Regulation of forkhead box O transcription factor by insulin signaling pathway controls the reproductive diapause of the lady beetle, Coccinella septempunctata.

In biological control programs, knowledge about diapause regulation in natural enemy insects provides important insight for improving long-term storage, transportation, and field adoption of these biological control agents. As a natural predator of agricultural pests, the lady beetle Coccinella septempunctata has been commercially mass-cultured and widely employed in pest management. In some insects, insulin signaling, in conjunction with the downstream transcription factor Forkhead box O (FoxO), are master regulators of multiple physiological processes involved in diapause, but it is unclear whether insulin signaling and FoxO affect the diapause of C. septempunctata. In this study, we use a combination of approaches to demonstrate that insulin signaling and FoxO mediate the diapause response in C. septempunctata. In diapausing beetles, application of exogenous insulin and knocking down expression of CsFoxo with RNA interference (RNAi) both rescued beetles from developmental arrest. In non-diapausing beetles, knocking down expression of the insulin receptor (CsInR) with RNA interference (RNAi) arrested ovarian development and decreased juvenile hormone (JH) content to levels comparable to the diapause state. Taken together, these results suggest that a shutdown of insulin signaling prompts the activation of the downstream FoxO gene, leading to the diapause phenotype.

RNA Interference in the Egg Parasitoid, Trichogramma dendrolimi Matsumura.

The egg parasitoids, Trichogramma spp, are recognized as efficient biological control agents against various lepidopteran pests in agriculture and forests. The immature stages of Trichogramma offspring develop within the host egg, exhibiting remarkable diminutiveness (approximately 0.5 mm in adult length). RNA-interference (RNAi) methodology has emerged as a crucial tool for elucidating gene functions in numerous organisms. However, manipulating RNAi in certain small parasitoid species, such as Trichogramma, has generally posed significant challenges. In this study, we present an efficient RNAi method in Trichogramma denrolimi. The outlined procedure encompasses the acquisition and isolation of individual T. dendrolimi specimens from host eggs, the design and synthesis of double-stranded RNA (dsRNA), the in vitro transplantation and cultivation of T. dendrolimi pupae, the micro-injection of dsRNA, and the subsequent assessment of target gene knockdown through RT-qPCR analysis. This study furnishes a comprehensive, visually detailed procedure for conducting RNAi experiments in T. dendrolimi, thereby enabling researchers to investigate the gene regulation in this species. Furthermore, this methodology is adaptable for RNAi studies or micro-injections in other Trichogramma species with minor adjustments, rendering it a valuable reference for conducting RNAi experiments in other endoparasitic species.

The identification and expression pattern of the sex determination genes and their sex-specific variants in the egg parasitoid Trichogramma dendrolimi Matsumura (Hymenoptera: Trichogrammatidae).

Introduction: Trichogramma wasps are egg parasitoids of agricultural lepidopteran pests. The sex of Trichogramma is determined by its ploidy as well as certain sex ratio distorters, such as the endosymbiotic bacteria Wolbachia spp. and the paternal sex ratio (PSR) chromosome. The sex determination systems of hymenopterans, such as Trichogramma spp., involve cascades of the genes transformer (tra), transformer-2 (tra2), and doublesex (dsx) and are associated with sex-specific tra and dsx splicing. First, these genes and their sex-specific variants must be identified to elucidate the interactions between the sex ratio disorders and the sex determination mechanism of Trichogramma. Methods: Here, we characterized the sex determination genes tra, tra2, and dsx in Trichogramma dendrolimi. Sex-specific tra and dsx variants were detected in cDNA samples obtained from both male and female Trichogramma wasps. They were observed in the early embryos (1-10 h), late embryos (12-20 h), larvae (32 h and 48 h), pre-pupae (96 h), and pupae (144 h, 168 h, 192 h, and 216 h) of both male and female T. dendrolimi offspring. Results: We detected female-specific tra variants throughout the entire early female offspring stage. The male-specific variant began to express at 9-10 h as the egg was not fertilized. However, we did not find any maternally derived, female-specific tra variant in the early male embryo. This observation suggests that the female-specific tra variant expressed in the female embryo at 1-9 h may not have originated from the maternal female wasp. Discussion: The present study might be the first to identify the sex determination genes and sex-specific gene splicing in Trichogramma wasps. The findings of this study lay the foundation for investigating the sex determination mechanisms of Trichogramma and other wasps. They also facilitate sex identification in immature T. dendrolimi and the application of this important egg parasitoid in biological insect pest control programs.

Posterior concentration of Wolbachia during the early embryogenesis of the host dynamically shapes the tissue tropism of Wolbachia in host Trichogramma wasps.

Introduction: The bacterial endosymbiont, Wolbachia spp. induce thelytokous parthenogenesis in certain parasitoid wasps, such as the egg parasitoid wasps Trichogramma spp. To complete the cycle of vertical transmission, Wolbachia displays efficient transovarial transmission by targeting the reproductive tissues and often exhibits strong tissue-specific tropism in their host. Method: The present study aimed to describe the basic Wolbachia distribution patterns that occur during the development of Wolbachia-infected, thelytokous Trichogramma dendrolimi, and T. pretiosum. We used fluorescence in situ hybridization (FISH) to investigate Wolbachia signal dynamics during early embryogenesis (from 30 to 120 min). Wolbachia titers and distributions from the embryo to adult stages of Trichogramma after early embryogenesis were detected by absolute quantitative polymerase chain reaction (AQ-PCR) and FISH. The symmetry ratios (SR) of the Wolbachia signals were calculated using the SR odds ratios in the anterior and posterior parts of the host. The SR was determined to describe Wolbachia tropism during early embryogenesis and various developmental stages of Trichogramma. Results: Wolbachia was concentrated in the posterior part of the embryo during early embryogenesis and the various developmental stages of both T. dendrolimi and T. pretiosum. Wolbachia density increased with the number of nuclei and the initial mitotic division frequency during early embryogenesis. The total Wolbachia titer increased with postembryogenesis development in both T. dendrolimi and T. pretiosum. However, the Wolbachia densities relative to body size were significantly lower at the adult and pupal stages than they were at the embryonic stage. Discussion: The present work revealed that posterior Wolbachia concentration during early host embryogenesis determined Wolbachia localization in adult wasps. By this mechanism, Wolbachia exhibits efficient vertical transmission across generations by depositing only female Wolbachia-infected offspring. The results of this study describe the dynamics of Wolbachia during the development of their Trichogramma host. The findings of this investigation helped clarify Wolbachia tropism in Trichogramma wasps.

Wolbachia-infected Trichogramma dendrolimi is outcompeted by its uninfected counterpart in superparasitism but does not have developmental delay.

BACKGROUND: Wolbachia infection increases the superparasitism frequency of Trichogramma females and provides an opportunity for horizontal intraspecific transmission. However, superparasitism may lead to interstrain competition between Wolbachia-infected Trichogramma offspring and their uninfected counterparts. This study investigated the outcome of interstrain intrinsic competition between Wolbachia-infected thelytokous strain (W) and uninfected bisexual strain (B) of Trichogramma dendrolimi. To determine the developmental rate of both strains, the sizes of immature stages of T. dendrolimi offspring at different times after parasitisation were measured in single parasitism and superparasitism conditions. RESULTS: The results reflect increased superparasitism by Wolbachia-infected females compared with uninfected females. Trichogramma females did not discriminate between host eggs previously parasitised by either B or W females. When the first oviposition was performed by B females, the B offspring outcompeted W offspring deposited later. Although when W offspring was deposited 8 h earlier than the B offspring, it gained no advantage over B offspring. Regardless of parasitism conditions, differences in the development rate between W and B offspring were not significant. CONCLUSION: The results reconfirmed that W females presented a higher tendency for superparasitism than B females, and showed that B offspring outcompeted W offspring even when the latter was deposited 8 h earlier. The inferiority of Wolbachia-infected Trichogramma compared with their uninfected counterparts is not due to the developmental delay. This study provides insights into the effects of intrinsic competition on the control efficacy of Wolbachia-infected Trichogramma against pests in biological control programs. © 2022 Society of Chemical Industry.

Wolbachia-Driven Memory Loss in a Parasitic Wasp Increases Superparasitism to Enhance Horizontal Transmission.

Horizontal transmission of the endosymbiont, Wolbachia, may occur during superparasitism when parasitoid females deposit a second clutch of eggs on a host. Wolbachia may increase the superparasitism tendency of Trichogramma wasps by depriving their memory. To test this hypothesis, we investigated the effects of conditioning experience and memory inhibitors (actinomycin D [ACD] and anisomycin [ANI]) on memory capacity, and expressions of memory-related genes (CREB1 and PKA), and superparasitism frequency of Wolbachia-infected (TDW) and uninfected (TD) lines of Trichogramma dendrolimi after conditioning with lemon or peppermint odor. We detected the presence of Wolbachia in eggs, larvae, pre-pupae, pupae, and adults of Trichogramma by using fluorescence in situ hybridization. The results showed that TDW females had a more reduced memory capacity than TD females after conditioning. Compared with TD females, TDW females showed a higher proportion of superparasitism and a downregulation of CREB1 and PKA genes after conditioning. TD females fed ACD or ANI showed a higher tendency for superparasitism and a downregulation of CREB1 and PKA, along with memory loss after conditioning than TD females fed honey solution only. The presence of Wolbachia was detected in the anterior region of the larva, pre-pupa, and pupa, but was not found in the head of the adult. The results provide evidence of host behavioral manipulation of Wolbachia by depriving memory of host Trichogramma wasps based on Poulin' s criteria. These host behavioral changes led by Wolbachia may be caused by the virulence of Wolbachia on the nervous system of the host. IMPORTANCE The endosymbiotic bacteria, Wolbachia, live widely within cells of arthropods. Wolbachia are not only transmitted vertically from host mother to offspring, but are also transmitted horizontally among host individuals. Horizontal transmission is expected to occur during superparasitism when host parasitoid females deposit a clutch of eggs on a host previously parasitized by the same parasitoid species. Thus, a question is proposed regarding whether superparasitism behavior is a behavior modification induced by the symbiont to favor symbiont transmission. This study highlights behavioral mechanisms of Wolbachia-induced superparasitism in Trichogramma wasps and the manipulation of symbionts on host parasitoids.

Penetrance during Wolbachia-mediated parthenogenesis of Trichogramma wasps is reduced by continuous oviposition, associated with exhaustion of Wolbachia titers in ovary and offspring eggs.

BACKGROUND: Thelytokous Wolbachia-infected Trichogramma wasps are superior to bisexual uninfected wasps regarding biological control programs. However, continuous oviposition weakens the parthenogenesis-inducing (PI) strength of Wolbachia. Whether this reduced PI strength relates to decreases in the titer of Wolbachia in the ovary and offspring eggs of Trichogramma remains unclear. Here, using fluorescence in situ hybridization (FISH) and reverse transcription quantitative polymerase chain reaction (RT-qPCR) methods, we investigated how the penetrance of Wolbachia-mediated parthenogenesis, Wolbachia density, and distributions of two Wolbachia-infected Trichogramma species, T. pretiosum (TP) and T. dendrolimi (TD), were influenced by different host access treatments [newly-emerged virgin females (NE), 7-day-old females without access to host eggs (NAH), and 7-day-old virgin females with access to host eggs (AH)]. RESULTS: Continuous oviposition decreased Wolbachia PI strength and titers in TP and TD. Continuous oviposition in AH decreased Wolbachia titers in abdomen and offspring eggs of TP and TD females, compared with NAH and NE; NAH had a lower thorax Wolbachia titer than NE. The numbers of parasitized host eggs and offspring wasps, and emergence rates of offspring deposited by AH were lower than those of NE and NAH, for either species. CONCLUSION: Weakened PI strength, driven by continuous oviposition in Trichogramma wasps, is associated with Wolbachia titer exhaustion in ovary and offspring eggs. Wolbachia density is dependent on PI strength in Trichogramma wasps, highlighting the side effects of continuous oviposition regarding thelytokous Wolbachia-infected Trichogramma in biological control programs. © 2022 Society of Chemical Industry.

Evaluation of Sensitivity to Phoxim and Cypermethrin in an Endoparasitoid, Meteorus pulchricornis (Wesmael) (Hymenoptera: Braconidae), and Its Parasitization Efficiency Under Insecticide Stress.

Insecticides can have consequences for beneficial arthropods. Insect parasitoids can contact insecticides through direct exposure spray droplets or residues on crop foliage. Here, we focus on better understand the response of Meteorus pulchricornis (Wesmael), a parasitoid wasp of lepidopteran pests, and its detoxification mechanisms on stress caused by phoxim and cypermethrin. Hence, we determined the dose-mortality curves and estimating the sublethal concentrations (LC30 and LC50). Then, we applied the sublethal concentrations against adult parasitoids to assess its survival, parasitism efficacy, and also developmental and morphometric parameters of their offspring. Simultaneously, we check the activities of glutathione S-transferase (GST), acetylcholinesterase (AChE), and peroxidase (POD) after sublethal exposure of both insecticides, which has measured until 48 h after treatment. Overall, phoxim and cypermethrin exhibited acute lethal activity toward the parasitoid with LC50 values 4.608 and 8.570 mg/liter, respectively. Also, we detect that LC30 was able to trigger the enzymatic activity of GST, AChE, and POD, suggesting a potential detoxification mechanism. However, even when subjected to sublethal exposure, our results indicate strong negatives effects, in particular for phoxim, which has affected the parasitism efficacy and also the developmental and morphometric parameters of M. pulchricornis offspring. Therefore, it can be concluded that both phoxim and cypermethrin have negative impacts on M. pulchricornis and we suggest cautioning their use and the need for semifield and field assessments to confirm such an impact.

Optimal clutch size for quality control of bisexual and Wolbachia-infected thelytokous lines of Trichogramma dendrolimi Matsumura (Hymenoptera: Trichogrammatidae) mass reared on eggs of a substitutive host, Antheraea pernyi Guérin-Méneville (Lepidoptera: Saturniidae).

BACKGROUND: Trichogramma dendrolimi has been widely used in augmentative biocontrol of lepidopteran pests in China. In mass production of T. dendrolimi using Antheraea pernyi eggs as substitutive hosts, which are large in size, as clutch size is a parameter of importance to produce high quality parasitoids. Here, we aimed to determine the optimal clutch size for the bisexual Wolbachia-uninfected line (TdB) and Wolbachia-infected thelytokous line (TdT) of T. dendrolimi reared on A. pernyi eggs. RESULTS: A medium clutch size of 42.75 to 62.27 for TdB and 52.93 to 57.14 for TdT was optimal to maximize fitness-correlated traits of parasitoid individual. The optimal clutch sizes with maximized parameters included adult emergence rate, adult body size, adult longevity, fecundity, and sum of fecundity of all females per brood were 58.31 (86.00%), 42.75 (231.11 µm), 50.92 (2.69 days), 62.27 (150.89 eggs), and 83.25 (7926.33 eggs) for TdB and 57.14 (94.54%), 52.93 (236.97 µm), 53.64 (2.62 days), 56.80 (161.01 eggs), and 70.10 (8579.71 eggs) for TdT. The TdT had a shorter adult longevity, longer development time, and higher adult emergence rate than did its non-infected bisexual counterpart. CONCLUSION: A medium brood size in a A. pernyi egg host was optimal to produce offspring parasitoids with higher fitness parameters for both bisexual Wolbachia-uninfected and thelytokous Wolbachia-infected lines of T. dendrolimi. The determination of optimal clutch size for T. dendrolimi will provide the reference for the quality control of T. dendrolimi production and improvement of the field performance of the wasps. © 2020 Society of Chemical Industry.

Phylogenetic signals in pest abundance and distribution range of spider mites.

BACKGROUND: Attributes of pest species like host range are frequently reported as being evolutionarily constrained and showing phylogenetic signal. Because these attributes in turn could influence the abundance and impact of species, phylogenetic information could be useful in predicting the likely status of pests. In this study, we used regional (China) and global datasets to investigate phylogenetic patterns in occurrence patterns and host ranges of spider mites, which constitute a pest group of many cropping systems worldwide. RESULTS: We found significant phylogenetic signal in relative abundance and distribution range both at the regional and global scales. Relative abundance and range size of spider mites were positively correlated with host range, although these correlations became weaker after controlling for phylogeny. CONCLUSIONS: The results suggest that pest impacts are evolutionarily constrained. Information that is easily obtainable - including the number of known hosts and phylogenetic position of the mites - could therefore be useful in predicting future pest risk of species.

Effects of temperature and superparasitism on quality and characteristics of thelytokous Wolbachia-infected Trichogramma dendrolimi Matsumura (Hymenoptera: Trichogrammatidae) during mass rearing.

Thelytokous Wolbachia-infected Trichogramma spp. are widely used egg parasitoids against lepidopteran pests in biological control programs. Wolbachia may manipulate host wasps for superparasitism and is sensitive to temperature. To explore effects of temperature and superparasitism, we compared fitness parameters and Wolbachia-mediated phenotype of thelytokous Wolbachia-infected Trichogramma dendrolimi between those emerging from superparasitised or single-parasitised hosts at 17, 21, 25, or 29 °C. Infected mothers of T. dendrolimi showed reduced superparasitism and parasitism increased with temperature. Wolbachia titre decreased with temperature when females emerged from singly-parasitised hosts, but there was no correlation in superparasitised hosts. Females showed higher Wolbachia titres at 21, 25, or 29 °C when developing from superparasitised hosts. The daily male ratio of offspring increased with temperature, and the day-age threshold for 5%, 50%, or 95% daily male ratio decreased with temperature in both parasitism forms. Females that emerged from superparasitised hosts had a shorter life span and reduced fecundity. These results indicate that Wolbachia may affect host behaviour by increasing superparasitism to enhance its spread, but this has negative effects on thelytokous Wolbachia-infected T. dendrolimi.

Co-occurrence of thelytokous and bisexual Trichogramma dendrolimi Matsumura (Hymenoptera: Trichogrammatidae) in a natural population.

Trichogramma dendrolimi is one of the most successful biocontrol agents in China. However, an inundative condition is necessary to obtain acceptable parasitism effect. A good solution to this is the application of its thelytokous counterparts which unfortunately are scarce in field. We here report the first case of a natural T. dendrolimi population in China comprising both bisexual wasps and an extremely low proportion of thelytokous wasps. These two forms of T. dendrolimi are phylogenetically related based on the reconstructions of ITS-2 and COI genes. Also, the phylogenetic results suggested a potentially Wolbachia-drived ITS-2 variation. The expression of thelytoky was hardly affected by temperature, which might help control Asian corn borer and Dendrolimus punctatus. Wolbachia are responsible for current thelytoky according to phylogenetic analyses, antibiotic treatment and introgression experiment. We also present the third case of paternal sex ratio chromosome that restrains the expansion of Wolbachia. Moreover, the low frequency of thelytoky may be common in natural populations. Consequently if for biological control it is determined that a thelytokous strain is to be preferred, then large number of field collected females should be set up as isofemale lines, to detect the rare thelytoky.

Gradual, temperature-induced change of secondary sexual characteristics in Trichogramma pretiosum infected with parthenogenesis-inducing Wolbachia.

Intersex is an intermediate stage of sexual differentiation in insects. Determining intersex morphology and the cause of its production will aid in the understanding of the mechanism of sexual differentiation in insects. In this paper, Wolbachia-infected Trichogramma pretiosum (T. preW+ ) that shows thelytokous parthenogenesis were used as subjects. In order to determine the causes of the T. preW+ intersex and the influence of parental generation temperature on gradual changes in secondary masculinization in intersex offspring, we examined the occurrence of intersex offspring (F1 and F2 generation) after the parental generations were treated with high temperature (27, 29, 31, and 33 °C) and described the external morphology of the intersexes. The results showed that the T. preW+ parental generation temperature is positively correlated with the probability of intersex offspring. The probability of F1 intersex is significantly higher than that of F2 intersex in different high temperature. The degree of secondary masculinization in T. preW+ intersexes increases as parental generation temperature increases. In addition, our study first identified 11 intersex types in T. preW+ and found that the primary and secondary sexual characteristics showed a regular distribution. We also found that the D type and H type of intersex have the highest frequency of appearance. The external genitalia of most intersexes were female, and only three intersex types have male external genitalia. Conclusions were ultimately obtained: Wolbachia is a direct factor that causes the occurrence of intersexes, while high temperature is an indirect factor that determines the external morphology of intersexes. The effects of high temperature on T. preW+ intersexes is passed through the parental generation to offspring, and this maternal effect weakens as the number of generations increases. In T. preW+ intersex individuals, most exhibit female primary sexual characteristics, and secondary sexual characteristics exhibit signs of masculinization.

Mitochondrial variation in small brown planthoppers linked to multiple traits and probably reflecting a complex evolutionary trajectory.

While it has been proposed in several taxa that the mitochondrial genome is associated with adaptive evolution to different climatic conditions, making links between mitochondrial haplotypes and organismal phenotypes remains a challenge. Mitonuclear discordance occurs in the small brown planthopper (SBPH), Laodelphax striatellus, with one mitochondrial haplogroup (HGI) more common in the cold climate region of China relative to another form (HGII) despite strong nuclear gene flow, providing a promising model to investigate climatic adaptation of mitochondrial genomes. We hypothesized that cold adaptation through HGI may be involved, and considered mitogenome evolution, population genetic analyses, and bioassays to test this hypothesis. In contrast to our hypothesis, chill-coma recovery tests and population genetic tests of selection both pointed to HGII being involved in cold adaptation. Phylogenetic analyses revealed that HGII is nested within HGI, and has three nonsynonymous changes in ND2, ND5 and CYTB in comparison to HGI. These molecular changes likely increased mtDNA copy number, cold tolerance and fecundity of SBPH, particularly through a function-altering amino acid change involving M114T in ND2. Nuclear background also influenced fecundity and chill recovery (i.e., mitonuclear epistasis) and protein modelling indicates possible nuclear interactions for the two nonsynonymous changes in ND2 and CYTB. The high occurrence frequency of HGI in the cold climate region of China remains unexplained, but several possible reasons are discussed. Overall, our study points to a link between mtDNA variation and organismal-level evolution and suggests a possible role of mitonuclear interactions in maintaining mtDNA diversity.

Incidence of Facultative Bacterial Endosymbionts in Spider Mites Associated with Local Environments and Host Plants.

Spider mites are frequently associated with multiple endosymbionts whose infection patterns often exhibit spatial and temporal variation. However, the association between endosymbiont prevalence and environmental factors remains unclear. Here, we surveyed endosymbionts in natural populations of the spider mite, Tetranychus truncatus, in China, screening 935 spider mites from 21 localities and 12 host plant species. Three facultative endosymbiont lineages, Wolbachia, Cardinium, and Spiroplasma, were detected at different infection frequencies (52.5%, 26.3%, and 8.6%, respectively). Multiple endosymbiont infections were observed in most local populations, and the incidence of individuals with the Wolbachia-Spiroplasma coinfection was higher than expected from the frequency of each infection within a population. Endosymbiont infection frequencies exhibited associations with environmental factors: Wolbachia infection rates increased at localities with higher annual mean temperatures, while Cardinium and Spiroplasma infection rates increased at localities from higher altitudes. Wolbachia was more common in mites from Lycopersicon esculentum and Glycine max compared to those from Zea mays This study highlights that host-endosymbiont interactions may be associated with environmental factors, including climate and other geographically linked factors, as well as the host's food plant.IMPORTANCE The aim of this study was to examine the incidence of endosymbiont distribution and the infection patterns in spider mites. The main findings are that multiple endosymbiont infections were more common than expected and that endosymbiont infection frequencies were associated with environmental factors. This work highlights that host-endosymbiont interactions need to be studied within an environmental and geographic context.

[Prokaryotic expression of trigeminy artificial fusion gene of Leptospira interrogans and the immunogenicity of its products].

OBJECTIVE: To construct lipL32/1-lipL21-OmpL1/2 fusion gene of Leptospira interrogans and its prokaryotic expression system, and to identify the immunogenicity of its products. METHODS: PCR using linking primers was applied to construct lipL32/1-lipL21-OmpL1/2 fusion gene and a prokaryotic expression system of the fusion gene was then established using routine genetic engineering technique. SDS-PAGE was used to examine output of the target recombinant protein rLipL32/1-LipL21-OmpL1/2. Double immunodiffusion and Western Blot assay were applied to identify immunogenicity of rLipL32/1-LipL21-OmpL1/2. RESULT: lipL32/1-lipL21-OmpL1/2 fusion gene with correct sequence and its prokaryotic expression system E.coli BL21DE3pET42a-lipL32/1-lipL21-ompL1/2 was obtained in this study. The output of rLipL32/1-LipL21- OmpL1/2 after optimisation was 37.78 mg/L. The immunodiffusion titer of rabbit antiserum against rLipL32/1-LipL21-OmpL1/2 was 1:4. The rLipL32/1-LipL21-OmpL1/2 antiserum was able to recognize rLipL32/1-LipL21-OmpL1/2, rLipL32/1, rLipL21 and rOmpL1/2. Positive Western hybridization signals were found among rLipL32/1-LipL21-OmpL1/2 and rabbit antiserum against whole cell of strain 56601 and serum from patients infected with L.interrogans serogroups Icterohaemorrhagiae, Grippotyphosa, Autumnalis and Pomona. CONCLUSION: The fusion gene lipL32/1-lipL21-OmpL1/2 and its prokaryotic expression system were successfully constructed in this study. The expressed fusion protein can be used as the antigen for developing universal genetic engineering vaccine and universal serological tests of leptospirosis.