Diversity and molecular identification of endosymbionts of the white-flies Bemisia tabaci and Trialeurodes vaporariorum

The infection of insects with symbiotic bacteria has significant implications for the evolution and ecology of the hosts. Maternally inherited symbionts associated with B. tabaci and T. vaporariorum white-flies play a vital role in their fitness and survival. Whitefly symbionts have been identified in many countries, but no study has been undertaken in Iraq and the UK. For the first time in both countries, the molecular identification and diversity of the symbionts of both white-flies have been investigated in the present study. Fourteen populations of B. tabaci from Iraq and twenty populations of T. vaporario-rum from the UK were used to detect and identify seven common endosymbiont bacteria associated with whitefly using the 16S rRNA and 23S rRNA nuclear markers. All females and males of B. tabaci harboured one primary symbiont, Portiera aleyrodidarum, and almost all of both sexes of all B. tabaci species have the two secondary symbionts Hamiltonella sp. and Rickettsia sp. The primary symbiont P. aleyrodidarum was also detected in both sexes of T. vaporariorum, whereas only one secondary symbiont, Arsenophonus sp., was detected in almost all females but not in the males. Additionally, an investigation into genetic diversity using three genes of the Ar-senophonus sp. populations showed no variation among different populations. The results supported the notion that Arsenophonus sp. might play an important role in the survival of T. vaporariorum females and maybe a killer of male whiteflies. Also, secondary symbi-onts Hamiltonella sp. and Rickettsia sp. with B. tabaci could support their host’s fitness and survival. These findings reveal the endosymbionts associated with B. tabaci and T. vaporariorum in Iraq and the UK, respectively. Further investigation is needed to understand the roles of these symbionts in both countries .


Introduction
Endosymbiosis is vital in insect-plant interactions, affecting numerous aspects of herbivorous ecology.[1] described hundreds of different bacterial endosymbionts of herbivores through their anatomy.Symbiotic bacteria have traditionally been classified as primary or secondary endosymbionts.Relations among hosts and primary symbionts are often ancient, with an expected history of 30-250 million years [2].Primary symbionts are inherited entirely vertically through the germline to offspring.They are normally considered mutualistic symbioses and are commonly required for host fitness, survival, and reproduction.The endosymbionts are adapted to the hosts' diet by supplying vital nutrients, which are obligated to both partners [3].Obligate symbionts are located in particular host cells that might constitute a larger organ-like structure called the bacteriome.It has been reported that 15% of insect species harbour a primary symbiont [1].
Secondary symbionts are considered facultative endosymbionts from the host's perspective and have a shorter coevolutionary history with the host species [4].Some secondary symbionts are uncommon, whereas others are fixed in their hosts [5,6].Facultative symbionts are usually located in specific host tissues, such as fat bodies, muscle, nervous tissue, and the gut, but they might also be found in the haemocoel of their host, and they occur at lower titres than primary endosymbionts [7,8].Secondary symbiotic bacteria are commonly transmitted vertically, but in some cases, horizontal transmission between hosts might occur [9,4,10].
Whiteflies are known to host the obligatory symbiont Portiera aleyrodidarum, which has a long coevolutionary history with all species of the Aleyrodinae subfamily [11].In addition to the primary endosymbiont, whiteflies contain a range of secondary symbionts, including species of Hamiltonella sp., Cardinium sp.(Bacteroidetes), Fritschea sp., Wolbachia sp., Arsenophonus sp., and Rickettsia sp.(Rickettsiales) [12,13].Both the endosymbiotic bacteria and mtDNA are vertically transmitted and are linked with the evolutionary history of their hosts, and consequently might be used to shed light on evolutionary processes relating to both sides of symbiosis [14,15].
Endosymbiotic bacteria have been reported to have effects on various aspects of host biology, including genetic diversity, nutrition, survival, reproduction, insecticide resistance, and the ability to cope with environmental factors [16,17].The primary symbiont Portiera s Supplements the hosts' diet with essential nutrients like amino acids and carotenoids that provide significant anti-oxidant action [18].Additionally, secondary symbionts contribute to pest hosts and may play negative or even decisive roles in the survival of their hosts.For instance, secondary symbionts such as Wolbachia sp. can provide nutrients [19], initially increase host resistance to parasitic wasps and pathogens [20], and may also increase tolerance to heat stress [21].However, at the same time, some secondary endosymbionts, such as Wolbachia sp., Arsenophonus sp., Cardinium sp. and Rickettsia sp., have been reported to be parasitic rather than useful to their hosts [22].Endosymbionts influence the reproductive sys-tems of insects by imposing asexuality, being male-killers, and feminising genetic males.Also, the endosymbionts encourage cytoplasmic incompatibility (CI) together with parthenogenesis; all these aspects help the symbionts to spread their infections in host populations [23,15,24].
In the case of whitefly, secondary endosymbionts have been found to affect several aspects of the performance of their hosts, for instance, in increased resistance to parasitoids [25], tolerance to high temperatures [26], the capacity to transmit viruses [27], and susceptibility to pesticides [28,29].[30] revealed that the MEAM1 genetic group of B. tabaci infected with Rickettsia in the US exhibited significantly increased fitness.Also, there was an increase in female bias in their host populations.The symbionts could perform two functions, being mutualistic and reproductive manipulators for their host insect, which could positively affect the host population size, and spread the symbiont in the field.Additionally, the secondary endosymbionts Cardinium and⁄ or Arsenophonus in B. tabaci might influence interbreeding among whitefly biotypes [31].
The secondary symbionts Rickettsia sp. and Hamiltonella sp. are known to be harboured by specific B. tabaci biotypes and play important roles in their fitness.For instance, Rickettsia sp.linked with B. tabaci MEAM1 genetic group has been reported as unable to synthesise some nutritional substances such as amino acids.Therefore, Rickettsia sp. in biotype B needs to obtain nutrition from its host [32].In addition, the secondary symbiont Hamiltonella sp. also increases its host's resistance to parasitoid wasps [33].Also, Hamiltonella sp.linked with B. tabaci MEAM1 might play an important role in assisting the invasion of MEAM1 throughout the world [34] and is suggested to increase the transmission capacity of plant viruses, especially TYLCV [27,35].
Bacterial diversity in whitefly has been studied in several regions of their distribution, but there is as yet no data concerning T. vaporariorum and B. tabaci symbionts in the UK and Iraq.Thus, this study aims to investigate the endosymbionts associated with T. vaporariorum and B. tabaci populations from the UK and Iraq, respectively.The results report the presence of primary and secondary symbionts of whitefly in both countries.The results might improve our understanding of the role of symbiotic bacteria in whitefly and may support the development of better whitefly management.

Field sampling
The locations and host plants of samples of whiteflies collected from Iraq and the UK are described and detailed in Table (1).

Confirming the identity of whitefly molecularly and morphologically
The morphological and molecular techniques used to identify the B. tabaci and T. vaporariorum are described [36,37,38].

Molecular identification and sequencing of endosymbionts
The total gDNA of 10 males and ten females from each population of B. tabaci and T. vaporariorum was used to detect the presence of obligate and facultative bacterial symbionts.The PCR was performed using species-specific markers for the 16S rRNA genes in Portiera sp., Wolbachia sp., Rickettsia sp., Hamiltonella sp., and Cardinium sp. and the 23S rRNA genes in Arsenophonus sp. and Fritschea sp.(Table 2).The protocol of PCR amplification, as in [36,37], was used.Additionally, to check the quality of DNA extraction, samples that tested negative for all symbiotic bacteria were cross-checked for the primary endosymbiont P. aleyrodidarum using primers 518f and 799r of the 16S rRNA gene to check the DNA quality.Also, adults of both B. tabaci and T. vaporariorum positive for secondary symbionts were included to test for the reliability of the PCR testing.The following conditions for PCR reactions were used: initial denaturation at 93 °C for 2 min, followed by 35 cycles of 93 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min.The PCR products were visualised on 2% agarose gels containing ethidium bromide and were purified using ExoSap as described in [39].

Characterisation of Arsenophonus sp. diversity
One to two Arsenophonus sp.positive individuals were randomly chosen from each T. vaporariorum sample (representing both UK mtCOI haplotypes and all geographic locations) for use in multi-locus sequence typing (MLST).The PCR and sequencing of three housekeeping genes of Arsenophonus sp.(ftsK, yaeT, and fbaA) were carried out using the primers described in Table 2 [40,41].The same PCR reac-tion was used as described in [39] (Kareem 2018), and the appropriate annealing temperature was used for each reaction, as indicated in Table 2.

Sequence alignment and phylogenetic analysis
All of the symbiont DNA sequencings were performed and visualised on a 3130XL Genetic Analyzer as described in the mtCOI sequencing procedure.The sequences obtained were checked using Geneious, version 6.1.4[42].All sequences were compared with those in the GenBank database using the NCBI BLAST algorithm.Single sequences of primary and secondary endosymbionts of B. tabaci were deposited in NCBI GenBank under accession numbers KY465885, KX679579, and KX679580, respectively.Also, sequences of a primary and a secondary endosymbiont of T. vaporariorum were deposited in GenBank under accession numbers KY457224 and KY243936.Additionally, the Arsenophonus sp.gene sequences were deposited in NCBI GenBank under the accession numbers KY626170-KY626172 for fbaA, ftsK and yaeT genes, respectively.The phylogenies were estimated using maximum likelihood (ML) using MEGA 6, as described in [39].

Table (2):
Primers are used to screen the primary and secondary symbionts in whitefly species [41].Ann.: annealing temperature.Amp.Size: amplification product size.

Results and Discussion Confirmation of the identity of specimens molecularly and morphologically
Both whitefly species have been confirmed morphologically and molecularly as T. vaporariorum and B. tabaci in the UK and Iraq, respectively.

Symbionts
The results for the symbionts of T. vaporariorum showed that the primary symbiont P. aleyrodidarum was identified in almost all samples of both sexes, indicating that the DNA extracts were good quality.The infection status of T. vaporariorum was 96.6% for one secondary symbiont, Arsenophonus sp., in the females, and it was not present in any of the males.At the same time, no PCR products were found for the other symbionts (Table 3).The PCR products were sequenced to confirm the genus and species of symbiotic bacteria using the corresponding NCBI GenBank databases, the sequence length of Arsenophonus sp.23S rRNA was 447 bp, whereas the sequence for the primary endosymbiont P. aleyrodidarum 16S rRNA was 784 bp in length.
The analyses of the P. aleyrodidarum and Arsenophonus sp.sequences from 20 T. vaporariorum populations showed no polymorphisms within species.All the P. aleyrodidarum and Arsenophonus sp.sequences obtained from 20 populations of T. vaporariorum are identical to those sequences deposited in GenBank under accession numbers KY457224 and KY243936, respectively.The P. aleyrodidarum sequence matched 100% with the GenBank sequences with accession numbers CP004358 and Z11928 [43], and secondary symbionts Arsenophonus sp.matched 99% with the Arsenophonus sp.isolated from India with the accession number KJ541957.
For Iraqi B. tabaci, the primary symbiont P. aleyrodidarum was identified in all samples in both sexes, again indicating that the DNA extracts were of good quality.The infection status of B. tabaci was 96.4% for the secondary symbionts Hamiltonella sp. and Rickettsia sp. in both sexes, while no PCR products were found for the other symbionts considered (Table 3).The PCR products on the gels were sequenced to confirm the secondary species of symbiotic bacteria.Sequences for P. aleyrodidarum, Hamiltonella sp. and Rickettsia sp.matched 100% to the corresponding sequences of each of the symbiont species available in NCBI GenBank [43,34].The analyses of the P. aleyrodidarum, Hamiltonella sp. and Rickettsia sp. from 14 B. tabaci populations showed no polymorphisms within species.All the 16S rRNA sequences of the P. aleyrodidarum, Hamiltonella sp. and Rickettsia sp. were identical to those sequences deposited in GenBank under accession numbers KY465885, KX679580 and KX679579 with total lengths 623, 676, and 768 bp, respectively.

Genetic characterisation of Arsenophonus sp.
The sequences of three housekeeping genes of the secondary endosymbiont Arsenophonus sp. of glasshouse whitefly T. vaporariorum showed a 100% match to the NCBI GenBank database sequences.In the three bacterial genes investigated for MLST analysis, with total lengths of 587, 382, and 335 bp for fbaA, yaeT, and ftsK, respectively, no polymorphism was detected in 20 populations of whitefly collected from the UK.For the first time, this study presents the identification of endosymbionts of T. vaporariorum in the UK and B. tabaci populations in Iraq, respectively.T. vaporariorum populations from the UK harboured just one secondary symbiont, Arsenophonus sp., in females but not males.This finding is identical to that of another study that showed that males of T. vaporariorum from Japan did not harbour Arsenophonus sp., even though females from this population in various countries were all infected [41] (Kapantaidaki et al., 2015).In contrast, populations of this species in Croatia, Bosnia, and Herzegovina harboured both Arsenophonus sp. and Hamiltonella bacterial symbionts found in females [44,45].A more diverse community of bacterial symbionts was recorded in T. vaporariorum populations from Montenegro, where the populations harboured Rickettsia, Hamiltonella, Arsenophonus, Wolbachia, and Cardinium [46].However, B. tabaci populations from Iraq harboured the same obligatory primary symbiont P. aleyrodidarum and the two secondary symbiotic bacteria Hamiltonella sp. and Rickettsia sp.This finding was similar to other studies [47,32].Other mtCOI biotypes of B. tabaci harboured different species of secondary symbionts.For example, the symbiotic bacteria of the Mediterranean (MED) species (including the common biotype (Q) vary among regions.French and Uruguayan populations of MED Q1 were infected with Cardinium sp. and Hamiltonella sp. at high frequencies [6].However, in Greek and West African populations and a laboratory population representing MED Q1 in China, approximately 100% infection was found with Hamiltonella sp. but not with Cardinium sp.[48,49,50].There are doubts about the role of Hamiltonella sp. with B. tabaci biotypes.For instance, [35] demonstrated a link between the capacity of B. tabaci biotypes to harbour Hamiltonella sp. and to transmit TYLCV.In contrast, the MED population in the same study without the secondary symbiont Hamiltonella sp. was ineffective in transmitting the virus.Therefore, it would be interesting to know the role of secondary symbionts since new strains of TYLCV have recently been recorded in Iraq and might be transmitted by new biotypes harbouring Hamiltonella sp.As a result, the primary role of Hamiltonella sp. in virus transmission in the various B. tabaci biotypes needs to be further investigated.
It has been reported that B. tabaci biotypes that harbour Rickettsia sp.might be linked to insecticide resistance, increased host resistance against parasitoid wasps, and increased whitefly fitness and female bias [28,25,30].Rickettsia sp. of B. tabaci MEAM1 isolated was confirmed to be linked with a reduced capacity of whitefly to resist pesticides and immunoreactions against parasitic wasps [28,25].Therefore, Rickettsia sp.linked with Iraqi B. tabaci could play the same role as above to make its host more fit and able to survive.
The results of a further investigation of the genetic diversity in secondary symbionts of the UK whitefly showed no genetic diversity within Arsenophonus sp.infecting T. vaporariorum, despite its prevalence in this species.The sequences obtained from the fbaA, ftsK, and YaeT housekeeping genes were identical for all our positive samples of Arsenophonus sp. in T. vaporariorum.On the other hand, the sequence analysis of fbaA, ftsK, and YaeT revealed genetic diversity within Arsenophonus infecting B. tabaci, but this diversity was highly correlated with the different B. tabaci biotypes [40] (Mouton et al., 2012).In the same study, almost no polymorphism was found in the Arsenophonus gene sequences from African T. vaporariorum samples, which was identical to the present finding.
The low polymorphism of the secondary symbiont Arsenophonus sp.within T. vaporariorum populations, alongside its high occurrence in T. vaporariorum, is consistent with an established and vertically transmitted endosymbiont.The information concerning the symbionts and mtCOI diversity of T. vaporariorum confirmed and supported the idea that there are no biotypes in T. vaporariorum.However, more sec-ondary symbionts and high mtCOI diversity reported from Iraqi whitefly are consistent with the complex species of B. tabaci [40,51,52,53].
These findings provide an initial database for further investigating symbiotic bacteria associated with whiteflies in the UK and Iraq.Further study of the role of these symbionts and their diversity is needed to update the status of T. vaporariorum and B. tabaci.The outcomes may potentially influence the management of Whitefly.
The presence of Portiera sp., an obligate endosymbiont in T. vaporariorum haplotypes H1 and H3 in the UK, was found in both sexes, whereas the facultative symbiont Arsenophonus sp. was detected in females but not males.analysis of the four B. tabaci biotypes in Iraq showed the presence of Portiera sp., an obligate endosymbiont.In contrast, the facultative symbionts Hamiltonella sp. and Rickettsia sp. were also detected in most individuals.

Table ‫المستند .
‬ ‫في‬ ‫المعين‬ ‫النمط‬ ‫من‬ ‫نص‬ ‫يوجد‬ ‫ال‬ ‫:)1(خطأ!‬Collection sites, population codes, dates of collection, host plants, and coordinates for the glasshouse whitefly T. vaporariorum and B. tabaci sampled from the UK and Iraq examined in this study.