The Existence of Deuterotokous Reproduction Mode in the T. tabaci (Thysanoptera: Thripidae) Cryptic Species Complex

To begin the experiment, 12 adult females with the unknown age was isolated from the culture of each lineage (12 females from L1 and 12 females from T) type. They were reared individually in 2-ml micro-centrifuge tubes with single leaf discs of their ideal host plants (cabbage leaf discs for L1 and tobacco leaf discs were provided for T) as a food and ovi-position substrate. The leaf discs were replaced every 24 hours. The rearing has been carried out under the environmental growth chamber at 23 °C with 16 : 8 (light : dark). The eggs in the leaf discs have checked with an under-light stereomicroscope. The dead mothers were preserved in 96% alcohol and their mitochondrial cytochrome c oxidase subunit I (MT-COI) gene was sequenced to confirm the lineage (Farkas et al. 2020). The hatched first and second instar larvae were individually transferred to new tubes with fresh leaf discs and raised to the pupal stage. To test the pupal insemination, 42 female pupae from L1 and 44 female pupae from T type were used. In order to investigate the response of immature pupa upon mating with an adult male, a single female pupa was confined to a single adult male in a 2-ml micro-centrifuge tube for 24 hours. The response of female pupa to the adult male during mating was checked using a stereomicroscope.

To avoid the mating between newly emerged female adult with male adult, a male from the tube that contained the female pupa was removed before the newly female adult emerge and the female pupa was left on the leaf discs until they became an adulthood. The newly emerged female adults were reared for 8–10 days to produce eggs and all their newly hatched first and second instar larvae were collected and preserved in 75–80% ethanol alcohol. This immature preservation method was suggested by Morison (1947). After that, the preserved first and second instars of L1 and T types were slide mounted to identify their sex.

At a later period, 439 first and 219 second in-stars of L1 and T lineages were mounted by pouring few drops of Berlese mount medium on the microscopic flattened rectangular glass. After adding the larvae specimens to the drop of Berlese mounting medium, the microscopic glass was covered with a glass coverslip. To dry the specimens, the slides were kept in an oven at 50 °C for 2 days.

For immature identification of the first and second instar larva, the technique described by Speyer and Parr (1941) was used. This identification key for Thripidae is based on the number of setae on the II to VII abdominal segments and the number of setae on their pronotum, and the differences between first and second instar larvae are listed below.

First instar larvae is small in size when compared with the second instar larvae, and it comprises two pairs of sternal setae on the abdominal segments II to VII and six pairs of setae on the pronotum (Vierbergen et al. 2010). It also contains one pair of spiracle on the abdominal segment II (Kirk 1987).

The sex of the first and second instar larvae in the family Thripidae is determined based on their abdominal segment IX. The first instar male larvae exhibit four pairs of setae and female larvae exhibit three pairs of setae on their abdominal segment IX, whereas the second instar male larvae exhibit five pairs of setae and female larvae exhibit four pairs of setae on their abdominal segment IX (Vierbergen et al. 2010). All the features and sex identification of the first and second instar larvae were examined using Leica DMLB binocular light microscope (Leica Microsystem GmbH, Wetzlar, Germany) with 400×, 600× and 1000× magnifications.

To determine the female pupal insemination, the sex of the progeny was identified at first and second in-star pre-pupa, pupa, and adult stages. Thus, the female pupa was considered as inseminated by adult male if there is a single female from her progenies, and the female pupa was considered as not inseminated by adult male if all the progenies are males.

T type lineage is reproducing through the arrhenotokous (sexual) mode of reproduction in which the virgin females produce only males and the mated females produce both males and females. The sex identification of adult progeny was the method used to determine the mode of reproduction in T type. A virgin female producing only male progeny was considered as arrhenotoky female, and a virgin female producing a combination of male and female progeny was considered as a deuterotoky female. To confirm the arrhenotokous and deuterotokous modes of reproduction in the tested individuals, the following procedure was used: 20 female adults of unknown age were isolated randomly from T type culture and reared separately in 2-ml micro-centrifuge tubes. The tobacco leaf discs were used to serving as a food source and oviposition site. Before the tobacco leaf discs were provided as a food and ovi-position object, all tobacco leaves were checked under a light microscope to ensure that the leaf discs were not contaminated by other thrips. The progeny from these mothers were raised to adulthood also in isolation after egg hatching and were used as F₀ generation. The reason to use these progenies as F₀ generation is that their mothers were taken from the culture and had been exposed to high densities of males and experienced to mate by any other males and it makes difficult to determine whether the females were produced either through the arrhenotokous or deuterotokous mode of reproduction.

To test whether a mother and son inbreeding is the factor to induce deuterotokous mode of reproduction in T lineage, 10 females of unknown age were isolated randomly from stock culture and reared separately in 2-ml micro-centrifuge tubes. The tobacco leaf discs were free of contamination by other thrips species. The newly hatched progeny from these mothers were raised to adulthood. Those progenies were considered as F₁ generation and used as a parental generation for the subsequent inbred line. Thus, all the tested inbred generations were assumed to be related. Mother and son inbreeding has tested for two subsequent generations such as (F₁) females were daughters of females taken from stock culture and used as a parental generation in the experiment and (F₂) females were daughters of F₁ inbred females.

To ensure a possibility to obtain a son from virgin female, 30 virgin female adults of T lineage were isolated from the F₁ females and reared separately on the tobacco leaf discs for 2 days at 23 °C. As they were virgins, their eggs would not be fertilized and only sons would arise. Then the reproducing females were placed at 15 °C. At this low temperature, the mother's longevity has increased, which increases the chance for mating with their sons. However, some mothers died within a relatively short period before mating with their sons. They were excluded from this test. When the son became adult, single mother and son were confined into the same micro-centrifuge tube for 48 h. Then the male was removed, and its mother was kept isolated individually for the rest of her life. The same procedure was used for F₂ mothers and sons inbreeding test. The relatedness of isolated individuals in the F₁ and F₂ progenies were recorded, and F₂ sisters were produced by the same F₁ inbred female (Fig. 1).

Fig. 1

MT-COI genes and sex determination at the adult and nymphal stages were the criteria applied to describe the lineage and mode of reproduction, respectively. A virgin inbred female produced a combination of male and female was considered as a deuterotokous female.

The morphological identifications of these two lineages larvae were based on the number of setae on their pronotum and the number of setae on the abdominal segments II to III. As described in the methodology, first instar larvae consisted of one pair of setae on the ventral abdominal segments II to III and six pairs of setae on the pronotum (Figs. 2 A, B and 3 A, B).

Fig. 2

Fig. 3

On the basis of the morphological identifications of first and second instar larvae, the sex of the immature progeny was determined based on their number of setae on the abdominal segment of IX. First instar male larvae consisted of four pairs of setae on both dorsal and ventral of abdominal segment IX (Fig. 4) and second instar male larvae consisted of five pairs of setae on both dorsal and ventral abdominal segment IX (Fig. 5).

Fig. 4

Fig. 5

According to this experiment result, pupal insemination is not possible in the T. tabaci lineages within 24 hours of mating. Thus, all the tested females produced only male progeny (Table 1).

Deuterotokous reproduction mode has encountered during mother and son inbreeding test. At F₂ generation, the inbred virgin female taken from F₁ generation produced a combination of male and female progenies, and this female was considered as a deuterotoky ones (Table 2).

The deuterotoky females produced from in-bred females for three consecutive generations. About 20 females and 75 males were produced for two consecutive generations. F_1D female has produced 10 deuterotoky females, and 2 of them died at the pupal stage; of the 8 deuterotoky females, only 1 was continued to produce both males and females and the rest 7 females were turned to their original arrhenotokous mode of reproduction. Similarly, at F_2D generation, of the eight deuterotoky females, only one virgin female yielded both male and female progenies and the rest seven females were turned to their original arrhenotokous reproduction mode. After three generations, all the deuterotoky females turned to their original arrhenotokous mode of reproduction (Table 3).