined, many from multiple, and different, origins. We summarize the present status of pupal pigmentation in the described species of Aphytis, according to the species groups, which could be used as an important supplementary diagnostic character for distinguishing species of Aphytis, especially closely-related species. Abstract Species identification of Aphytis on the basis of adult morphology is extremely difficult, especially in the A. lingnanensis group, with several cryptic species. Pupal pigmentation could be used as one of the taxonomic characters for Aphytis species, and in some instances, pupal pigmentation actually provided the first clue to the distinctness of cryptic Aphytis species. The present study investigated the full-grown larvae or younger pupae of Aphytis species, and pupal pigmentation and pupal development were observed and photographed. Four characteristic pigmentation patterns of Aphytis pupae were summarized including: entirely yellow, partly dark brown, entirely or predominantly black, and partly black. The species in the chilensis and mytilaspidis groups, and some unassigned species, generally have entirely, or predominantly and or partly black pupae. The species in the chrysomphali, funicularis, and proclia groups generally have the pupae entirely yellow. The species of the lingnanensis group have the pupae both entirely yellow, e.g., A. fisheri, and partly dark brown pupae, e.g., A. lingnanensis, A. holoxanthus and A. melinus. The pupae of Aphytis species in this study had a developmental duration of about 5–8 days at 27 ± 1 °C, 70 ± 5% RH and a photoperiod of 10L: 14D. It was found that the pupal skin was always melanized at the beginning stage, generally in the first day, among the pigmented pupae of Aphytis species. As development continued, the pigmentation became darker and the eye colour changed from pale red/brown to green. No significant intra-specific variation in pupal colour pattern was detected despite relatively high numbers of specimens examined, many from multiple, and different, origins. Overall, our study indicates that pupal pigmentation could be more helpful in species identification of Aphytis.


Introduction
The species of Aphytis Howard (Hymenoptera: Aphelinidae) are among the most important ectoparasitoids of armoured scale insects (Hemiptera: Diaspididae), and many of them were utilized successfully in biological control projects of important armoured scale pests around the world. For instance, A. lingnanensis Compere from Guangdong, China in 1947 and A. melinus DeBach from India and Pakistan in 1956-1957 were introduced into California, USA against the California red scale, Aonidiella aurantii (Maskell); A. holoxanthus DeBach was introduced into Israel in 1956-1957 from Hong Kong, China against the Florida red scale, Chrysomphalus aonidum (L.), and A. yanonensis DeBach & Rosen was introduced into Japan in 1980 from Sichuan, China against the arrowhead scale, Unaspis yanonensis (Kuwana) [1][2][3].
However, species identification of Aphytis is extremely difficult on the basis of adult morphology, especially in the A. lingnanensis group, with several cryptic species. This difficulty is due to their minute size (rarely exceeding one millimeter in length), the propodeal crenulae not easy to observe, and the relative scarcity of reliable distinguishing characters, as well as often unsatisfactory slide-mounted specimens without clearly visible characters [1,4].
Taylor [5] was the first to notice differences in pupal pigmentation, the yellow pupae and the dark pupae, between the closely related species A. chrysomphali (Mercet) and what was probably A. holoxanthus. Pupal pigmentation was consequently used as one of the taxonomic characters for Aphytis species not readily separable using adult morphology [1,[6][7][8][9][10][11][12]. In some instances, pupal pigmentation actually provided the first clue to the distinctness of cryptic Aphytis species. A. lingnanensis, for example, was first recognized as distinct from A. chrysomphali on the basis of this character [7]. In the A. lingnanensis group, A. fisheri DeBach, A. holoxanthus and A. melinus are considered sibling or near-sibling species, almost inseparable at the adult stage, but they are different in pupal pigmentation. The pupae of A. holoxanthus exhibit dark brown pigmentation on both the mesosomal sterna and the base of the metasomal sterna, those of A. melinus are pigmented only on the mesosomal sterna, whereas the pupae of A. fisheri are entirely yellow [1]. Yasnosh [13] provided a key for identification of the Aphytis species from the pupae and meconia (in the former USSR), and Prinsloo [14] gave a key to distinguishing the pupae of Aphytis parasitic on the California red scale on citrus in South Africa.
In southern China there is a major Aphytis parasitoid associated with the Florida red scale on citrus, which was mistakenly identified as A. chrysomphali for long time, and later was clarified as A. holoxanthus on the basis of the pupal pigmentation (the former with yellow pupa and the latter with partly dark brown pupa), as well as the characters of propodeal crenulae (the former belonging to A. chrysomphali group and the latter to A. lingnanensis group) and other adult characters [3,15].
Therefore, the aim of this study is to observe and document the taxonomic characters of pupal pigmentation and pupal development, in order to provide information useful for the separation of Aphytis species. We are not aware of any between-species or species group diagnostic studies in other Aphelinidae, nor indeed any other Hymenoptera genera.

Culture of Aphytis Pupae
Several species of armoured scale insects were collected from citrus and ornamental plants in the fields. The full-grown larvae or younger pupae of Aphytis species were removed individually from parasitized scale insect hosts, and placed each in an individually numbered plastic box (3 cm in diameter, 1.5 cm high), in which a filter paper was placed. The boxes were put into a climatic chamber for pupa culture, under the controlled conditions at 27± 1 • C, 70 ± 5% RH and a photoperiod of 10L: 14D. The pupal pigmentation and pupal development were observed and photographed daily, also adding a drop of water on the filter paper to maintain humidity. Pigmentation of mature, green-eyed pupae was recorded and compared for Aphytis species. Length of pupal development was measured Insects 2021, 12, 399 3 of 11 from the time of larval pupation until the formation of the mature pupa, indicated by the change in eye pigment to green.

Identification of Aphytis Species
The body colour of Aphytis species after emergence was initially photographed, and the specimens were preserved in 100% ethanol. Later the adult Aphytis specimens were photographed for details of the propodeal crenulae and thoracic tergum, and slide-mounted for species identification following the method outlined by Noyes [4].

Photography of Specimens
Specimens were photographed with a DS-Ri2 camera (Nikon, Tokyo, Japan) attached to a Nikon SMZ18 microscope with NIS-Elements D software and a DSC-T900 camera (Sony, Tokyo, Japan). The slide-mounted specimens were photographed using the Nikon DS-Ri2 camera and the same software attached to a Nikon Ni microscope equipped with differential interference contrast.

Results
Four characteristic pigmentation patterns of Aphytis pupae were recorded in this study, including: 1. entirely yellow; 2. partly dark brown; 3. entirely or predominantly black, and 4. partly black.

Type 1, Entirely Yellow Pupa
In this category, four Aphytis species with entirely yellow pupae were from the funicularis, chrysomphali, lingnanensis and proclia groups.    The pupa of A. lepidosaphes is entirely yellow, with pupal development lasting 7 days ( Figure 2).

Aphytis fisheri DeBach (lingnanensis Group)
The pupa of A. fisheri is entirely yellow, with pupal development lasting 5 days (    The pupa of A. hispanicus is actually entirely yellow. The melanization seen in the late stage of pupal development (Figure 4e-g) is that of the developing adult, evident through the integument of the fully developed pupa. The pupal development duration is 7 days (Figure 4).

Aphytis hispanicus (Mercet) (proclia Group)
The pupa of A. hispanicus is actually entirely yellow. The melanization seen in the late stage of pupal development (Figure 4e-g) is that of the developing adult, evident through the integument of the fully developed pupa. The pupal development duration is 7 days (Figure 4).

Type 2, Partly Dark Brown Pupa
In this type, the Aphytis species observed were characterized by dark brown pigmentation on both the thoracic and abdominal sterna, or on the thoracic and basal abdominal sterna, or only on the thoracic sterna of the pupae.

Type 2, Partly Dark Brown Pupa
In this type, the Aphytis species observed were characterized by dark brown pigmentation on both the thoracic and abdominal sterna, or on the thoracic and basal abdominal sterna, or only on the thoracic sterna of the pupae.

Aphytis lingnanensis Compere (lingnanensis Group)
A. lingnanensis is characterized by dark brown pigmentation on both the thoracic and abdominal sterna of the pupa, with pupal development lasting 6 days ( Figure 5

Aphytis holoxanthus DeBach (lingnanensis Group)
A. holoxanthus is characterized by dark brown pigmentationon on the thoracic and basal abdominal sterna of the pupa, with pupal development lasting 6 days ( Figure 6

Aphytis holoxanthus DeBach (lingnanensis Group)
A. holoxanthus is characterized by dark brown pigmentationon on the thoracic and basal abdominal sterna of the pupa, with pupal development lasting 6 days ( Figure 6).

Aphytis melinus DeBach (lingnanensis Group)
A. melinus is characterized by dark brown pigmentation only on the thoracic sterna of the pupa, with pupal development lasting 6 days (Figure 7).

Aphytis melinus DeBach (lingnanensis Group)
A. melinus is characterized by dark brown pigmentation only on the thoracic sterna of the pupa, with pupal development lasting 6 days (Figure 7).
A. melinus is characterized by dark brown pigmentation only on the thoracic sterna of the pupa, with pupal development lasting 6 days (Figure 7).

Aphytis longicaudus Rosen & DeBach (Unassigned Species)
The pupa of A. longicaudus is entirely black, with pupal development lasting 8 days (Figure 8).  The pupa of A. sankarani is predominantly black, except for being pale on the distad  The pupa of A. sankarani is predominantly black, except for being pale on the distad abdominal sterna, with pupal development lasting 6 days (Figure 9).

Pupal Development
The pupae of Aphytis species in this study had a developmental duration of about 5-8 days at 27 ± 1 °C, 70 ± 5% RH and a photoperiod of 10L: 14D. The experiments showed that the pupal skin was always melanized at beginning stage, generally in the first day, among the pigmented pupae of Aphytis species. As development continued, the pigmentation became darker and the eye colour from pale red/brown to green/black brown.

Discussion
Until now less than one-third of the known species of Aphytis have had their pupal pigmentation described (Table 1).

Pupal Development
The pupae of Aphytis species in this study had a developmental duration of about 5-8 days at 27 ± 1 • C, 70 ± 5% RH and a photoperiod of 10L: 14D. The experiments showed that the pupal skin was always melanized at beginning stage, generally in the first day, among the pigmented pupae of Aphytis species. As development continued, the pigmentation became darker and the eye colour from pale red/brown to green/black brown.

Discussion
Until now less than one-third of the known species of Aphytis have had their pupal pigmentation described (Table 1). Notes: (1) According to the record by Rosen and DeBach [1] and the authors following them. (2) Summary comment on the pupal pigmentation of proclia group by Rosen and DeBach [1]. (3) The 27 species in the vittatus group of Aphytis were transferred to the genus Paraphytis [32].
Of the known species of Aphytis (Table 1), the species in the chilensis and mytilaspidis groups, and some unassigned species, generally have entirely, or predominantly and or partly black pupae.
The species in the chrysomphali, funicularis, and proclia groups generally have the pupae entirely yellow. However, the pupal pigmentation of the proclia group seems to be generally mottled with fuscous; wing pads and appendages appear fuscous to black; dark areas or furcae are visible on both dorsal and ventral surfaces of mesosoma and metasoma; metasomal segments with dark patches dorso-laterally. Actually the pupal skin itself is usually not melanized at all. It is straw-yellow, and the melanization seen is that of the developing adult. Coarse, black spines are also usually evident through the integument of the fully developed pupa [1,8,9]. By comparison, the pupal skins of other types are melanized.
The species of the lingnanensis group have the pupae entirely yellow, e.g., A. fisheri, and partly dark brown pupae, e.g., A. lingnanensis, A. holoxanthus and A. melinus, which are almost inseparable by adult characters, but their differences in pupal pigmentation provides a method to separate these species.

Conclusions
This study summarized four characteristic pigmentation patterns of Aphytis pupae, including entirely yellow, partly dark brown, entirely or predominantly black, and partly black. No significant intra-specific variation in pupal colour pattern was detected despite relatively high numbers of specimens examined, many from multiple, and different, origins. Although one type of pupal pigmentation is shared with more than one species group, and pupal pigmentation is somewhat variable at times, for example the new pigmentation patterns were reported in some individuals of A. melinus and A. chrysomphali species respectively [33], the pupal pigmentation certainly may be regarded as an important supplementary diagnostic character, based on the morphological characters, and may even serve as a convenient shortcut to the separation of certain closely related species of Aphytis.