Wolbachia与昆虫精卵细胞质不亲和

Wolbachia是广泛分布在昆虫体内的一类共生菌,能通过多种机制调节宿主的生殖方式,包括诱导宿主精卵细胞质不亲和(CI)、孤雌生殖、雌性化、杀雄等,其中细胞质不亲和为最普遍的表型,即感染Wolbachia的雄性和未感染或感染不同品系Wolbachia的雌性宿主交配后,受精卵不能正常发育,在胚胎期死亡。多数CI胚胎在第1次分裂时,来自父本的染色质浓缩缺陷,导致父本遗传物质无法正常分配到子细胞中,因而引起胚胎死亡。守门员模型认为,产生CI可能需要有两种因子,其中之一使得精子发生修饰改变,导致受精后雄性原核发育滞后。第2种因子可能与Wolbachia的原噬菌体有关,在胚胎发育后期导致胚胎死亡。近期的研究已发现,在Wolbachia感染的宿主中,一些与生殖细胞发生和繁殖相关基因的表达发生了显著改变,Wolbachia可能因此对宿主的生殖产生重大影响,进而导致CI的产生。本文主要综述了CI的细胞学表型、解释CI的模型及其分子机理,向读者展示一个小小的细菌是如何通过精妙的策略影响昆虫宿主的繁殖,从而实现其自身的生存和传播的。     

Wolbachia引起黑腹果蝇产生“修饰-挽救”因子的初步探究

沃尔巴克氏体(Wolbachia)是一种广泛分布于昆虫体内的共生细菌,对宿主的生殖具有多种调控作用,其中,最常见的调控方式称为细胞质不亲和(Cytoplasmic Incompatibility,CI),即感染了Wolbachia的雄性宿主与未感染或感染不同品系Wolbachia的雌性宿主交配后,胚胎在发育早期死亡。关于CI产生的原因,目前人们认为是一种"精子修饰-卵子挽救"机制,本研究初步探索了黑腹果蝇中参与"修饰-挽救"机制的重要因子。本研究采用定量RT-PCR方法,检测了3种基因:yem-α(yemanuclein)、uif(uninflatable)、Prosα4T1(proteasomeα4 subunit,Testis-specific 1)在感染和未感染果蝇中的表达差异。结果发现,Wolbachia感染能够显著上调雌蝇中yem-α和uif的表达,并且显著下调雄蝇中yem-α的表达。尽管Prosα4T1在雄性果蝇体内特异表达,但是Wolbachia感染对Prosα4T1表达无明显影响。因此,yem-α可能是Wolbachia产生"修饰-挽救"机制的重要因子之一,Wolbachia可能通过调控果蝇体内yem-α的表达,影响卵子发生和精子发生进程,导致CI的产生。     

沃尔巴克氏体感染对黑腹果蝇性腺和早期胚胎DNA 6mA去甲基化酶基因DMAD表达的影响

【目的】DNA甲基化是基因修饰的一种重要方式,主要可以形成5-甲基胞嘧啶(5m C)和6-甲基腺嘌呤(6m A)等。目前关于5m C的研究比较多,而关于6m A在真核生物中的研究则较少。沃尔巴克氏体Wolbachia是昆虫中最常见的共生菌之一,可通过多种方式操纵宿主生殖,其中最常见的就是引起精卵细胞质不亲和(cytoplasmic incompatibility,CI),即感染Wolbachia的雄性与未感染的雌性宿主交配后,胚胎致死,但其机制还不清楚。本研究拟从6m A甲基化的角度,探讨Wolbachia影响果蝇生殖的分子机制。【方法】以模式生物黑腹果蝇Drosophila melanogaster为材料,通过实时荧光定量PCR方法,检测Wolbachia感染对果蝇精巢、卵巢以及3个交配组[TT(对照,父母本都未感染),TW(父本未感染,母本感染,胚胎感染,可发育)和WT(CI,致死)]早期胚胎中DNA 6m A去甲基化酶基因DMAD的表达变化。【结果】Wolbachia感染可显著上调1日龄果蝇精巢中DMAD的表达水平,而对卵巢中DMAD的表达没有显著影响。在产卵后0.5 h(中囊胚过渡前)的胚胎中,CI胚胎的DMAD表达水平极显著高于可正常发育的胚胎(TT和TW);在3 h的胚胎(中囊胚过渡期)中,TW和CI胚胎中DMAD的表达量都极显著高于对照组;在6 h的胚胎(中囊胚过渡后)中,CI胚胎中DMAD的表达量相对最低。【结论】Wolbachia感染可能通过干扰宿主果蝇精巢中6m A甲基化水平对精子产生修饰,导致其与正常未感染的卵子受精后胚胎致死。     

黑腹果蝇Mst84Db敲降引起的父本缺陷类似Wolbachia诱导的细胞质不亲和

【目的】Wolbachia 是广泛存在于昆虫体内的一类通过母系传递的共生菌,能够通过多种方式影响宿主的生殖。细胞质不亲和（CI）是Wolbachia 引起的最普遍的一种表型,即感染Wolbachia的雄性和未感染的雌性宿主交配后,胚胎发育停滞于早期阶段。但目前有关CI的分子机理还不清楚。本研究组前期实验表明,Wolbachia感染引起黑腹果蝇Drosophila melanogaster 3龄幼虫精巢中Mst84Db基因的表达显著下调。本研究的目的是进一步研究Mst84Db与CI的关系。【方法】我们体外合成了Mst84Db的双链RNA（dsRNA）,注射雄性果蝇,将注射过的雄果蝇与野生雌果蝇交配,检测其繁殖力。基因表达采用定量RT-PCR方法进行检测。胚胎表型采用DAPI染色进行分析。【结果】注射dsRNA 24 h后,Mst84Db基因的表达水平发生显著下调。注射后72 h,基因表达下调幅度最大。与对照组相比,基因敲降后的雄蝇繁殖能力显著下降,与雌果蝇交配后胚胎孵化率显著低于对照组,这与Wolbachia诱导的CI现象类似。未孵化胚胎的表皮上没有体节出现,说明胚胎停滞于发育的早期。部分胚胎细胞核分裂不同步,且有染色质间桥出现,这也与CI胚胎中的细胞学表型一致。【结论】Wolbachia感染可能抑制果蝇精子发生过程中Mst84Db基因的表达,从而使精子失去正常功能,最终导致与雌性果蝇交配后,胚胎发育停滞,并最终死亡。Mst84Db基因在雄性果蝇中表达下调可能是产生CI的重要原因之一。     

二斑叶螨两种群中Wolbachia诱导的胞质不亲和作用的影响因子比较研究

Wolbachia诱导胞质不亲和（cytoplasmic incompatibility, CI）是对寄主的生殖调控中最常见的一种方式,在不同种群中CI表达的差异较大。以二斑叶螨Tetranychus urticae辽宁兴城（LN）和江苏徐州（JS）两个地理种群为实验材料,经筛选获得100%感染Wolbachia和不感染Wolbachia的品系,通过杂交实验和实时定量PCR的方法研究寄主遗传背景、雄螨日龄、温度以及雄螨体内Wolbachia菌量等因子对我国二斑叶螨体内Wolbachia诱导CI能力的影响。结果表明：1,3,5和7日龄的雄螨诱导的CI的强度没有差异,表明雄螨日龄对我国二斑叶螨体内Wolbachia诱导CI的能力没有影响。二斑叶螨分别放在20℃的低温、 25℃的适温和30℃的高温条件下饲养时,Wolbachia诱导CI的能力也没有任何变化,表明温度对我国二斑叶螨体内Wolbachia诱导CI的能力也没有影响。江苏徐州种群所感染Wolbachia菌量显著高于辽宁兴城种群,并且这两个种群感染Wolbachia菌量都随着雄螨日龄的增大而显著增加,表明Wolbachia菌量对我国二斑叶螨体内Wolbachia诱导CI的能力没有影响;江苏徐州种群内Wolbachia不能诱导CI可能是Wolbachia株系与寄主的遗传背景共同作用的结果。研究结果为进一步了解Wolbachia的生殖调控机理提供了重要依据。     

Wolbachia和Cardinium对皮氏叶螨生殖的影响及在寄主体内的定位

Wolbachia和Cardinium均为母系遗传的胞内共生菌, 它们能够通过诱导胞质不亲和（cytoplasmic incompatibility, CI）以调控寄主的生殖。目前, 关于Wolbachia和Cardinium共同对同一寄主进行生殖操控的机制还不清楚。本研究以皮氏叶螨Tetranychus piercei McGregor广州种群为实验材料, 通过杂交实验和荧光原位杂交的方法, 研究Wolbachia和Cardinium单感染和双感染对寄主生殖的影响。结果表明： 单感染Wolbachia诱导较弱的CI, 不亲和组合的未孵化率为17.8%±1.6%。单感染Cardinium及双感染Wolbachia和Cardinium能诱导高强度的CI, 不亲和组合的未孵化率分别为70.3%±1.3%和72.9%±1.2%。同时双感染Wolbachia和Cardinium雌螨的平均产卵量为35.2±1.2, 显著高于单感染和不感染的雌螨的产卵量。Wolbachia 和Cardinium分别诱导以及共同诱导CI的水平与精子形成过程中的感染情况有关。Wolbachia和Cardinium的垂直传播模式结果显示, 在卵的不同发育阶段, Wolbachia和Cardinium主要伴随着营养物质从滋养细胞、 中肠、 输卵管进入发育中的卵。研究结果为进一步了解 Wolbachia和Cardinium的母系遗传机制提供了重要依据。     

Wolbachia对苹果蠹蛾生殖调控的作用

【背景】苹果蠹蛾是重要的果树害虫和检疫对象。研究显示,苹果蠹蛾体内所含的Wolbachia属于具有诱导胞质不亲和(CI)现象的A组中Dor亚组。本研究目的在于摸清苹果蠹蛾体内的Wolbachia对宿主的生殖调控是否具有诱导胞质不亲和的功能。【方法】通过饲喂抗生素消除苹果蠹蛾体内的Wolbachia,比较感染与消除Wolbachia的苹果蠹蛾生态适合度以及Wolbachia感染对苹果蠹蛾生殖的影响。【结果】消除Wolbachia后,苹果蠹蛾F_1、F_2代各虫态的发育历期、产卵量、孵化率和蛹的存活率与对照无显著差异;Wolbachia对苹果蠹蛾成虫的寿命、产卵前期、平均产卵量和性别比例均无影响。不同处理苹果蠹蛾之间的杂交试验结果表明,4种杂交的苹果蠹蛾都能产卵,且卵可以正常孵化。【结论与意义】在自然状态下苹果蠹蛾体内的Wolbachia不能诱导其宿主的胞质不亲和现象。     

共生菌Wolbachia引起宿主细胞质不亲和的研究进展

Wolbachia 是一类广泛存在于节肢动物以及线虫体内细胞质中呈母系遗传的共生细菌,能够在宿主中产生细胞质不亲和、孤雌生殖、雌性化及杀雄等多种生殖调控作用,其中细胞质不亲和是指被 Wolbachia 感染的雄性个体与未感染的雌性个体（单向不亲和）,或者感染不同株系 Wolbachia 的雌性个体（双向不亲和）交配后不能或很少产生后代,或者后代偏雄性的现象。细胞质不亲和作用使感染的雌性个体在种群中具有很大的生殖优势,凭借这种生殖优势,Wolbachia 能够迅速在宿主种群中扩张。细胞质不亲和的机理探索主要集中在细胞学水平上,其中广为接受的精子“修饰”和“拯救”理论认为,精巢中的 Wolbachia 能够修饰宿主的精细胞,使其不能和卵细胞正常融合,但是当母本感染相同的 Wolbachia 时,就能够将“修饰”过的精子细胞“拯救”过来,使其恢复与卵细胞的正常融合。而分子机理上的探索也开始在转录组、基因组和miRNA水平上对部分昆虫展开了研究。影响细胞质不亲和的因素有很多,包括宿主遗传背景、 Wolbachia 株系、Wolbachia 基因型、共生菌密度（浓度、滴度）、雄虫年龄、环境因素以及共生菌在宿主生殖组织的分布等。近年来,人类也应用细胞质不亲和控制害虫（主要是蚊虫）和人类疾病,取得了较好的进展。     

我国三地区黑腹果蝇中Wolbachia的系统发育关系及其对宿主生殖的影响

【目的】Wolbachia 是广泛存在于节肢动物和丝状线虫体内的一类共生菌, 能够以多种方式对宿主产生影响。精卵细胞质不亲和（CI）是其引起的最普遍的表型, 即感染Wolbachia的雄性宿主与未感染或感染不同品系的雌性宿主交配后, 不能产生后代或后代极少, 而感染同品系Wolbachia的雌雄宿主交配后则能正常产生后代。我们前期研究发现, 湖北武汉、 云南六库和天津3个地区黑腹果蝇Drosophila melanogaster被Wolbachia感染。本研究旨在明确这3个地区黑腹果蝇中Wolbachia的系统发育关系及其对宿主生殖的影响。【方法】利用Clustal X软件对Wolbachia的wsp基因序列进行比对, 利用MEGA软件构建系统发育树。采用多位点序列分型（MLST）的方法对Wolbachia进行分型。通过区内交配和区之间杂交的方式研究不同地区黑腹果蝇体内Wolbachia 的关系及其对果蝇生殖的影响。【结果】湖北武汉、 云南六库和天津3个地区黑腹果蝇中感染的Wolbachia都是属于A大组的Mel亚群。这3个地区果蝇感染的Wolbachia的序列类型（ST）不同, Wolbachia之间存在一定的差异。湖北武汉和天津果蝇中的Wolbachia能引起强烈的CI表型, 而云南六库果蝇中的Wolbachia引起的CI强度相对较弱。武汉果蝇中Wolbachia不能完全挽救天津果蝇中Wolbachia引起的CI表型, 而天津果蝇中Wolbachia也不能完全挽救武汉果蝇中Wolbachia引起的CI表型。【结论】武汉和天津地区黑腹果蝇中的Wolbachia可能距离较远。Wolbachia的长期共生可能对黑腹果蝇的进化产生了一定的影响, 湖北武汉与云南六库的黑腹果蝇中感染的Wolbachia属于不同的序列类型, 这2个地区的黑腹果蝇已发生了一定的分歧, 产生了一定的生殖隔离。     

Wolbachia在熊蜂中的双重感染

Wolbachia是一类广泛存在于节肢动物体内细胞质遗传的细菌,它可以通过诱导产雌孤雌生殖、引起细胞质不亲和、遗传雄性的雌性化、雄性致死和增强生殖力等作用方式引起其寄主生殖行为的改变.本文以16S rDNA为标记检测了3种熊蜂不同组织(头,胸,足,卵巢或雄外生殖器)的Wolbachia感染.其中明亮熊蜂Bombus l...     

Independent cytoplasmic incompatibility induced by Cardinium and Wolbachia maintains endosymbiont coinfections in haplodiploid thrips populations

Cardinium and Wolbachia are common maternally inherited reproductive parasites that can coinfect arthropods, yet interactions between both bacterial endosymbionts are rarely studied. For the first time, we report their independent expression of complete cytoplasmic incompatibility (CI) in a coinfected host, and CI in a species of the haplodiploid insect order Thysanoptera. In Pezothrips kellyanus, Cardinium‐induced CI resulted in a combination of male development (MD) and embryonic female mortality (FM) of fertilized eggs. In contrast, Wolbachia‐induced CI resulted in FM together with postembryonic mortality not previously reported as a CI outcome. Both endosymbionts appeared to not influence fecundity but virgins produced more offspring than mated females. In coinfected individuals, Wolbachia density was higher than Cardinium. Wolbachia removal did not impact Cardinium density, suggesting a lack of competition within hosts. Maternal transmission was complete for Wolbachia and high for Cardinium. Our data support theoretical predictions and empirical detection of high endosymbiont prevalence in field populations of the native range of this pest thrips. However, previous findings of more frequent loss of Wolbachia than Cardinium, particularly in field populations of the host's invasive range, suggest that genetic diversity or varying environmental factors between field populations also play a role in shaping host‐endosymbiont dynamics.     

Maintenance of adaptive differentiation by Wolbachia induced bidirectional cytoplasmic incompatibility: the importance of sib-mating and genetic systems

Background  

Bacteria of the genus Wolbachia are reproductive parasites widespread among arthropods. The most common effect arising from the presence of Wolbachia in a population is Cytoplasmic Incompatibility (CI), whereby postmating reproductive isolation occurs in crosses between an infected male and an uninfected female, or when a male is infected with a different strain of Wolbachia to that of the female (bidirectional CI). Previous theoretical models have demonstrated that bidirectional CI can contribute to the genetic divergence of populations in haploid and diploid organisms. However, haplodiploid organisms were not considered in these models even though they include Nasonia parasitoid wasps – the best example of the implication of Wolbachia in ongoing speciation. Moreover, previous work did not investigate inbreeding mating systems, which are frequently observed in arthropod species.     

Incidence of Wolbachia in aquatic insects

Wolbachia is a genus of intracellular bacteria typically found within the reproductive systems of insects that manipulates those systems of their hosts. While current estimates of Wolbachia incidence suggest that it infects approximately half of all arthropod species, these estimates are based almost entirely on terrestrial insects. No systematic survey of Wolbachia in aquatic insects has been performed. To estimate Wolbachia incidence among aquatic insect species, we combined field‐collected samples from the Missouri River (251 samples from 58 species) with a global database from previously published surveys. The final database contained 5,598 samples of 2,687 total species (228 aquatic and 2,459 terrestrial). We estimate that 52% (95% CrIs: 44%–60%) of aquatic insect species carry Wolbachia, compared to 60% (58%–63%) of terrestrial insects. Among aquatic insects, infected orders included Odonata, Coleoptera, Trichoptera, Ephemeroptera, Diptera, Hemiptera, and Plecoptera. Incidence was highest within aquatic Diptera and Hemiptera (69%), Odonata (50%), and Coleoptera (53%), and was lowest within Ephemeroptera (13%). These results indicate that Wolbachia is common among aquatic insects, but incidence varies widely across orders and is especially uncertain in those orders with low sample sizes such as Ephemeroptera, Plecoptera, and Trichoptera.     

Contrasting effects of Wolbachia on cytoplasmic incompatibility and fecundity in the haplodiploid mite Tetranychus urticae

Recent studies on Wolbachia‐induced incompatibility in haplodiploid insects and mites have revealed a diversity of cytoplasmic incompatibility (CI) patterns among host species. Here, we report intraspecific diversity in CI expression among four strains of the arrhenotokous mite Tetranychus urticae and in T. turkestani. Variability of CI expression within T. urticae ranged from no CI to complete CI, and included either female embryonic mortality or male conversion types of CI. A fecundity cost attributed to the infection with the high‐CI Wolbachia strain was the highest ever recorded for Wolbachia (?80 to ?100% decrease). Sequence polymorphism at a 550‐bp‐portion of Wolbachia wsp gene revealed two clusters distant by 21%, one of which included three Wolbachia strains infecting mite populations sampled from the same host‐plant species, but showing distinct CI patterns. These data are discussed in the light of theoretical predictions on the evolutionary pathways followed in this symbiotic interaction.     

Artificial selection on ant female caste ratio uncovers a link between female‐biased sex ratios and infection by Wolbachia endosymbionts

Social insect sex and caste ratios are well‐studied targets of evolutionary conflicts, but the heritable factors affecting these traits remain unknown. To elucidate these factors, we carried out a short‐term artificial selection study on female caste ratio in the ant Monomorium pharaonis. Across three generations of bidirectional selection, we observed no response for caste ratio, but sex ratios rapidly became more female‐biased in the two replicate high selection lines and less female‐biased in the two replicate low selection lines. We hypothesized that this rapid divergence for sex ratio was caused by changes in the frequency of infection by the heritable bacterial endosymbiont Wolbachia, because the initial breeding stock varied for Wolbachia infection, and Wolbachia is known to cause female‐biased sex ratios in other insects. Consistent with this hypothesis, the proportions of Wolbachia‐infected colonies in the selection lines changed rapidly, mirroring the sex ratio changes. Moreover, the estimated effect of Wolbachia on sex ratio (~13% female bias) was similar in colonies before and during artificial selection, indicating that this Wolbachia effect is likely independent of the effects of artificial selection on other heritable factors. Our study provides evidence for the first case of endosymbiont sex ratio manipulation in a social insect.     

Wolbachia in scale insects: a distinct pattern of infection frequencies and potential transfer routes via ant associates

Wolbachia is one of the most successful endosymbiotic bacteria of arthropods. Known as the ‘master of manipulation’, Wolbachia can induce a wide range of phenotypes in its host that can have far-reaching ecological and evolutionary consequences and may be exploited for disease and pest control. However, our knowledge of Wolbachia's distribution and the infection rate is unevenly distributed across arthropod groups such as scale insects. We fitted a distribution of within-species prevalence of Wolbachia to our data and compared it to distributions fitted to an up-to-date dataset compiled from surveys across all arthropods. The estimated distribution parameters indicate a Wolbachia infection frequency of 43.6% (at a 10% prevalence threshold) in scale insects. Prevalence of Wolbachia in scale insects follows a distribution similar to exponential decline (most species are predicted to have low prevalence infections), in contrast to the U-shaped distribution estimated for other taxa (most species have a very low or very high prevalence). We observed no significant associations between Wolbachia infection and scale insect traits. Finally, we screened for Wolbachia in scale insect's ecological associates. We found a positive correlation between Wolbachia infection in scale insects and their ant associates, pointing to a possible route of horizontal transfer of Wolbachia.     

灰飞虱核心共生菌的鉴定

媒介昆虫的核心共生菌能被用作基因工程菌发挥抗病毒功能.灰飞虱是一种重要的农业害虫,其传播的水稻条纹病毒造成水稻的大面积减产甚至绝收.[目的]本研究对不同稻区及温室饲养的灰飞虱进行菌群组成分析并初步鉴定灰飞虱的核心共生菌.[方法]通过16SrDNA介导的二代测序技术,分析了 2018-2020年间采集自云南昆明、河南开封...     

The role of Wolbachia and the environment on sex determination of the Indian lac insect,Kerria lacca (Coccoidea: Tachardiidae)

The Indian lac insect, Kerria lacca (Kerr) exhibits a highly variable sex ratio, apparently influenced by various environmental factors, in the absence of a chromosomal sex determination mechanism. The endosymbiont Wolbachia, well known as sex ratio distorter, was reported in this species but its role in the sex determination of the host has hitherto remained unexplored. Distinct variation in the presence of Wolbachia was observed in relation to the developmental stages and the sexes of K. lacca as well as the host plant species; adult males of W + whereas adult females are W-. The post-settlement environmental factors such as host plant, season and genetic makeup affect the sex ratio in this insect, probably mediated through Wolbachia dynamics. Denser settlement of the crawlers led to comparatively higher Wolbachia presence with an elevated sex ratio. Suppression of Wolbachia through antibiotic administration resulted in a nearly threefold increase in the sex ratio. Distinct transitions in the proportion of insects with heterochromatic genome (cytological males) have been observed during the first instar on different host plants, indicating that changes in the sex ratio occur during early development phase. Environmental factors appear to influence Wolbachia, which in turn brings about sex ratio changes, probably mediated through changes in juvenile hormone levels, as the male and female lac insects show distinct modes of metamorphosis.     

Frequency of infection with A and B supergroup <Emphasis Type="Italic">Wolbachia</Emphasis> in insects and pests associated with mulberry and silkworm

Wolbachia is a ubiquitous, Gram-negative, vertically transmitted, alpha-proteobacterium that causes an array of reproductive abnormalities including cytoplasmic incompatibility, feminization of genetic males, parthenogenesis in a number of insect species, among others. Wolbachia is now being exploited as an agent for pest and vector control. Previous surveys indicated that it is commonly seen in 16–76% of arthropods. In this paper, using polymerase chain reaction assay based on specific amplification of the ftsZ-A and-B supergroup Wolbachia gene fragments, we found that 30% of insects and pests screened were positive for Wolbachia. Among them 66.7% harbour double Wolbachia infection, while 33.3% harbour single Wolbachia infection. These results indicate widespread infection with both double and single Wolbachia, and provide a wealth of information to exploit this endobacterium for the management of pests and vectors.     

Factors Affecting the Strength of Cardinium-Induced Cytoplasmic Incompatibility in the Parasitic Wasp Encarsia pergandiella (Hymenoptera: Aphelinidae)

Bacteria that cause cytoplasmic incompatibility (CI) are among the most common maternally transmitted parasites of insects. In CI, uninfected females produce few or no offspring when they mate with infected males and, as a result, are often at a reproductive disadvantage relative to infected females. Two different bacteria are known to cause CI, Wolbachia and Cardinium. CI Cardinium was discovered more recently and has been little studied. Here, factors that could influence the reduction in reproductive output in a CI cross, or CI “strength,” were explored in the parasitic wasp Encarsia pergandiella. Cardinium in this wasp exhibits variable CI strength. Experiments tested the effect of male age, male size, male host species, Cardinium density, and male development time on CI strength. We found a striking effect of male development time, with males that took longer to develop exhibiting stronger CI when mated to uninfected females. Male age had little effect; although in one experiment, the oldest males exhibited stronger CI. Male size, host species, and bacterial density had no effect on the strength of CI. Identifying the factors that control CI are crucial for understanding the dynamics of infection, as well as the success of strategies that aim to use CI microbes to control insect pests and disease vectors.