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Courtship, Mating Behavior, and Ovary Histology of the Nymph Parasitoid Psyllaephagus bliteus (Hymenoptera: Encyrtidae)

Abstract The potential of the parasitoid Psyllaephagus bliteus Riek for the biological control of the eucalyptus pest Glycaspis brimblecombei Moore (Hemiptera: Aphalaridae) nymphs is high. This research sought to analyze the courtship, male competition, and mating behavior of P. bliteus at the proportions of 1:1 and 2:1 males to female in a Petri dish (5 cm diameter), and to describe the ovary histology of virgin and mated females of this parasitoid. At 1:1, males touch the antennae and thorax–abdomen of females during courtship, but females avoid mate attempts before they are 48 h old. At 2:1, the competition between male parasitoids inhibits mating. The histology of ovaries of virgin and mated P. bliteus females is similar, with two well-defined germarium and vitellarium regions, with oocytes at different developmental stages, including mature ones rich in yolk and with eggshell. A clearer understanding of the reproductive behavior and histology of P. bliteus aids in the use of this parasitoid for the biological control of G. brimblecombei.

Psyllaephagus bliteus was accidentally introduced with G. brimblecombei in Mogi-Guaçú region, São Paulo state, Brazil in 2003(Berti-Filho et al. 2003. This natural enemy parasitizes all the five nymphal instars of the red gum lerp psyllid, although the third and fourth instars are the main target. The parasitoid lays one or two eggs in the ventral region of the thorax or abdomen per nymph of the pest (Daane et al. 2005), but only one parasitoid emerges from each nymph. Psyllaephagus bliteus modifies the physiology of G. brimblecombei nymphs, increasing carbohydrate and protein excretion for its self-protection. Parasitoid development begins when the G. brimblecombei nymph reaches the fifth instar (Sullivan et al. 2006).
The parasitized nymphs of G. brimblecombei, 'mummies', are dilated with opaque colors, and the parasitoid larvae consume the internal organs in the abdominal cavity of the host (Plascencia-Gonzalez et al. 2005) with a development period of 12.6 d at 26°C (Daane et al. 2005). Psyllaephagus bliteus pupae are exarate and black and remain inside the host nymphs. The sharp or rounded genitalia of the females and males, respectively, allow to distinguish the sex of the parasitoid (Riek 1962). The parasitoid adult opens a hole at the posterior part of the G. brimblecombei nymph abdomen with its mandibles to emerge (Plascencia-Gonzalez et al. 2005).
Psyllaephagus bliteus controlled about 64% of G. brimblecombei nymphs (Margiotta et al. 2017), which is the most important strategy for the biological control of the pest in eucalyptus plantations worldwide. Environmental factors affect the populations and parasitism rates of P. bliteus, with values ranging from 0.21 to 7.03% in Brazil (Ferreira-Filho et al. 2015), 1.67 to 30.00% in California (Daane et al. 2012), and less than 15.00% in Italy (Caleca et al. 2018) and Portugal (Boavida et al. 2016). The longevity of adult parasitoids was negatively correlated with the temperature, from 15 to 5 d at field conditions (Daane et al. 2012). Its females lived longer than males under laboratory conditions (Daane et al. 2005; A. L. Favoreto, personal information). Mass releases of P. bliteus increase the parasitism rate on G. brimblecombei in the field (Ferreira-Filho et al. 2015, Tuller et al. 2017) and reduce the yield losses by this insect (Paine et al. 2015).
The efficient use of P. bliteus to manage G. brimblecombei requires knowledge on its reproduction, including the courtship and mating behavior ritual as the most important tasks (Cade 1985). The mating success varies with many factors, such as age, male competition, longevity, size (Joyce et al. 2009, Avila et al. 2016, maturation, and fecundity to mass produce the parasitoid in the laboratory.
The aim of this study was to describe, in laboratory conditions, the courtship, mating, male competition, and postmating behaviors of couples, as well as the ovary histology of virgin and mated P. bliteus females.

Glycaspis brimblecombei and Psyllaephagus bliteus Rearing
The experiments were conducted at the Laboratory of Biological Control of Forest Pests (LCBPF) in São Paulo State University (UNESP), School of Agricultural Sciences in Botucatu, São Paulo state, Brazil. Glycaspis brimblecombei was caged (40 cm × 45 cm × 80 cm) with Eucalyptus camaldulensis and/or Eucalyptus grandis × E. camaldulensis seedlings at 25 ± 2°C, 60 ± 10% RH, and 12:12-h light: dark photoperiod (Toctagon Tubular LED T8-9W, 50/60 HZ). One hundred G. brimblecombei adults were released per cage and, after 7 d, the eggs found on the eucalyptus seedlings were counted. These seedlings were monitored every 3 d to follow G. brimblecombei nymph development until the adult stage (Firmino-Winckler et al. 2009, Wilcken et al. 2010. Psyllaephagus bliteus adults were obtained from mummies of G. brimblecombei collected in E. camaldulensis plantation at UNESP Teaching, Research and Extension Farm. These adults were collected using a manual insect aspirator, sexed (Riek 1962), fed on bee honey drops, and reared with G. brimblecombei under the aforementioned conditions. Ten to 15 pairs of P. bliteus were released into each cage when 50% of G. brimblecombei nymphs reached the third instar. After 15 d, nymphs of G. brimblecombei were verified and its parasitized reserved, parasitoid pupae individualized in Petri dishes (3 cm diameter) until adult emergence (Wilcken et al. 2010), which were individually placed and fed ad libitum with bee honey inside similar Petri dishes.

Description of Psyllaephagus bliteus Courtship and Mating Sequence
Psyllaephagus bliteus courtship and mating behavior were analyzed through direct observation under stereoscopic microscope ZEISS, Leica Application Suite (version 2.1.0) software. Adults parasitoids were sexed after emergence (Riek 1962) and fed with honey bee. After this, the behavior of 50 unmated couples (1 male: 1 female) of P. bliteus with different ages and 20 combinations (2 males: 1 female) were analyzed. Then at 1:1, fifty couples were formed to 10 couples of newly-emerged parasitoids, 10 of 24 h-old females and newly-emerged males, 10 of newly-emerged females and 24 h old males, 10 of 48 h old females and newly-emerged and males, and 10 of newly-emerged females and 48 h old males. At 2:1, all 20 combinations of parasitoids were newly emerged. Pairs of parasitoids were formed and analyzed daily for 15 d, without replacing the dead males or females (Table 1). The experiments were carried out at 25 ± 2°C with one pair or combination per Petri dish (5 cm diameter) for 20 min. Males and females were individualized until the moment of the test and after the analysis period. The behavioral sequences (identification, courtship, mating and postmating) associate with 474 observations in the laboratory, and successful and unsuccessful mating attempts were analyzed every day until one died, as well the period for the male and female to find each other, number of matings (duration), and postmating behaviors were recorded.
Preliminary assays did not indicate variations in parasitoid behavior and mating activity in the morning, afternoon, twilight, or night during observations for 60, 40, 30, or 20 min. The male parasitoid was not more active at any period, to find the females and to try to mate.

Ovary Histology
Ten virgin females of P. bliteus younger than 24 h and 10-dold mated females, kept together with males, were transferred to Carnoy's fixative (chloroform: ethanol: acetic acid, 6: 3: 1) and were then sent to the Cell Ultrastructure Laboratory of the Federal University of Viçosa in Viçosa, Minas Gerais state, Brazil. These females were dehydrated in a graded ethanol series (70, 80, 90, and 95%) for 10 min each and embedded in historesin (Leica). Samples were sectioned with 2 µm thickness in a rotative microtome, stained with hematoxylin (12 min) and eosin (30 s), and analyzed with a light microscope Olympus BX-60.

Statistical Analyses
Before the analysis, data were submitted to normality test. The statistical analysis were performed using Kruskal-Wallis, t-test (significant P-value < 0.05) for data period time for male to find the female, and Mann-Whitney U statistic (significant P-value < 0.05) to compare the frequency of male behavior between unmated and mated couples and those of mating to the nonparametric since its dataset was not normally distributed. Data were analysed using Sigma Plot v. 11.0. for Windows software.

Results
The conditional behavioral sequences were divided into the following phases: identification, courtship, mating, and postmating in ethogram (Table 2; Fig. 1).
Psyllaephagus bliteus males were active during courtship, whereas females remained passive with low response movements. Male parasitoid wasps that were released into Petri dish arena ran around the arena (latency stage) to recognize the area to begun courtship just after finding the female, whereas females stood still or, sometimes, also surveyed the arena. The male found the female in 14 s (H = 1.322; P = 0.858), regardless of its age (Table 3), approached, paired in front of it, touched it with its antennae, and, in some cases, intertwined its antennae with that of the female with a slight back and forth movement. Afterwards, the frequency of male antennae tapping the female thorax and abdomen, followed by chasing as a mating attempt, did not differ between matted or unmated males (Mann-Whitney U = 28.50, P = 0.318; Table 4). Nonreceptive females repelled males, walking off or flying away. These events were the same for newly emerged, >24 h old or for pairs with different ages. Males released into the arena immediately started courtship, repeating the whole process-antennae drum the female antennae (t(18) = 1.365, P = 0.191; Table 3) and thorax and abdomen (t(18) = 1.442, P = 0.169; Table 3). Females, younger than 48 h old or older, rejected male courtship, but some of them, 48 h old or older, were receptive, fanning their wings and abdomen (Mann-Whitney U = 0.00, P < 0.001; Table 4). In this case, the male prepared to mount or aligned its body on the opposite side, below the female body (Mann-Whitney U = 0.00, P < 0.001) ( Table  4; Fig. 2A).
Eight matings were observed with a mean duration of 15.4 s, varying from 5 to 32 s (5,7,9,13,15,18,24, and 32 s) (Table 4). Males mounted from the posterior (five of eight) or aligned under the female body (three of eight). The receptive females, 48 h old (four of eight) or older (one of 72 h old and 144 h old, one of 96 h old), fanned their wings and slowly abdomen movements. Newly emerged males initiated courtship and mated (Fig. 2B). Postmating, the parasitoids again ran or stayed still in the arena.
Males, in the 2 males: 1 female combination per arena, walked (latency stage) and, when they met each other, confronted one another with antennal movements (aggressive display 1), sometimes moving their wings (aggressive display 2) and returned to find their rival for a subsequent antennal confrontation. In this case, no courtship or mating occurred (Table 2; Fig. 1).
The reproductive tracts of virgin or mated P. bliteus females were similar (Figs. 3A-D and 4A-D). Their ovarioles were of polytrophic meroistic type with evident germarium and vitellarium regions. Large and undifferentiated oogonia and small prefollicular cells were found in the germarium. The cells undergo differentiation in the basal portion of the germarium with the nurse chambers larger than the oocytic ones, both surrounded by a single layer of follicular cells (Figs. 3A and 4A). As the oocytes moved to the vitellarium region, they increased in size and were surrounded by columnar follicular cells, resulting in an ovocytic chamber larger than the nurse ones (Figs. 3B and 4C). A total degeneration of nurse and follicular cells with a flattened aspect followed the chorion synthesis in mature oocytes (Figs. 3B, 3C, and 4D).

Discussion
The male P. bliteus started courtship after recognizing the female with antennal tapping on female antennae and/or thoraxabdomen, a behavior crucial for Encyrtidae parasitoids, such as Leptomastidea abnormis Girault and Anagyrus pseudococci Girault (Hymenoptera: Encyrtidae) (Romano et al. 2016(Romano et al. , 2018. Courtship behavior can stimulate female receptivity, as suggested for Ibalia japonica Matsumura (Hymenoptera: Ibaliidae) and  Means followed by the same letter do not differ (P < 0.05). Sphex ingens Smith (Hymenoptera: Sphecidae), reducing courtship period and increasing reproductive success (Souza et al. 2015(Souza et al. , 2016Kuramitsu et al. 2019). The behavior of P. bliteus males intertwining females with antennae did not guarantee mating, but the females of the parasitoid Cephalonomia tarsalis Ashmead (Hymenoptera: Bethylidae) allowed mating after similar courtship behavior (Cheng et al. 2004). Psyllaephagus bliteus females, 48 h old or older, are receptive to fanning wings and abdomen movements, preparing males for mating, as described for the parasitoids L. abnormis and A. pseudococci. These females bend their abdomen dorsally and spread their wings, preparing to copulate (Romano et al. 2016(Romano et al. , 2018. Cleruchoides noackae Lin & Huber (Hymenoptera: Mymaridae) mated shortly after emergence, without courtship or female receptivity for additional copulations (Mutitu et al. 2013, Becchi et al. 2020. Rejection of mating attempts is a common behavior among females, wherein they walk or fly away from the male (Avila et al. 2016, Romano et al. 2016, such as that observed for P. bliteus. However, S. ingens females are more aggressive, partially opening their mandibles and tilting the body vertically (Souza et al. 2015).
The presence of the male competitor results in antennae-antennae fighting in P. bliteus as found for Ceratosolen gravelyi Grandi (Hymenoptera: Agaonidae) males with violent combat with biting on their mid or hindleg . Cotesia urabae Austin & Allen (Hymenoptera: Braconidae) males try to interrupt mating by pulling the successful male away from the female (Avila et al. 2016). The mating failure by S. ingens male manipulated the female exposure time and reduced the opportunity for other competing males (Souza et al. 2016).  The duration of 5-32 s for the P. bliteus mating when females were receptive to males with fanning wings and abdomen movements is also found for the other parasitoids. The behavior in which the female 'dances', reported for L. abnormis, results in a 5.7-s mating duration (Romano et al. 2016). Nevertheless, mating period varies with species, with 2-50 s for Cl. noackae (Mutitu et al. 2013, Becchi et al. 2020) and 23.3 s for C. tarsalis (Cheng et al. 2004). Furthermore, P. bliteus males court virgin or mated females, similar to Anastatus disparis Ruschka (Hymenoptera: Eupelmidae) Hao 2019, Liu et al. 2020), whereas Co. urabae was attracted only by virgin females (Avila et al. 2016).
The oocytes in the ovarioles of both virgin or mated P. bliteus females were at different maturation stages, including mature ones, characterizing this species as synovigenic, whereby newly emerged females present both young and mature oocytes (Jervis et al. 2001) (Souna et al. 2017), and Eibesfeldtphora tonhascai Brown (Diptera: Phoridae) (Farder-Gomes et al. 2019). The synchrony between egg maturation and P. bliteus longevity allows their females to oviposit in several host nymphs shortly after emergence, with a higher oviposition rate in the first 22 d of the parasitoid lifespan in the laboratory (Daane et al. 2005). However, P. bliteus longevity in the field is lower (Daane et al. 2012) but with a high parasitism rate immediately after its mass release and reaching more than 70% in the first 15 d and drops to 37% after 30 d (Ferreira-Filho et al. 2015). The decreased parasitism rate of P. bliteus has been correlated with its egg production and longevity, but it varies with arrhenotokous parthenogenesis (C. F. Wilcken, personal information), where males develop from unfertilized eggs (Godfray 1994, Heimpel andde Boer 2008). This is a disadvantage for the biological control because females are responsible for parasitism.
Psyllaephagus bliteus females and males are solitary parasitoids, i.e., females and male do not emerge at the same time. Mating is a social behavior and this natural enemy copulates with various partners. Females of this parasitoid emerge with mature eggs and present parasitism and mating capacity, mainly, after they are 48 h old. Individuals of both sexes should remain together in laboratory rearing for at least 48 h to ensure that they mate.